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5-2009 DEVELOPMENT OF STRATEGIES FOR ENHANCED IN SITU BIOREMEDIATION OF HIGH CONCENTRATIONS OF HALOGENATED METHANES Huifeng Shan Clemson University, [email protected]

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DEVELOPMENT OF STRATEGIES FOR ENHANCED IN SITU BIOREMEDIATION OF HIGH CONCENTRATIONS OF HALOGENATED METHANES

A Dissertation Presented to the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Environmental Engineering and Earth Science

by Huifeng Shan May 2009

Accepted by: Dr. David L. Freedman, Committee Chair Dr. Harry D. Kurtz, Jr. Dr. Cindy M. Lee Dr. Elizabeth R. Carraway

ABSTRACT

Bioremediation of high concentrations of halomethanes e.g., carbon tetrachloride

(CT), trichlorofluoromethane (CFC-11) and chloroform (CF) has seldom been addressed

before and remains highly challenging. A microcosm study was conducted to investigate

bioremediation strategies for groundwater contaminated with CT (6 to 10 mg/L), CFC-11

(1 to 26 mg/L), CF (3-500 mg/L), and 1,1-dichloroethene (1,1-DCE; up to 9 mg/L) at a

former industrial site in California. Biostimulation with corn syrup and catalytic amounts

of vitamin B 12 was demonstrated as a feasible remedial strategy and bioaugmentation with B 12 represents the most promising bioremediation method of the ones studied.

Halomethane transformation occurred sequentially, i.e., CT was transformed first, followed by CFC-11, then CF. 14 C analyses revealed that the dominant end products of

CT and CF transformation were CO, CO 2 and organic acids. Bioaugmentation with enriched indigenous sulfate-reducing cultures grown on lactate or ethanol without prior acclimation to halomethanes was effective in enhancing transformation of CT and CFC-

11, although limitations existed. In addition, a fermentative enrichment culture appeared to be a promising bioaugmentation culture for transforming high concentrations of CF.

Bioremediation of high concentrations of CF in groundwater is challenging because of its high toxicity and inhibitory effect on most anaerobic prokaryotes.

Enrichment and field applications of cultures that can transform high concentrations of

CF (>100 mg/L) are not currently available. DHM-1, a fermentative enrichment culture grown on corn syrup, was developed in this study by using soil samples from the

California industrial site. DHM-1 was demonstrated to transform over 500 mg/L CF in

ii the presence of catalytic amounts of vitamin B12 at a rate as high as 22 mg/L/d in a mineral salts medium. Production of non-toxic end products, i.e., CO, CO 2, and organic acids indicates hydrolytic reactions as the dominating pathway. DHM-1 is facultative and can grow on corn syrup while transforming high concentrations of CF, which makes it a promising culture for bioaugmentation. The two strains isolated from DHM-1 are related to Pantoea spp., and both possess equal or even slightly better CF transforming capability than the DHM-1 enrichment. Development of DHM-1 and isolation of the two

Pantoea spp. makes bioremediation of source zone CF contamination possible for the first time.

Following the development and characterization of DHM-1, the capacity of

DHM-1 for biotransformation of high concentrations of halogenated methanes was explored. With the addition of catalytic amounts of vitamin B 12 , DHM-1 can readily transform at least 2000 mg/L CF, which is equal to one fourth of the CF solubility in water. In addition, DHM-1 is able to transform CT and CFC-11 as well. Complete transformation of a mixture of 11 mg/L CT, 24 mg/L CFC-11 and 500 mg/L CF was achieved in less than four months by DHM-1 in a mineral salts medium. This study also demonstrated the capability of SDC-9, a commercially available mixed culture, to transform high concentrations of halomethanes. DHM-1 has an advantage over SDC-9 in terms of the rate of CF transformation, while SDC-9 exhibited better kinetics with CFC-

11. A second microcosm study with soil and water from the California industrial site further demonstrated the use of these two cultures for bioaugmentation to enhance in situ bioremediation of groundwater contaminated by high concentrations of halomethanes.

iii This study for the first time makes source zone bioremediation of halomethanes

contamination possible. DHM-1 and the two isolates (patent pending) developed in this

study are the first mixed and pure cultures, respectively, reported to biotransform high

concentrations of CF and other halomethanes to non-toxic end products. DHM-1 is a

promising bioaugmentation culture for in situ bioremediation of high concentrations of halomethanes owing to its distinctive characteristics.

iv DEDICATION

This dissertation is dedicated to my wife, Yue Luo; my parents, Xiaoquan Shan and Longzhu Jin; and my son, Allen Yitian Shan. This work cannot be done without their consistent encouragement and love.

v ACKNOWLEDGEMENTS

I wish to express my sincere thanks to my advisor Dr. David L. Freedman for his advice, patience and guidance through my pursuit of the Ph.D. degree. I would also like to acknowledge my committee members, Dr. Harry Kurtz, Dr. Cindy Lee, and Dr.

Elizabeth Carraway for their valuable input into this work. I would like also thank Dr.

Yanru Yang for her assistance and advice with molecular methods.

I gratefully acknowledge Dr. Harry Kurtz for his important contribution to the molecular work involved in this study and identification of the isolates; MACTEC, Inc., for providing financial and logistical support to conduct the microcosm study reported in

Chapter 2; and Dr. Rob Steffan at The Shaw Group, Inc., for providing samples of SDC-9.

I would like to thank my lab mates during my time at Clemson University, i.e.,

James Henderson, Ramona Darlington, Tess Brotherson, Vijay Elango, Ashley Eaddy,

Jessica High, Rong Yu, etc. for making the environment in our lab friendly and collaborative. I would like to thank Audrey Bone and Bridget O ′Donoghue for their assistance in sample preparation and analyses.

vi TABLE OF CONTENTS

Page

TITLE PAGE ...... i

ABSTRACT ...... ii

DEDICATION ...... v

ACKNOWLEDGEMENTS ...... vi

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

LIST OF ABBREVIATIONS ...... xiv

1. INTRODUCTION AND OBJECTIVES ...... 1

2. EVALUATION OF STRATEGIES FOR ANAEROBIC BIOREMEDIATION OF HIGH CONCENTRATIONS OF HALOMETHANES ...... 12

2.1 Abstract ...... 12 2.2 Introduction ...... 12 2.3 Materials and methods ...... 15 2.3.1 Chemicals and radioisotopes ...... 15 2.3.2 Soil and groundwater ...... 16 2.3.3 Microcosms ...... 17 2.3.4 Analysis of volatile organic compounds ...... 19 2.3.5 Analysis of 14 C-labeled compounds ...... 20 2.3.6 Development of enrichment cultures ...... 22 2.3.7 Denaturing gradient gel electrophoresis (DGGE) and protein analysis ...... 25 2.3.8 Dissolved organic carbon, organic acids, and anions ...... 25 2.4 Results ...... 26 2.4.1 Microcosm response to biostimulation and bioaugmentation ...... 26 2.4.2 First order rate constants of halomethane removal ...... 32 2.4.3 Distribution of products from [ 14 C]CT and [ 14 C]CF ...... 33 2.4.4 Electron donor utilization and sulfate consumption ...... 35 2.4.5 DGGE ...... 37 2.5 Discussion ...... 37 2.6 Tables and Figures ...... 45

vii 3. ANAEROBIC BIOTRANSFORMATION OF HIGH CONCENTRATIONS OF CHLOROFORM BY AN ENRICHMENT CULTURE AND TWO ISOLATES ...... 55

3.1 Abstract ...... 55 3.2 Introduction ...... 55 3.3 Materials and methods ...... 57 3.3.1 Chemicals, radioisotopes and culture media ...... 57 3.3.2 Development of an enrichment culture acclimated to CF ...... 58 3.3.3 Pure culture isolation and acclimation to CF ...... 59 3.3.4 CF biotransformation ...... 60 3.3.5 Analytical methods and 14 C distribution ...... 61 3.3.6 Denaturing gradient gel electrophoresis (DGGE) and protein analysis ...... 63 3.3.7 Identification of isolates ...... 64 3.4 Results ...... 64 3.4.1 Development of DHM-1 ...... 64 3.4.2 Product distribution from [ 14 C]CF ...... 65 3.4.3 Growth of DHM-1 while transforming CF ...... 65 3.4.4 Importance of sulfide to CF transformation ...... 66 3.4.5 DGGE Analysis of DHM-1 ...... 67 3.4.6 Isolate identification and CF transformation ...... 67 3.5 Discussion ...... 68 3.6 Figures...... 74

4. EVALUATION OF CAPACITY OF DHM-1 FOR TRANSFORMING HIGH CONCENTRATIONS OF HALOMETHANES IN MEDIA AND MICROCOSM AND COMPARISON TO A COMMERCIAL CULTURE .... 79

4.1 Abstract ...... 79 4.2 Introduction ...... 80 4.3 Materials and methods ...... 81 4.3.1 Chemicals, radioisotopes, enrichment cultures and culture media ...... 81 4.3.2 Transformation experiments with the enrichment cultures ...... 83 4.3.3 Microcosm evaluation of bioaugmentation with DHM-1 and SDC-9 ...... 84 4.3.4 Analytical methods and 14 C distribution ...... 87 4.3.5 Sulfide demand test and dissolved sulfide measurement ...... 88 4.4 Results ...... 88 4.4.1 Biotransformation of CT, CFC-11 and CF by DHM-1...... 88 4.4.2 Biotransformation of halomethane mixtures by DHM-1 ...... 90 4.4.3 Transformation rates ...... 93 4.4.4 14 C product distribution ...... 94 4.4.5 Microcosm evaluation of bioaugmentation ...... 94 4.5 Discussion ...... 97 4.6 Tables and figures ...... 101

viii 5. CONCLUSIONS...... 110

6. APPENDICES ...... 114

A. Supplementary results of Chapter 2 ...... 115 A.1 GC headspace results of individual microcosms in Chapter 2 ...... 115 A.2 Data used to determine first order rate constants ...... 150 A.3 Sample data used to determine the first order transformation rate constants ...... 168 A.4 Percent removal of halogenated compounds in the microcosm study in Chapter 2 ...... 170 A.5 Characteristics of the soils used to prepare the microcosms...... 171 A.6 Characteristics of the precipitates formed by pH adjustment ...... 172 A.7 Organic acids analysis for the microcosms in Chapter 2 at the end of incubation ...... 173 A.8 Fate of lactate in bioaugmentation microcosms ...... 174 B. Supplementary data for Chapter 3 ...... 175 B.1 Comparison of growth pattern of DHM-1 with and without CF ...... 175 B.2 Effect of addition of sulfide on CF transformation by DHM-1 in MSM ..... 176 B.3 CF-transformation ability of DHM-1 after 24 h exposure to air and then returning them to anaerobic MSM ...... 177 B.4 Sequences of partial 16S rRNA gene of the six bands of DHM-1 (Figure 3-4) cut off from DGGE ...... 178 B.5 Complete sequences of 16S rRNA gene of the two Pantoea strains ...... 180 B.6 Growth curves of the two Pantoea strains ...... 181 C. Supplementary data for Chapter 4 ...... 182 C.1 Media controls (without B 12 ) for CT, CFC-11 and CF ...... 182 C.2 Transformation of individual halomethane by SDC-9 ...... 183 D. Protocols ...... 184 D.1 PCR protocol using Eppendorf Master Mix ...... 184 D.2 DGGE protocol ...... 185 D.3 Preparation of the medium for DHM-1 ...... 188 D.4 Preparation of RAMM for SDC-9 ...... 189 D.5 Transferring DHM-1 ...... 190 D.6 Dissolved sulfide measurement ...... 191 E. Supplementary experimental results ...... 192 E.1 Amount of corn syrup added in each transfer of DHM-1 to transform 500 mg/L CF ...... 192 E.2 Ability of a culture that grows anaerobically on DCM to use chlorofluoromethane ...... 193 E.3 Response factors of GC ...... 195

7. REFERENCES ...... 196

ix LIST OF TABLES

Table Page

Table 2-1 Groundwater pH adjustment...... 45

Table 2-2 Target concentrations of halogenated contaminants in the microcosms ...... 46

Table 2-3 Products from transformation of [ 14 C]CT ...... 47

Table 2-4 Products from transformation of [ 14 C]CF...... 48

Table 2-5 DOC and sulfate removal for different treatment strategies ...... 49

Table 2-6 Reactions involved in the transformation of halomethanes and associated Gibbs free energy changes a ...... 50

Table 4-1 Experimental design for the microcosm study ...... 101

Table 4-2 Halomethane transformation rates (mg/L/d) ...... 102

Table 4-3 Comparison of end product distribution of CT and CF transformation by DHM-1 and SDC-9 ...... 103

Table 6-1 Percent removal of parent contaminants in the microcosm study in Chapter 2 ...... 170

Table 6-2 Characteristics of the soils used to prepare the microcosms a ...... 171

Table 6-3 Composition of the precipitates (%) ...... 172

Table 6-4 Organic acids analysis for the microcosms in Chapter 2 at the end of incubation ...... 173

Table 6-5 Minimal salts medium for DHM-1 ...... 188

Table 6-6 Response factors of GC for 160 mL serum bottles with 50 mL liquid phase and 99 mL headspace ...... 195

Table 6-7 Response factors of GC for 160 mL serum bottles with 100 mL liquid phase and 60 mL headspace ...... 195

x LIST OF FIGURES

Figure Page

Figure 1-1 Pathways for biotransformation of CT, CF, and CFC-11 under low redox conditions. Further reduction of DCM to chloromethane and methane is possible, but occurs at very slow rates. Numbers in parentheses represent oxidation states of the carbon, with the exception of organic acids; i.e., not all of these have carbon with an overall oxidation state of zero. Dotted lines indicate presumed pathways...... 11

Figure 2-1 Representative performance of microcosms with high concentrations of halomethanes, treated by biostimulation ( a) and bioaugmentation ( b); arrow= addition of electron donor (↓=corn syrup, ↓= lactate, ↓= ethanol); =addition of culture (C1= lactate SRB, C2= ethanol SRB, C3=corn syrup fermentative, and C4=1,1-DCE respiring culture)...... 51

Figure 2-2 Representative performance of microcosms with medium concentrations of halomethanes, treated by biostimulation (a) and bioaugmentation (b); arrow= addition of electron donor ( ↓=corn syrup, ↓= lactate, ↓= ethanol; =addition of culture (C1=SRB lactate, C2=SRB ethanol, C3=corn syrup fermentative, and C4=1,1-DCE respiring culture)...... 52

Figure 2-3 Comparison of first-order rate constants for CT (a), CFC-11 (b), and CF (c) with different treatments, for high, medium and low concentrations. Error bars are one standard deviation of six microcosms...... 53

Figure 2-4 DGGE results for biostimulation microcosms. Lanes 1-3 are for microcosms with low concentrations of halomethanes, 4-6 for medium concentrations, and 7-9 for high concentrations; lanes 1, 4 and 7 are for the as-is treatment; lane 2, 5 and 8 are for biostimulation with corn syrup; and land 3, 6, and 9 are for biostimulation with corn syrup + B 12 . Bands that were identified are labeled a-i...... 54

Figure 3-1 Increase in the rate of CF transformation during initial development of the DHM-1 enrichment culture (a) and following a transfer (1% v/v) in MSM (b). Results in panel b are the average of duplicate bottles; error bars represent the data range...... 74

Figure 3-2 Effect of CF on growth of DHM-1 based on protein yields after 35-39 days of incubation (a); and associated CF transformation in the three live treatments and media control with B 12 but no culture (MC+B 12 ) (b); blank=live culture + MSM only; CS=corn syrup; error bars are the

xi standard deviation for duplicate measurements from duplicate bottles. Corn syrup was added on days 0 and 22...... 75

Figure 3-3 Growth of DHM-1 while transforming CF. The average of duplicate bottles are shown. Error bars are the standard deviation for duplicate measurements from duplicate bottles. Corn syrup was added only on day 0...... 76

Figure 3-4 Variation of microbial structure of DHM-1 along with transfers and comparison among the mixed culture and its isolates by DGGE. Lane 1, 2, and 3 are the fourth, sixth, and tenth transfers of DHM-1, respectively; lane 4 is strain DHM-1B, lane 5 is strain DHM-1T...... 77

Figure 3-5 CF transformation by the two Pantoea strains. Formation of DCM and CM was minor (< 1% of CF transformed). Results are the average of duplicate bottles; error bars represent the data range...... 78

Figure 4-1 Transformation of CT (a), CFC-11 (b) and CF (c) by DHM-1 in MSM with corn syrup and B 12 added. LC=live control, MC+B 12 =media control with B12 . Daughter products formed only in the DHM-1 + B 12 treatments. ↓=addition of corn syrup; B 12 was added only at t=0. Error bars are the data range for duplicate bottles...... 104

Figure 4-2 CF transformation by DHM-1 in MSM with corn syrup and B 12 added (at t=0), at varying initial CF concentrations. Arrows indicate addition of corn syrup. Error bars represent the data range for duplicate bottles...... 105

Figure 4-3 Performance of DHM-1 in transforming mixtures of CT + CF (a), CFC-11 + CF (b) and CT + CFC-11 (c) in MSM and B 12 added (t=0). Arrows indicate addition of corn syrup...... 106

Figure 4-4 Behavior of a mixture of halomethanes in controls (a, b and c) and live bottles with DHM-1 and corn syrup + B 12 in MSM (d). ↓ = addition of corn syrup; = addition of B 12 ; = reinoculation with DHM-1; WC=water controls, MC+B 12 = media control with B 12 , and AC=autoclaved control with B 12 . Error bars are the data range for duplicate bottles...... 107

Figure 4-5 Performance of biostimulation (a-c) and bioaugmentation with DHM-1 (d-f) and SDC-9 (g-i) in microcosms. ↓ = addition of electron donor; =addition of B 12 ; =reinoculation; = addition of sulfide...... 108

Figure 4-6 Sulfide demand of the soil used in the microcosms. The dashed line at 0.5 mM represents the target concentration for microcosms...... 109

xii Figure 6-1 GC headspace results of all individual microcosms in the microcosm study in Chapter 2 ...... 149

Figure 6-2 Data used to determine first order rate constants for the microcosm study in Chapter 2 ...... 167

Figure 6-3 Sample data of first order transformation following a lag phase for a biostimulation microcosm with corn syrup plus B 12 (CF-H-B12 -1) ...... 168

Figure 6-4 Sample data of first order transformation following a lag phase for a bioaugmentation microcosm (CF-H-SRB-1) ...... 169

Figure 6-5 Analysis of the precipitate generated during pH adjustment of groundwater172

Figure 6-6 Fate of lactate in the high (a) and medium (b) concentration bioaugmentation microcosms (in chapter 2) ...... 174

Figure 6-7 Comparison of growth pattern of DHM-1 with and without CF ...... 175

Figure 6-8 Effect of sulfide (0.5 mM) on CF transformation by DHM-1 in MSM ...... 176

Figure 6-9 CF-transformation ability of DHM-1 after 24 h exposure to air and then returning them to anaerobic MSM, in comparison with no air exposure 177

Figure 6-10 Growth curves of the two DHM-1 isolates, i.e., DHM-1 B and DHM-1T, in MSM + corn syrup (960 mg COD/L) and B 12 (3% mole ratio), with and without CF (500 mg/L) ...... 181

Figure 6-11 Media controls (without B 12 ) for CT, CFC-11 and CF ...... 182

Figure 6-12 Transformation of individual halomethane by SDC-9 in RAMM, LC=live control, MC+B 12 =media control with B 12 . Daughter products formed only in the DHM-1 + B 12 treatments...... 183

Figure 6-13 Test of ability of a culture that grows anaerobically on DCM to use chlorofluoromethane. Where, DCM-A: CH 2ClF was added together with DCM after growth on DCM was established; DCM-B: only CH 2ClF was added after growth on DCM was established ...... 194

xiii LIST OF ABBREVIATIONS

Acronym Definition

AC autoclaved control(s) CF chloroform CFC-11 trichlorofluoromethane CM chloromethane COD chemical oxygen demand CT carbon tetrachloride 1,1-DCE 1,1-dichloroethene DCM dichloromethane DGGE denaturing gradient gel electrophoresis DOC dissolved organic carbon dpm disintegrations per minute GC gas chromatograph HCFC-21 dichlorofluoromethane HCFC-31 chlorofluoromethane HPLC high performance liquid chromatograph LSC liquid scintillation cocktail MCL maximum contamination limit MSM mineral salts medium NSR nonstrippable residue PCE tetrachloroethene RAMM revised anaerobic mineral medium SRB sulfate reducing bacteria VC vinyl chloride WC water control(s)

xiv 1. INTRODUCTION AND OBJECTIVES

In spite of major recent advances in bioremediation of contaminated groundwater, significant challenges remain. One of these challenges involves developing effective methods to bioremediate halogenated methanes, including carbon tetrachloride (CT), chloroform (CF), and trichlorofluoromethane (CFC-11). Although halogenated methanes receive less attention than chlorinated ethenes, they are among the most significant groundwater contaminants. The Superfund Act requires preparation of a list each year of substances found at facilities on the National Priority List that pose the most significant potential threat to human health. In the 2007 Priority List of Hazardous Substances of the

Environmental Protection Agency, CF ranks the third highest among chlorinated organics, after vinyl chloride and polychlorinated biphenyls (8). This is a reflection of how frequently CF is detected in groundwater and its high level of toxicity. Although not ranked as highly, CT is also on the list, and CFC-11 is often a co-contaminant with CT and CF. CT had a long history of use as an industrial solvent and as a dry-cleaning agent before it was banned in 1970 in consumer products in the U.S., followed by a complete ban in 1996 according to the Montreal Protocol. Improper disposal, spills and leaks from underground storage tanks have resulted in widespread contamination of groundwater.

CFC-11, also known as Freon ®-11, has been extensively used as a foam-blowing agent, an aerosol propellant and a refrigerant. Its well-known adverse environmental impacts include ozone layer depletion (5) and contribution to global warming (29).

All of these contaminants can be transformed by subsurface anaerobic microorganisms to nonhazardous products, although some of the pathways involved for

1 halogenated methanes are considerably different from the reductive dechlorination process that predominates with chlorinated ethenes. Among these compounds, CF is the focal point for evaluating the feasibility of bioremediation, since it is very toxic to most obligate anaerobic prokaryotes. For example, only 1 mg/L of CF completely inhibited tetrachloroethene dechlorination by a mixed chlororespiring anaerobic culture (73).

Inhibition of reductive dechlorination of chloroethenes by CF is a general problem for sites co-contaminated with CF, and can only be overcome by removing the CF first (73).

In other words, until a substantial decrease in CF is achieved, little biological activity is likely to occur on the other contaminants. Inhibitory effects of CT (20) and CFC-11 (57) on anaerobes at low concentrations have been reported as well.

At this point in time, it appears that anaerobic biotransformation of CT, CF and

CFC-11 is strictly a cometabolic process. No cultures have been shown capable of using these compounds as growth-linked substrates. Use of CT and CF as terminal electron acceptors is theoretically possible, as has been demonstrated with chlorinated ethenes via chlororespiration (49, 72). Use of dichloromethane (DCM) as a sole carbon and energy source (i.e., electron donor) under fermentative conditions is well established (14).

Although this is theoretically possible with CF, it has not been shown (12) and is unlikely due to the much higher toxicity of CF compared to DCM. Therefore, bioremediation of

CT, CFC-11 and CF is more challenging than that of chloroethenes and DCM because cometabolic transformations are more difficult to engineer than metabolic processes.

Aerobic transformation of CT and CFC-11 is unfavorable because of their fully oxidized carbons. Although aerobic biotransformation of CF is possible (e.g.,

2 cometabolism by a butane-grown strain (30)), CF is more difficult to cometabolize than trichloroethene (88), in part due to its more toxic transformation intermediate (i.e., phosgene versus TCE-epoxide). Thus, most studies on biotransformation and bioremediation of these halomethanes are limited to anaerobic conditions.

The most economical bioremediation strategy is monitored natural attenuation

(MNA). A premise of MNA is the presence of high background levels of organic compounds, i.e., electron donors, either within the contaminant plume or the aquifer through which the plume passes. Moreover, MNA usually would not be an option for highly contaminated sites because it could take an unreasonably long period of time to clean them up. It is, therefore, unrealistic to rely on MNA to remediate any plumes contaminated with high concentrations of halomethanes. More aggressive strategies are needed.

The only way known to date to enhance anaerobic biotransformation of tetra- and trihalomethanes is by biostimulation. Many organic substrates have proven effective at biostimulating CT and CF transformation, including alcohols (e.g., methanol (9)), sugars

(e.g., fructose (47)) and organic acids (e.g., acetate (24, 36)). Hydrogen is also effective, although it is not considered a “universal donor” for CT and CF dechlorination the way it is for chlorinated ethenes. One of the objectives of this dissertation was to compare the effectiveness of several electron donors for anaerobic transformation of halomethanes.

Corrinoids such as cyanocobalamin (i.e., vitamin B 12 ) are effective catalysts (even at micromolar concentrations) for increasing the rate of CT and CF biotransformation.

The effect of B 12 on accelerating transformation of CFC-11, however, has not yet been

3 investigated. Addition of cyanocobalamin also shifts the pathway away from reductive

dechlorination and towards formation of environmentally benign products such as CO,

CO 2 and organic acids (12, 47, 48). Although the high unit cost of cyanocobalamin is a concern, its effectiveness has been demonstrated at the field scale with in situ dechlorination of CT and 1,1,1,2-tetrachloroethane. Cyanocobalamin was added with titanium citrate and glucose in a recirculating well at Aberdeen Proving Grounds, MD, with promising results (63). Delivery of cyanocobalamin along with an electron donor is likely to be technically feasible because it is highly soluble and does not adsorb significantly to most aquifer solids (46).

The potential for using bioremediation to remediate groundwater contaminated with high concentrations of halomethanes is being considered for an industrial site in

California. This site represents a complicated contamination scenario, with halomethanes at concentrations ranging from 6-10 mg/L of CT, 1-26 mg/L of CFC-11, and 3-500 mg/L of CF, and up to 9 mg/L 1,1-dichloroethene. Current groundwater conditions are highly unfavorable to bioremediation, including pH levels as low as 3, total halomethanes concentrations above 500 mg/L, and positive E h levels. Because these conditions have existed for several decades, it seems likely that the population of indigenous microorganisms is quite low and, therefore, bioaugmentation may be required, although bioaugmentation cultures have not been evaluated for this purpose. One of the objectives of this dissertation was to develop a bioaugmentation culture that may be used for treating high concentrations of halomethanes.

4 The highest concentrations listed above for the California site suggest the

presence of dense nonaqueous phase liquids, based on aqueous phase concentrations that

exceed one percent of water saturation (i.e., approximately 800 mg/L for CT; 8000 mg/L

for CF, and 1100 mg/L for CFC-11). At concentrations this high, it may seem

impractical to consider bioremediation, due to the inherent toxicity of these lipophilic

compounds. However, a growing body of evidence indicates that bioremediation at

concentrations approaching those found near source zones (i.e., close to water saturation)

is feasible with chlorinated ethenes (51). Indeed, reductive dechlorination of the

chlorinated ethenes enhances dissolution of the nonaqueous phase. It is not yet known if

microbes capable of transforming halomethanes can also function at such high

concentrations. A key difference is the fact that chlorinated ethenes are typically used as

growth-linked terminal electron acceptors during chlororespiration, while transformation

of CT, CFC-11 and CF is cometabolic. This means there may be less “incentive” for the

microbes to transform the contaminants at the high concentrations found near source

zones. One of the objectives of this dissertation was to evaluate the tolerance of an

enrichment culture that transforms CF to concentrations as high as 50% of water

saturation.

Among the tetrahalo- and trihalomethanes, CT appears to undergo

biotransformation most readily. Acetobacterium woodii (47) and sulfate reducing enrichment cultures developed by Freedman et al. (36) (using anaerobic digester sludge from a municipal wastewater treatment plant as inoculum) can transform 92 and 350 mg/L CT in the presence of catalytic amounts of hydroxocobalamin or cyanocobalamin,

5 respectively. Nonetheless, transformation of CT by Acetobacterium woodii was tested at

35 °C and no studies were found that evaluated the ability of A. woodii to transform CF.

The sulfate reducing culture is not suitable for in situ application because the main

product formed was carbon disulfide (CS 2) (73%); although CS 2 is not regulated as a

drinking water contaminant, it is a potent neurotoxin and is, therefore, an undesirable

transformation product. Few studies have investigated anaerobic biotransformation of

CFC-11 or CF close to the very high concentrations reported at the California industrial

site mentioned above. Becker and Freedman (12) were able to biotransform up to 260

mg/L of CF in an enrichment culture (grown on DCM as the sole substrate) when vitamin

B12 was also added. Under these conditions, less than 1% of the CF accumulated as

DCM, chloromethane, or methane. Corresponding experiments with [ 14 C]CF identified

14 14 the main product as CO 2, followed by CO and soluble products (presumptively

organic acids). However, use of this culture is not practical for in situ bioremediation,

since it requires DCM as a growth substrate and only grows well at 35 oC. The above-

mentioned sulfate reducing culture can also transform up to 270 mg/L CF, although the

predominant product is DCM, which is not an acceptable endpoint (36). The highest

concentration of CFC-11 evaluated in previous biotransformation studies is 2.2 mg/L, an

order of magnitude lower than the highest concentration found at the industrial site

mentioned above. At low concentrations, Methanosarcina barkeri transformed CFC-11

via reductive dechlorination to dichlorofluoromethane (HCFC-21) (66%) as the dominant

end product (57). Halogenated methanes are not acceptable endpoints for successful

bioremediation.

6 A summary of the biotic and non-enzymatic pathways involved in transformation of CT, CF and CFC-11 under low redox anaerobic conditions (i.e., E h < -100 mV) is shown in Figure 1-1. Reductive dechlorination of CT to CF is often a significant pathway, but unlike the chlorinated ethenes, reductive dechlorination of CF may not be the dominant pathway. Depending on environmental conditions, net hydrolysis of CF to

CO can be extensive, with subsequent oxidation of CO to CO 2 and use of the resulting equivalents to form organic acids. Likewise, CT can undergo reductive hydrolysis to CO.

In the presence of sulfide and a coenzyme, e.g., vitamin B 12 , or surface catalysts, e.g., biotite (56) and pyrite (55), significant conversion of CT to CS 2 is also possible. Abiotic transformation of CS 2 to CO 2 is possible under alkaline conditions.

Reductive dechlorination of CF to DCM is also an important pathway, but it rarely occurs stoichiometrically. Furthermore, reductive dechlorination of DCM to chloromethane is a minor pathway, and methane is a trivial overall reductive dechlorination product. Instead, under low redox conditions, DCM may undergo fermentation (as a sole source of carbon and energy) to acetate and formate. Formate can subsequently be fermented to CO 2 + H 2 and other organic acids (14, 33, 34, 70-72).

The pathways shown in Figure 1-1 indicate that completing a mass balance for the daughter products from CT and CF is more complicated that simply monitoring halogenated methanes. A complete mass balance is feasible only under controlled conditions, such as in a laboratory microcosm study, using 14 C-labeled parent compounds.

[14 C]CT and [ 14 C]CF are commercially available at a reasonable cost while [ 14 C]CFC-11 is available only by custom synthesis, making its use prohibitively expensive for typical

7 microcosm studies. Using 14 C-labeled parent compounds makes it possible to monitor

14 14 14 14 products such as CO, CO 2, C-labeled organic acids, and C assimilated into biomass. Each of these products can be formed by fermentation and anaerobic respiration of nonhalogenated substrates, so the use of 14 C-labeled parent compounds is essential to delineate their origin.

CFC-11 undergoes similar transformations to CT. However, since dechlorination is more thermodynamically favorable than defluorination, HCFC-21 and chlorofluoromethane (HCFC-31) tend to accumulate. As with CT, reductive hydrolysis of CFC-11 to CO is well documented (58), as shown in Figure 1-1. Net hydrolysis of

HCFC-21 to CO is also possible. While fermentation of DCM to organic acids is well established, it is not yet known if the same types of microbes (e.g., Dehalobacterium formicoaceticum ) can also use chlorofluoromethane as a substrate (hence the question mark on Figure 1.1 for this transformation).

The overall objective of this dissertation was to develop improved strategies for in situ bioremediation of high concentrations of halomethanes that occur as co-contaminants with chlorinated ethenes. A key component of this approach was the development of an enrichment culture for use in bioaugmentation. The specific objectives were:

1. To determine if in-situ biostimulation with substrates such as corn syrup, lactate

and emulsified vegetable oil can stimulate the growth of microbes that transform

halomethanes and evaluate the microbial community response to biostimulation.

This objective is addressed in Chapter 2.

8 2. To determine if adding catalytic amounts of vitamin B 12 along with an electron

donor can significantly enhance the rate of halomethane transformation and

minimize the accumulation of halogenated daughter products. This objective is

addressed in Chapter 2.

3. To determine if bioaugmentation with sulfate-reducing or fermentative

enrichment cultures (developed using indigenous microbes from the contaminated

industrial site in California) with vitamin B 12 addition can result in a faster rate of

biotransformation without accumulation of toxic daughter products. This

objective is addressed in Chapter 2.

4. To develop and characterize an enrichment culture that is relevant to use in

bioaugmentation, including its ability to transform CF at concentrations as high as

50% of water saturation. This objective is addressed in Chapters 3 and 4.

5. To isolate and characterize the microbes that are responsible for CF

transformation from the enrichment culture. This objective is addressed in

Chapter 3.

6. To determine the capacity of the enrichment culture for transforming high

concentrations of halomethanes. This objective is addressed in Chapter 4.

7. To compare the enrichment culture developed during this research with a

commercially available culture in terms of their ability to transform high

concentrations of halomethanes, in both defined media and in microcosms. This

objective is addressed in Chapter 4.

9 Chapters 2, 3 and 4 are “stand-alone”, i.e., each presents an introduction, materials and methods, results and discussion section that fully address one or several of the specific objectives. Chapter 5 provides a comprehensive list of conclusions for this dissertation.

10

4HCl or - - 2H 2S 3HCl + HF 2OH 2HS

CCl 3F CCl 4 CS 2 CO 2 (+4) 2[H] 2[H] 2[H] + H 2O 2[H] + H 2O

HF + 2[H] HCl 3HCl HCl 4HCl

CHCl 2F CHCl 3 CO H O 3HCl or 2[H] 2[H] 2 4[H] 2HCl + HF

HCl HCl ? organic (+0) CH ClF CH 2Cl 2 2 acids H O 2HCl 2 (e.g., acetate, (HF?) formate )

Figure 1-1 Pathways for biotransformation of CT, CF, and CFC-11 under low redox conditions. Further reduction of DCM to chloromethane and methane is possible, but occurs at very slow rates. Numbers in parentheses represent oxidation states of the carbon, with the exception of organic acids; i.e., not all of these have carbon with an overall oxidation state of zero. Dotted lines indicate presumed pathways.

11 2. EVALUATION OF STRATEGIES FOR ANAEROBIC BIOREMEDIATION OF

HIGH CONCENTRATIONS OF HALOMETHANES

2.1 Abstract

Bioremediation of high concentrations of halomethanes e.g., >10 mg/L of

trichlorofluoromethane (CFC-11) or >100 mg/L chloroform (CF) has very seldom been

addressed before and remains highly challenging. This microcosm study is the first to

investigate bioremediation strategies to clean up groundwater contaminated with carbon

tetrachloride (CT) up to 10 mg/L, CFC-11 up to 26 mg/L, CF up to 500 mg/L, and 1,1-

dichloroethene (1,1-DCE; up to 9 mg/L). Biostimulation with corn syrup and catalytic

amounts of vitamin B 12 appeared to be a practical remedial method, while bioaugmentation with B 12 proved the most promising bioremediation strategy.

Halomethane transformation occurred sequentially, i.e., CT, then CFC-11, then CF. 14 C analyses revealed that the dominant end products of CT and CF transformation are CO,

CO 2 and organic acids. Bioaugmentation with enriched indigenous sulfate-reducing bacteria (SRB) grown on lactate and ethanol without prior acclimation to halomethanes were effective in enhancing transformation of CT and CFC-11, although limitations existed. A fermentative enrichment culture acclimated to 500 mg/L of CF appeared to be a promising bioaugmentation culture for treatment of this toxic halomethane.

2.2 Introduction

In spite of recent advances in bioremediation of contaminated groundwater, significant challenges remain. One of these involves developing effective methods to bioremediate halogenated methanes, including CT, CF, and CFC-11. In the 2007

12 CERCLA Priority List of Hazardous Substances, CF ranks number 11, the third highest among chlorinated organics, after vinyl chloride (VC) and polychlorinated biphenyls.

This is a reflection of how frequently CF is detected in groundwater and its high level of toxicity. Although not ranked as highly, CT is also on the list, and CFC-11 is often a co- contaminant with CT and CF (8).

All of these contaminants can be transformed by subsurface anaerobic microorganisms to nonhazardous products, although some of the pathways involved for halogenated methanes are considerably different from the reductive dechlorination process that predominates with chlorinated ethenes. Among these compounds, CF is often the focal point for evaluating the feasibility of bioremediation, since it is very toxic to most obligate anaerobic prokaryotes. For example, only 1 mg/L of CF completely inhibited reductive dechlorination of tetrachloroethene (73). Inhibition of reductive dechlorination of chloroethenes by CF is a general problem for sites co-contaminated with CF, and can only be overcome by removing the CF first (73). Inhibitory effects of

CT (20) and CFC-11 (57) on anaerobes at low concentrations have been reported as well.

Aerobic transformation of CT and CFC-11 is unfavorable because of their fully oxidized carbons. Although aerobic biotransformation of CF is possible (e.g., cometabolism by a butane-grown strain (30)), CF is even more difficult to cometabolize than trichloroethene (88). Thus, most studies on biotransformation and bioremediation of these halomethanes are limited to anaerobic conditions. Anaerobic biotransformation of

CT, CF and CFC-11 appears to be achievable only by cometabolic processes.

Cometabolic transformation of high concentrations of nongrowth compounds is

13 especially challenging. The highest concentrations demonstrated with biotransformation

are 270 mg/L for CF using a dichloromethane (DCM)-grown culture (12) or lactate-

grown sulfate-reducing culture (36); 350 mg/L for CT using a lactate-grown sulfate-

reducing enrichment culture (36); and 2.2 mg/L for CFC-11 by Methanosarcina barkeri

(57). The cultures used with CT and CF are not generally applicable to groundwater

remediation (i.e., DCM was used as the growth substrate at 35 °C) or the products formed are not acceptable (i.e., CS 2 from CT or DCM from CF with the sulfate-reducing enrichment). Although studies have demonstrated that in situ biotransformation of CT

via bioaugmentation is achievable, the concentrations evaluated were considerably lower,

e.g., less than 5 mg/L (23, 24). Methods to achieve in situ biotransformation of high

concentrations of halomethanes to nonhazardous products have not yet been reported.

Addition of electron donor (i.e., biostimulation) is the only way known to enhance

anaerobic biotransformation of tetra- and trihalomethanes. Many organic substrates have

proven effective for this purpose, including alcohols (e.g., methanol) (9), sugars (e.g.,

fructose) (47) and organic acids (e.g., acetate) (36). Hydrogen is also effective, although

it is not considered a “universal donor” for halomethanes the way it is for chlorinated

ethenes. In addition to providing electron donors, addition of corrinoids such as

cyanocobalamin (i.e., vitamin B 12 ) has been shown to be advantageous. Catalytic levels

of B 12 increase the rate of halomethane biotransformation and shift the pathway away

from reductive dechlorination and towards hydrolytic and substitutive reactions forming

CO, CO 2, and organic acids (12, 47, 48). The importance of B 12 for chlororespiration of

chlorinated ethenes is well established (4) and B 12 is increasingly being included in

14 electron donor formulations used for in situ bioremediation (e.g., http://eosremediation.com/ ). Addition of B12 has also been used to enhance in situ abiotic transformation of CT (63).

The potential for bioremediation of groundwater contaminated with halomethanes is being considered for an industrial site in California. Concentrations range from 6-10 mg/L of CT, 1-26 mg/L of CFC-11, and 3-500 mg/L of CF. 1,1-Dichloroethene (DCE) is also present (10 mg/L), presumably from dehydrohalogenation of 1,1,1-trichloroethane.

Geochemical conditions are unfavorable to anaerobic bioremediation (e.g., acidic pH, high halomethanes concentrations, and positive E h levels) and have existed for decades.

The overall objective of this research was to develop a bioremediation strategy for remediation of high concentrations of halomethanes. This entailed evaluating several electron donors; testing the effectiveness of B 12 to enhance biotransformation; and evaluating several enrichment cultures for their use in bioaugmentation. The results revealed that corn syrup was the most effective of the substrates tested, catalytic levels of

B12 significantly enhanced the rates of halomethane transformation, bioaugmentation further enhanced the rates of transformation, and removal of halomethanes enabled subsequent reductive dechlorination of 1,1-DCE.

2.3 Materials and methods

2.3.1 Chemicals and radioisotopes

CT (99.9%, Sigma-Aldrich), CF (99.7%, Shelton Scientific), DCM (99.9%,

AlliedSignal), CFC-11 (99%, Aldrich), 1,1-DCE (99%, TCI America), and CS 2 (100%,

J.T.Baker) were obtained as neat liquids. Chloromethane (CM; 99%, Praxair),

15 dichlorofluoromethane (HCFC-21) (99%, SynQuest Labs), chlorofluoromethane (HCFC-

31) (99%, SynQuest Labs), VC (99.9%, Matheson), and methane (99.99%, Matheson)

were obtained as neat gases. Sodium lactate (60% syrup, EM Science), corn syrup

(regular type, Sweetener Products Company), and emulsified vegetable oil (standard type,

Newman Zone), were used as electron donors. The chemical oxygen demand (COD) for

corn syrup and emulsified vegetable oil was calculated based on their presumed

composition, i.e., C 6H12 O6 and C 8H16 O, respectively. Cyanocobalamin (i.e., vitamin B 12 ,

USP grade) was obtained from Research Organics, Inc. All other reagents were ACS grade or higher. Neat 14 C-labeled CT (1.0 mCi/mmol) and CF (0.5 mCi/mmol) were purchased from American Radiolabeled Chemicals, Inc. Stock solutions were prepared in deionized distilled water with approximately 10 7 disintegrations per minute (dpm) per mL.

2.3.2 Soil and groundwater

Soil and groundwater samples were obtained from three areas of the plumes at the former industrial site in California, corresponding to high, medium and low concentrations of halomethanes. Core samples of soil from the saturated zone were delivered in stainless steel sleeves (6 cm in diameter by 46 cm long) from 23-32 m below grade. Groundwater samples were obtained from monitoring wells closest to the core samples. Groundwater from the low and medium concentration areas had pH levels of

3.3-3.5 (Table 2-1). Anticipating that this would not be favorable to biostimulation or bioaugmentation, the pH was adjusted to near neutral using sodium carbonate. Notable amounts of an orange/brown precipitate formed (Table 2-1). This was consistent with the

16 high level of calcium (1300-2000 ppm), aluminum (210-470 ppm) and iron (87-170 ppm)

in the soils. Soil organic matter was below 0.2%, indicating a low background level of

electron donor needed for biotransformation of the halogenated contaminants. Nitrate in

the groundwater was below detection (1 mg/L); sulfate levels were approximately 3800,

2800 and 420 mg/L in the groundwater with low, medium and high concentrations of

halomethanes.

2.3.3 Microcosms

Eight types of treatments were evaluated (all were pH-adjusted): live with no

electron donor added (i.e., as-is); biostimulation with lactate; biostimulation with

emulsified vegetable oil; biostimulation with corn syrup; biostimulation with corn syrup

+ B 12 ; bioaugmentation (which also includes addition of lactate, ethanol, corn syrup and

B12 ); autoclaved controls (AC); and water controls (WC). Each treatment at each concentration range (Table 2-2) included two sets of triplicate microcosms; one set was prepared with [ 14 C]CF and another set with [ 14 C]CT, thereby permitting an evaluation of the fate of each compound based on 14 C.

Microcosms were prepared in 160 mL serum bottles with 20 g of soil and 50 mL of liquid (46.8-48.4 mL of pH-adjusted groundwater from the appropriate locations + 1.6-

3.2 mL of the halogenated compounds), in an anaerobic chamber (Coy Laboratories) containing an atmosphere of approximately 98% N 2 and 2% H 2, and sealed with 20-mm

Teflon-faced red rubber septa and aluminum crimp caps. The precipitate that formed in the groundwater due to pH adjustment was not separated from the groundwater, as would occur if the pH was adjusted in situ . Resazurin was added to the groundwater (1 mg/L) to

17 serve as a redox indicator. The volume of 20 g of soil was determined by volumetric displacement to be 11 mL. Consequently, the gas volume in the microcosms was approximately 99 mL. AC bottles contained soil and pH-adjusted groundwater and were autoclaved for 1 h on three consecutive days; halogenated compounds were added after autoclaving. WC bottles received only distilled deionized water (50 mL) and the halogenated compounds.

Analyses of groundwater as received from each plume location indicated that the concentrations of the halomethanes and 1,1-DCE in were below the target levels, which were based on the highest concentration of contaminants reported. To achieve the target initial concentrations (Table 2-2), water saturated solutions were used (1.6-3.2 mL/bottle) except for CF and CFC-11 in the high concentration bottles; in this case, neat compounds were added (5 µL CFC-11, 18 µL CF). Due to the high vapor pressure of CFC-11, it was necessary to cool the syringe and the neat compound to 4ºC prior to adding it to the microcosms.

The initial amount of electron donor provided was 500 mg/L as COD.

Subsequent additions of electron donors (250-500 mg COD/L per dose) were made at approximately 2-3 month intervals for the biostimulation microcosms, while more frequent additions were applied to bioaugmentation microcosms, especially after adding the cultures. [14 C]CT and [ 14 C]CF were added using stock solutions such that the initial level of activity was approximately 1.3 x 10 6 dpm per bottle. Subsequently, the microcosms were placed on a shaker table for approximately 15 min, incubated

18 quiescently overnight, and then analyzed for the initial total amount of dpm and volatile organic compounds.

The pH of the microcosms was tested approximately once per month using 20 µL samples and pH strips. When the pH dropped below 6.5, 8M NaOH was added to raise the pH to 7.0-7.7. When the microcosms were not being sampled, they were stored in an inverted position in boxes quiescently at room temperature in the anaerobic chamber.

First order transformation rates for each halomethane were calculated using data following the lag phase prior to the onset of transformation, i.e., the period of time when there was no significant decrease in the amounts per bottle. Data from triplicate bottles were pooled and fit with a logarithmic curve to determine the first order rate for each compound and treatment in each concentration range.

2.3.4 Analysis of volatile organic compounds

Halogenated (CT, CF, DCM, CM, CFC-11, HCFC-21, HCFC-31) and nonhalogenated volatile compounds (methane, ethane, ethene, CS 2) were monitored by gas chromatographic analysis of 0.5 mL headspace samples, as previously described (35).

A Hewlett Packard 5890 Series II gas chromatograph (GC) equipped with a flame ionization detector and a 2.44 m × 3.175 mm column packed with 1% SP-1000 on 60/80

Carbopack B (Supelco) was used. A GC response factor for each volatile organic compound was measured to relate the total mass of the compound to the GC peak area

(38). Results are reported in terms of the µmoles per bottle, in order to easily reveal the stoichiometry of the transformations. In the text, aqueous phase concentrations are also reported, based on partitioning of the compounds between the headspace and aqueous

19 phases (47). Previously reported values for Henry’s Law constants (Hc) at 23°C were

used (7, 38). The detection limit of this method is lower than the maximum contaminant

level (MCL) for each of the chlorinated compounds.

2.3.5 Analysis of 14 C-labeled compounds

The initial total amount of 14 C activity present was determined by counting a sample of the headspace (0.5 mL) and liquid (0.1 mL) in 10 mL liquid scintillation cocktail (LSC). At the point when a bottle was ready to be sacrificed for analysis of the

14 C distribution, the amount of 14 C activity remaining was determined after raising the pH

of the liquid phase (0.75 mL 10 M NaOH) in order to drive CO 2 in the headspace into the

liquid phase. 14 C activity remaining was determined by counting a headspace sample

(0.5 mL) and slurry sample (1 mL), to account for any 14 C activity that may have

partitioned to the soil. A WALLAC 1415 liquid scintillation counter was used to

quantify 14 C activity. Corrections for counting efficiency were made according to a quench curve (sample spectral Quench Parameter-Eternal (i.e., SQP(E)) versus efficiency) after incubating them overnight in the dark.

14 Volatile C-labeled compounds (primarily CT, CF, DCM, CO, and CS 2) were analyzed with a GC-combustion technique, as previously described (35). A 0.5-mL headspace sample from the microcosms (prior to adding NaOH) was injected onto the

Carbopack B column and the well-separated compounds were routed to a catalytic combustion tube (containing CuO), where the compounds were oxidized at 800°C to CO 2.

Each fraction was then trapped in 3 mL of 0.5 M NaOH and added to 15 mL LSC.

Whenever 14 C activity exceeded 200 dpm in the first fraction (0-1.1 min), which was

20 associated with the poorly-separated methane, CO and CO 2, a second headspace sample was injected onto a Carbosieve SII column (2.44-m by 3.175-mm; Supelco), which efficiently separates the one carbon gases. Fractions eluting from the Carbosieve column

14 14 14 14 were used to determine CO and CH 4. CO 2 was determined during analysis of C in the liquid phase (see below). The efficiency of the combustion technique ( Σ14 C in the fractions divided by the 14 C in a headspace sample) averaged 123 ±30% for the

Carbopack column and 106 ±20% for the Carbosieve column. Apparently higher recoveries occurred when CO was a major product because of the poor dissolution of CO in LSC (thereby underestimating the total activity present in the direct sample). The procedure used to convert the 14 C activity in each fraction to total 14 C activity for each volatile compound in a microcosm required the use of Henry’s Law constants, as previously described (19, 35).

The 14 C liquid analyses were done using a modified version of a previously described protocol (33). At the end of the incubation period and after adding NaOH to the microcosms, a 10 mL sample of well-mixed slurry was transferred to a 25 mL glass test tube (“stripping chamber”), which was connected by latex tubing to another 25 mL glass test tube (“trapping chamber”) containing 10 mL of 0.5 M NaOH. To initiate the analysis,

N2 was bubbled through the stripping chamber and into the trapping chamber. The contents of the stripping chamber were acidified by adding 0.25 mL of 6 M HCl, lowering the pH to below 4.

14 14 C activity in the trapping chamber corresponded to CO 2. This was confirmed

14 14 by precipitation of the CO 2 with barium hydroxide (35). C activity left in the

21 stripping chamber was referred to as nonstrippable residue (NSR), corresponding to

products of [ 14 C]CT and [ 14 C]CF that were not purged at acidic pH. The total NSR was separated into soluble (sNSR) and particulate (pNSR) fractions by centrifugation (26,600 g for 10 min; SORVALL® Evolution™ RC superspeed centrifuge). If sNSR represented more than 10% of the total initial 14 C activity, its composition was evaluated by injecting filtered samples (200 µL) onto a WATERS high performance liquid chromatograph

(HPLC) using 0.01 N H 2SO 4 as the eluant and an Aminex® HPX-87H column (300 mm x 7.8 mm) for separation of organic acids. As fractions eluted they were collected in 10 mL of LSC. Identification of organic acids was confirmed based on matching of their retention times with the retention time of authentic material. The recovery of sNSR through HPLC fractionation (i.e., total dpm in the fractions divided by dpm injected) was

74 ±8.4%.

The percentage of 14 C attributable to each compound or category of compounds, the physical losses, and the amount of 14 C in the “unaccounted for” category was calculated as described elsewhere (19).

2.3.6 Development of enrichment cultures

Given the high concentration of sulfate in the groundwater and the potential for

SRB to cometabolize halomethanes (36), two SRB enrichment cultures were developed.

A fermentative culture was later developed with the intent of further enhancing biotransformation. A previously described (32) mineral salts medium (MSM) was used, with the following modifications: NaCl and KH 2PO 4 were excluded; the concentration of

K2HPO 4 and NaHCO 3 was changed to 0.41 g/L and 4.0 g/L, respectively; and the

22 headspace consisted of 30% CO 2 and 70% N 2 instead of helium, resulting a pH of approximately 7.5. The bottles were prepared in an anaerobic chamber containing an atmosphere of approximately 98% N 2 and 2% H 2.

The lactate-grown SRB enrichment was started in a 250 mL glass bottle containing 200 mL of MSM, 37.1 mM sulfate, 3 mM sodium lactate and 20 g of soil from an uncontaminated area of the same industrial site described above. Growth was monitored based on consumption of sulfate and lactate. When the lactate was consumed, more was added. The maximum dose of lactate was kept at 5 mM in order to avoid enriching for a fermentation culture that converts lactate to propionate plus acetate (39) rather than an SRB culture. Once a consistent level of lactate and sulfate consumption was demonstrated, the enrichment was transferred (1% v/v) to MSM. After consuming approximately 30 mM lactate without significant propionate formation, the protein concentration exceeded 100 mg/L and the culture was considered ready for use in bioaugmentation.

A second SRB enrichment culture was developed using ethanol as the carbon and energy source to enhance the development of sulfate-reducing conditions because ethanol was not fermentable. Using the lactate enrichment as inoculum, a transfer was made to

MSM (1% v/v) containing 3 mM ethanol and 1.5 mM ferrous sulfate (to precipitate sulfides and minimize the opportunity for sulfide inhibition). Consumption of ethanol was followed indirectly based on accumulation of acetate. Repeated additions of 3-6 mM ethanol were made and sulfate reducing conditions were confirmed based on the consumption of sulfate. After consuming a total of 30-40 mM ethanol, the protein

23 concentration exceeded 100 mg/L and the enrichment culture was considered ready for

use in bioaugmentation.

A fermentative enrichment culture was started by combining soil (20 g) from an uncontaminated area of the industrial site as inoculum, MSM (100 mL) and corn syrup

(approximately 7680 mg COD/L) as the primary substrate in 160 mL serum bottles.

After 1-2 weeks of incubation, the protein level reached 200 µg/mL and a 1% transfer was made to MSM and corn syrup. When the protein concentration reached 200 µg/mL, the culture was concentrated by centrifugation (5500 g) and resuspended in MSM (200 mL) plus corn syrup in a 250 mL bottle sealed with a Teflon-lined rubber septum and screw cap. Acclimation to CF began by adding 2.8 µmol per bottle. Taking into account partitioning to the headspace (see above), the aqueous phase concentration was 1.5 mg/L.

The initial dose of B 12 was 10 µM. As CF was transformed, increasing amounts of CF were added until a final dose of 500 mg/L. Additional B 12 was provided to maintain a molar ratio of B 12 to cumulative CF at 3%. Further additions of corn syrup were based on pH, i.e., a decrease in pH indicated that fermentation was occurring due to accumulation of organic acids (predominantly formic, acetic and propionic). When the pH decreased to

6.7-7.0, it was raised to 7.5-7.7 with 8 M NaOH. When the pH stopped decreasing, suggesting a cessation in fermentation, more corn syrup was added (approximately 960 mg COD/L per dose).

Prior to adding the SRB enrichments to the microcosms, they were concentrated

100 fold by centrifugation. Adding 0.5 mL of the concentrate provided the microcosms with the same concentration of cells that were in the enrichment cultures. The

24 fermentative enrichment culture was concentrated 10 fold by centrifugation and 0.2 mL

of the concentrate was added per dose.

Several of the microcosms were bioaugmented with a chloroethene respiring

enrichment culture for the purpose of enhancing reductive dechlorination of 1,1-DCE.

This culture was originally enriched on tetrachloroethene and trichloroethene and sub-

samples were subsequently enriched on other chlorinated ethenes, including 1,1-DCE

(25). Samples of the 1,1-DCE enrichment culture were used for bioaugmentation of the

microcosms (1 mL per dose, without concentration, containing approximately 10 9 cells of

Dehalococcoides per mL).

2.3.7 Denaturing gradient gel electrophoresis (DGGE) and protein analysis

After lysing samples of the enrichment culture (15), protein levels were

determined using a Compat-Able TM assay preparation reagent set and a BCA TM protein assay kit (Pierce Chemical Company), following the manufacturer’s enhanced protocol.

Total community DNA was extracted from homogeneous samples of groundwater and soil from selected microcosms using an UltraClean® soil DNA isolation kit (MO BIO

Laboratories, Inc.) by following the manufacturer’s maximum yield protocol. A segment of the 16S rRNA gene of the bacteria domain was amplified with universal primer 1055F and 1406R with a 40 base GC clamp through an adapted touch-down polymerase chain reaction program (18) prior to separation by DGGE (74), with a 0-100% gradient.

2.3.8 Dissolved organic carbon, organic acids, and anions

Dissolved organic carbon (DOC) was quantified using a Shimadzu TOC –VCSH

TOC analyzer with a combustion catalytic oxidation method (680°C). Organic acids

25 were analyzed on a Waters 600E HPLC system equipped with a UV/Vis detector (Model

490E) set at 210 nm and an Aminex ® HPX-87H ion exclusion column (300 mm x 7.8

mm; BioRad). Eluant (0.01N H 2SO 4) was delivered at 0.6 mL per min. Sulfate was measured on a Dionex AS50 ion chromatography system equipped a CD25 Conductivity detector and a Dionex guard column (AG9-HC, 4 mm x 50 mm) followed by an IonPac ®

AS9-HC anion-exchange column (4 mm x 250 mm). Eluant (9 mM Na 2CO 3) was

delivered at 1.0 mL per min.

2.4 Results

2.4.1 Microcosm response to biostimulation and bioaugmentation

Bioaugmentation was the most effective treatment strategy for the high

concentration plume, followed by biostimulation with corn syrup + B 12 . Biostimulation

without B 12 was much less effective. The performance of representative microcosms for

the two treatments with B 12 added is shown in Figure 2-1. Complete results for all of the

microcosms are provided in Appendix A.

In the biostimulation treatments with high halomethane levels (Figure 2-1a), B 12

was added on days 0, 321, 442 (10 µM/dose), and days 679 and 1016 (30 µM/dose); the

total amount added was 1.3 mole percent of the total halomethanes added. CT

transformation started after a lag phase of 48 days and was completed by day 180. Close

to that time, transformation of CFC-11 started, decreasing from 90.3 µmol/bottle (29.1

mg/L) to 10.9 µmol/bottle (3.5 mg/L) on day 897, with relatively little further decrease

thereafter. A minor proportion (approximately 1.5%) of the CFC-11 was converted to

HCFC-21. A notably faster rate of CF transformation started close to day 803, when

26 CFC-11 had decreased to 4.4 mg/L. As a result, the remaining 124 µmol/bottle (234

mg/L) of CF was removed to below its MCL, i.e., 80 µg/L, by day 1052. Formation of

DCM, CM, methane and CS 2 was minor. Although there was a gradual decrease in 1,1-

DCE, this was not accompanied by an increase in dechlorination products, suggesting the

loss occurred predominantly via diffusion (see control results below).

The high concentration bioaugmentation microcosms initially received only corn

syrup (without B 12 ) and were incubated for 193 days prior to bioaugmentation (Figure 2-

1b). During this interval, the extent of halomethane and 1,1-DCE removal was similar to the microcosms with corn syrup added (Appendix A.1), i.e., some losses of CT, CF and

1,1-DCE occurred, while there was virtually no loss of CFC-11. The first bioaugmentation of the SRB lactate culture substantially improved the rate of CT removal. The rate of CT removal increased further in response to the first dose of B 12 (10

µM) on day 230, completing transformation of CT by day 255. During this interval, the

lactate added was converted to acetate in a nearly one to one molar ratio. Thereafter,

propionate started to become the dominant product from lactate, rather than acetate

(Appendix A.8), indicating the onset of a fermentation pathway. Biotransformation of the

CFC-11 began once the CT was nearly gone, accompanied by an increase in CS 2, an indirect indication of the availability of sulfide. HCFC-21 also increased via hydrogenolysis. The amount of CS 2 and HCFC-21 formed accounted for approximately

17% of the CFC-11 consumed until a second bioaugmentation using an ethanol-grown

SRB enrichment culture was applied on day 293. Although the increase of CS 2 and

27 accumulation of propionate ceased following the additions of ethanol and ferrous chloride, it failed to restore a high transformation rate.

Transformation of CFC-11 and CF either diminished or stopped until the third bioaugmentation with a fermentative enrichment culture together with the second dose of

B12 (10 µM) on day 490, which had an immediate effect. The remaining 1.6 µmol/bottle

(2.4 mg/L) of CFC-11 was removed by day 613. Two more doses of the fermentative culture were added to sustain a high rate of CF transformation. Transformation of the

143 µmol/bottle (271 mg/L) of CF remaining on day 490 was completed by day 675.

During this interval three more doses of B 12 were added, resulting in a total B 12 dose of

1.4 mole percent of the total halomethanes added. After the CF was consumed, the previously accumulated DCM and HCFC-21 decreased. Unlike the treatments without bioaugmentation (Figure 2-1a), the remaining 6.7 µmol/bottle (4.5 mg/L) of 1,1-DCE was removed by bioaugmentation with an approximately stoichiometric amount of ethene formation; a low level of VC appeared transiently. A notable amount of methane was produced in this interval (148-242 µmol/bottle; not shown in Figure 2-1b); prior to removal of the halomethanes, methanogenesis was completely inhibited.

Both treatments receiving B 12 achieved complete transformation of CT and CF.

However, biostimulation with corn syrup + B 12 was less efficient in terms of the total amount of CFC-11 removed, i.e., 93% versus 100% by bioaugmentation. Biostimulation with corn syrup, lactate, and vegetable oil without B 12 was less effective, but all achieved complete transformation of CT, 37-45% removal of CFC-11 and 80-84% removal of CF.

The 80% decrease of CT achieved in the as-is treatment (no substrate added) is

28 significantly higher than those in the controls (AC and WC), i.e., 18%. However, the decreases of CFC-11 (7%), CF (14%), and 1,1-DCE (26%) observed in the as-is treatment were comparable to those in the controls, indicating the losses were mostly diffusive. In contrast to the complete removal of 1,1-DCE achieved in bioaugmentation, approximately two thirds of 1,1-DCE was removed in all of the biostimulation treatments.

Lack of formation of daughter products, however, suggests the loss occurred predominantly via diffusion.

Representative results for microcosms prepared with medium concentrations of halomethanes that were subjected to biostimulation with corn syrup + B 12 and bioaugmentation are shown in Figure 2-2. The biostimulation treatments received B 12

(10 µM) on days 0 and 448. The total amount of B 12 added was 2.6 mole percent of the total halomethanes added. As Figure 2-2a shows, transformation of the 8 µmol/bottle

(7.5 mg/L) CT started after a lag phase of 53 days and was completed on day 146.

Transformation of the CFC-11 followed immediately once CT was gone. The 13

µmol/bottle (4.5 mg/L) of CFC-11 decreased to 0.23 µmol/bottle (73 µg/L) by day 502.

A minor amount of HCFC-21 (2% molar ratio of the CFC-11 transformed) accumulated.

There was no accumulation of HCFC-31 or CS 2. Activity on CF started along with the

CFC-11 transformation and accelerated on day 445 when the CFC-11 was nearly gone.

The 1,1-DCE respiring culture was bioaugmented once the CF dropped below its MCL on day 544, and the remaining 0.18 µmol/bottle (118 µg/L) of 1,1-DCE was removed below its MCL (i.e., 7 µg/L) in 55 days. A low level of VC appeared transiently. The performance of this microcosm suggests that the plume with medium concentration levels

29 of halomethanes may be remediated by biostimulation with corn syrup + B12 in

approximately 600 days.

The medium concentration bioaugmentation microcosms initially received only

corn syrup and were incubated for 143 days prior to bioaugmentation with the SRB

lactate enrichment culture along with the first dose of B12 (10 µM) (Figure 2-2b). The

response was immediate. The rate of CT removal increased substantially and removal

was complete by day 196. CS 2 started to accumulate. Acetate was the predominant

product of the lactate consumption during this interval, although propionate started to

increase (Appendix A.8). CFC-11 biodegradation began as CT levels decreased and the

rate of removal significantly accelerated in the interval of day 216 to 292, after CT was

completely consumed. In this interval, CFC-11 decreased from 7.7 µmol/bottle (2.5

mg/L) to 0.54 µmol/bottle (0.2 mg/L). CS 2 increased during this interval and a minor

amount of HCFC-21 also accumulated. The amount of CS 2 formed accounted for 34% of the CT and CFC-11 consumed. Lactate was increasingly converted to propionate rather than acetate (Appendix A.8). The second bioaugmentation dose using the SRB ethanol culture was implemented on day 293 to prevent any further increases in CS 2, enhance sulfate reducing activity, and accelerate CF removal. Addition of ferrous chloride along with ethanol curbed the increase in CS 2 and the ethanol-enriched culture stopped producing propionate. Nevertheless, the rate of CF removal did not change demonstrably and close to day 367 the rate of removal started to decline until the third bioaugmentation dose with the fermentative culture grown on corn syrup, plus a second dose of B 12 (10

µM) on day 490. The remaining 3.5 µmol/bottle (6.6 mg/L) of CF was reduced to below

30 the MCL by day 613. The total amount of B 12 added was 3.1 mole percent of the total

halomethanes added. Similar to the biostimulation with corn syrup + B 12 microcosms, the

residual 1,1-DCE was removed by bioaugmentation with the chloroethene respiring

culture, which also corresponded to the onset of methanogenic activity.

The two treatments that received B12 achieved complete transformation of the three halomethanes, with the exception of 98% removal of CFC-11 in the biostimulation treatment with corn syrup + B 12 . Among the biostimulation treatments without B 12 , the microcosms with corn syrup achieved complete removal of CT and approximately 35%,

57%, and 66% removal of CFC-11, CF and 1,1-DCE, respectively. In the microcosms with lactate and with emulsified vegetable oil, the percent removals for CT, CFC-11, and

1,1-DCE were approximately 10% lower. In the as-is treatment, CT decreased by 54%,

CF by 27%, , 1,1-DCE by 72%, and no significant decrease in CFC-11 was observed.

Losses of all compounds from the medium concentration AC microcosms were 18% or lower.

The microcosms with low concentrations of halomethanes (but no 1,1-DCE) behaved similarly to the high and medium concentration treatments. Bioaugmentation achieved complete transformation of the three halomethanes in approximately 300 days while biostimulation with corn syrup + B 12 required 300-600 days. Biostimulation with corn syrup was overall the best among the three treatments without B 12 , i.e., 88% and

21% decreases in CT and CFC-11, respectively, but no activity on CF. Losses of

halomethanes from the low concentration AC microcosms were 18% or lower.

31 2.4.2 First order rate constants of halomethane removal

CT transformation rates in the bioaugmentation microcosms were three to nine

times faster than in the biostimulation treatment with corn syrup + B 12 (Figure 2-3a).

Among the biostimulation treatments without B 12 , corn syrup showed the best overall

performance, although these rates were notably less than the ones for biostimulation with

corn syrup + B 12 . The initial concentration of total halomethanes (i.e., low, medium and

high) did not have a significant effect on the CT transformation rate, perhaps because the

initial CT concentration among the three plumes was similar. The exception was the

bioaugmentation treatment with low levels of halomethanes, which had the highest rate

of CT transformation. This suggests that the medium and high concentrations of CFC-11

and CF may have had an inhibitory effect on the rate of CT transformation.

For CFC-11, only the two treatment strategies with B 12 added were effective.

Removal rates in the bioaugmentation treatment in each concentration range for the

bioaugmentation treatment were approximately two to three times faster than for the

biostimulation treatment with corn syrup + B 12 (Figure 2-3b). That the highest rate occurred in the low concentration plume may indicate an inhibitory effect from the increasing concentrations of CFC-11 and/or CF.

With CF, the transformation rates for bioaugmentation were significantly higher than for the biostimulation treatment with corn syrup + B 12 for the high and low concentration microcosms, while they were equivalent for the medium concentration microcosms (Student t-test, α=0.05). CF transformation rates in the other live treatments were much lower. The initial concentration of halomethanes appears to have had a

32 modest inhibitory effect on the CF transformation rate in the bioaugmentation treatment,

while there was no apparent trend in the biostimulation treatment with corn syrup + B 12 .

2.4.3 Distribution of products from [ 14 C]CT and [ 14 C]CF

14 CO 2, sNSR, and pNSR were the main products of [ C]CT transformation in the bioaugmentation treatment in microcosms for all three concentration ranges (Table 2-3).

In addition, a significant amount of CS 2 was formed in the low concentration microcosms, but decreased in the medium and high concentration bottles. In the biostimulation with corn syrup + B 12 treatment, CO 2, sNSR, pNSR were predominant in the low and medium concentration microcosms, with an even larger percentage of CO 2 than for the bioaugmentation treatment. CO, however, was predominant in the high concentration microcosms (i.e., 35%), followed by sNSR. In the biostimulation treatments without B12 added, the main 14 C compounds recovered were CT, CF, and NSR, reflecting both incomplete conversion of CT and the prevalence of reductive dechlorination of CT to CF.

Losses of 14 C ranged from 21-55%, reflecting the long incubation times (i.e., up to 1200 days).

The predominant products of [14 C]CF transformation in the bioaugmentation treatment were CO 2 and sNSR in microcosms for all halomethane concentrations (Table

2-4). DCM was notable only in the low concentration bioaugmentation microcosms, most likely due to the use of the lactate-grown SRB culture, which tended to favor reductive

14 dechlorination. In the biostimulation treatment with corn syrup + B12 , the main C

compounds recovered were CO, CO 2 and sNSR, with CO predominant in the low and high concentration microcosms. Unconverted CF and sNSR were the main fractions for

33 14 the biostimulation treatments without B 12 added. Losses of C ranged from 22-68%, reflecting the long incubation times (i.e., up to 1250 days).

Untransformed [14 C]CT or CF was the main component in both as-is and AC

14 treatments, followed by NSR. Significant amounts of CO 2 (24%) formed from [ C]CT in high concentration as-is treatment, reflecting the potential for a limited level of natural attenuation. The relatively lower losses in as-is and AC treatments in the medium and high concentration microcosms is most likely due to less frequent sampling and, therefore, fewer puncture holes in the septa, thereby reducing the diffusive losses.

The composition of sNSR recovered in microcosms that received [14 C]CT and

[14 C]CF was similar in the bioaugmentation treatment, i.e., 22-44% of the sNSR was propionate and 19-26% was acetate, at each halomethane concentration. Minor amounts of formate, ethanol, iso-butyrate and butyrate were detected (i.e., less than 9% of sNSR).

In the biostimulation treatment with corn syrup + B 12 for the medium concentration

microcosms, acetate was the principal fraction of the sNSR recovered from [14 C]CT (53-

60% of sNSR), followed by propionate (11-15% of sNSR). Formate was the

predominant organic acid formed in the high concentration microcosms (38% of sNSR),

followed by lactate (20% of sNSR) and propionate (11% of sNSR). Acetate was

predominant in the sNSR recovered from [ 14 C]CF in the medium concentration

biostimulation treatment with corn syrup + B 12 (50% of sNSR), while propionate

dominated in the high concentration microcosms (28% of sNSR), followed by formate

(13% of sNSR) and acetate (10% of sNSR). The organic acid composition of sNSR

34 14 formed from [ C]CF in the biostimulation treatments without B 12 in the medium and high concentration microcosms were similar, with acetate and propionate predominant.

2.4.4 Electron donor utilization and sulfate consumption

DOC consumption was used as a surrogate for electron donor utilization. The background DOC level in the groundwater for the three plumes was less than 100 mg/L, so nearly all of the DOC present in the microcosm was from the substrate added. Table

2-5 shows the amount of DOC added and consumed. The highest decrease in DOC occurred with emulsified vegetable oil in all plumes, representing 80-84% of the DOC added. However, this was most likely due to adsorption of the oil to the soil, leaving very low amounts of substrate in the aqueous phase. DOC consumption in the treatments with corn syrup only and corn syrup + B 12 was similar, representing up to one third

consumption of the DOC added in the low and medium concentration microcosms, and

12-20% consumption in the high concentration microcosms. DOC utilization in the

bioaugmentation treatment ranged from 7-25% of the DOC added. Lactate was the least

efficiently used electron donor for biostimulation, with negligible use in both low and

high concentration microcosms and 20% decrease in DOC in the medium concentration

bottles.

Table 2-5 also lists the amount of DOC removed per amount of halomethanes

initially present. The treatments with corn syrup added had the highest ratios, reflecting

better utilization of the substrate in the presence of the halomethanes. The highest ratios

shown, for emulsified vegetable oil, are a consequence of loss of DOC to adsorption.

35 The highest amount of organic acid accumulation occurred in the bioaugmentation treatment in each concentration range, with similar amounts of acetate and propionate present (35-38% of the COD added, respectively). The total accumulation of organic acids accounted for 85-90% of COD added in the low concentration microcosms, and approximately 67% in the medium and high concentration bottles. Accumulation of propionate following addition of the SRB lactate culture suggested that sulfate reduction was no longer predominant and was replaced by fermentation of lactate to propionate. In the treatments with corn syrup added (with and without B 12 ), the COD of the organic acids accounted for 47-57% of the total COD added in the medium and high concentration microcosms and 22-60% in the low concentration bottles. Acetate was almost the only organic acid formed in the medium and low concentration microcosms (with a minor amount of propionate also present in the low concentration bottles); in the high concentration microcosms, acetate also predominated but other organic acids were also detected. In the treatment with lactate added, nearly all of the lactate remained at the end of the incubation period in most of the microcosms. In the treatments biostimulated with emulsified vegetable oil, there was no significant accumulation of organic acids at any halomethane concentrations, indicating that the decrease in DOC was mainly a consequence of adsorption of the oil.

The sulfate initially present in the groundwater was completely removed (i.e., 98-

100%) in the bioaugmentation treatment for the high and medium concentration microcosms, with a lower percent removal in the low concentration microcosms (42%)

(Table 2-5). In the biostimulation treatments with corn syrup + B 12 , sulfate removal

36 averaged 13%, 42%, and 14% in the high, medium and low concentration bottles, respectively. In the biostimulation treatments with corn syrup alone, sulfate removal averaged 100%, 33%, and 18%, in the high, medium and low concentration microcosms, respectively. Sulfate consumption in the biostimulation with lactate or emulsified oil treatments was the lowest, varying from zero to 20%. The lower level of sulfate consumption in the lactate treatments coincided with insignificant amounts of DOC removal (Table 2-5).

2.4.5 DGGE

When incubation of the microcosms was complete, DNA was extracted and analyzed by DGGE (Figure 2-4). In the as-is treatment (lanes 1, 4 and 7), bands e, f and i were common. At the higher concentration of halomethanes, two additional bands were also evident in the as-is treatment (g and h). At the low and medium concentrations, biostimulation with corn syrup (lanes 2 and 5) and with corn syrup + B 12 (lanes 3 and 6) significantly changed the microbial structure in comparison to the as-is treatments (lanes

1 and 4), i.e., bands a, b, c, and d became prominent while bands e and f were no longer evident (except in the corn syrup treatment with low halomethanes, lane 2). In contrast, the biostimulated treatments for the high concentration microcosms (lanes 8 and 9) remained similar to the corresponding as-is treatment (lane 7); the only notable exception was the appearance of band b in the corn syrup + B 12 treatment (lane 9).

2.5 Discussion

This microcosm study demonstrated for the first time that biostimulation and bioaugmentation are feasible remediation strategies for in situ treatment of high

37 concentrations of halomethanes and subsequent removal of 1,1-DCE by bioaugmentation.

All previous microcosm or field studies on bioremediation of halomethanes examined concentrations in the low mg/L level (21, 23, 24, 31, 63, 64, 79). Complete biotransformation of halomethanes in the microcosms with high concentrations was achieved in approximately 500 days using bioaugmentation (from time that the first bioaugmentation dose was added; Figure 2-2b). The time required for biostimulation with corn syrup + vitamin B 12 was approximately twice as long (Figure 2-2a).

Unlike chlororespiration, biotransformation of halomethanes is a cometabolic process, resulting in less efficient use of the electron donors (67). Prediction of primary substrate requirements is, therefore, less certain. This is especially true for anaerobic systems, since the concept of a transformation capacity used with aerobic cometabolism has not been widely adapted to cometabolism under anaerobic conditions. In this study, it appears that the amount of primary substrate provided was well in excess of that required based on the considerable excess of DOC remaining at the end of incubation

(Table 2-5). The ratio of primary substrate added to the initial amount of halomethanes present for the high concentration microcosms ranged from 247 mg DOC/mmol halomethanes for biostimulation without B 12 to 564 mg DOC/mmol halomethanes for bioaugmentation. Using data from Hashsham and Freedman (47), this ratio was 1,810 mg DOC/mmol halomethanes for biotransformation of approximately 100 mg/L of CT by fructose-grown A. woodii . The ratio of mg DOC added per mmol halomethanes present used in this study was higher for the medium and low concentration microcosms, suggesting that much more primary substrate was added to these treatments than what

38 was required. Additional work is needed to better define the minimum amount of

primary substrate needed to achieve high rates of halomethane transformation. However,

in terms of in situ remediation, this may not be a major concern due to the relatively low cost of corn syrup.

The bioaugmentation treatments had consistently higher rates of halomethane transformation (Figure 2-3). Since higher amounts of substrate were added to the bioaugmentation treatments than to the biostimulation treatments with corn syrup + B 12

(Table 2-5), the question arises as to whether the higher transformation rates are attributable to addition of the cultures or the higher level of primary substrate added. One of the complicating factors was the initial amount of sulfate, which was a potentially significant source of demand for primary substrate in addition to halomethane transformation, especially for the low and medium concentration microcosms. The higher dose of primary substrate given to these microcosms was based in part on the expected higher demand for sulfate reduction. The significant amount of DOC that remained at the end of the incubation period suggests that all of the treatments were saturated with substrate. For the bioaugmentation treatments and the biostimulation ones with corn syrup added, the ratios of DOC removed (due in part to sulfate reduction) to the initial amount of halomethanes present were similar (i.e., approximately 45, 920, and

1,500 for the high, medium and low concentration bottles, respectively). This suggests that the higher rates observed for bioaugmentation compared to biostimulation were due to the cultures rather than the higher amount of primary substrate addition. For the biostimulation treatments that received corn syrup alone versus corn syrup + B 12 , the

39 amount of substrate added was similar and, therefore, the higher rates observed with B 12

are clearly attributable to the presence of B 12 . Higher rates of halomethane

transformation with B 12 addition have been shown previously in several studies (12, 47,

48).

The first-order rate constants for CT biotransformation via bioaugmentation and

biostimulation with B 12 observed in this study were comparable to those reported in a

methanogenic sludge consortium (0.5 g VSS/L) with 1-2 mol% B 12 in transforming 15 mg/L CT (i.e., 0.51 – 0.81 d -1) (41), and those by Pseudomonas sp. strain KC (i.e.,

approximately 0.05-0.15 d -1) (86). The significantly higher (by approximately two orders of magnitude) first-order rate constants of three cultures grown on propanediol, dextrose or acetate, respectively, in transforming g/L levels of CT with 10 mg/L B 12 (89), was likely due to the very high ratios of B 12 to CT as well as the low initial CT concentrations.

The first-order rate constants for aerobic cometabolic transformation of CF by a methanotroph in media in the absence of methane (83) was over 100 times higher than what were observed via bioaugmentation and biostimulation with B 12 in this study.

However, the initial concentration of CF tested by Speitel et al. (83) was only 100 g/L.

The first-order rate constants for CFC-11 biotransformation via bioaugmentation and

biostimulation with B 12 observed in this study were comparable to those reported in

anaerobic soil in a peat land (11).

The effect of B 12 on the products formed from transformation of CT and CF in

this study was similar to previous findings (12, 47, 48). With B 12 present CO, CO 2 and

NSR were the predominant products. No previous studies were found that evaluated the

40 effect of B 12 on CFC-11 anaerobic transformation. At low mg/L concentrations of CFC-

11 and in the absence of B 12 , the predominant transformation product formed by

Methanoscarcina barkeri (57) and sulfate reducing bacteria (82) was HCFC-21. In this study, biotransformation of CFC-11 (at initial concentrations that were one order of magnitude higher) was fastest in the bioaugmented and biostimulated treatments with B 12 added, although HCFC-21 was a minor product. This suggests that B 12 had the same effect on altering the transformation pathway for CFC-11 as it did for CT and CF, i.e., by favoring substitutive and hydrolytic pathways over hydrogenolysis.

Among the biostimulation treatments without B 12 added, corn syrup was slightly more effective for CT transformation. Metabolism of corn syrup was confirmed by DOC removal, sulfate reduction, and accumulation of organic acids. Formation of acetate, propionate, formate, and lactate, was consistent with mixed acid fermentation (53, 69), while formation of propionate may have been a consequence of succinate decarboxylation (44). Low redox conditions were also established fastest with corn syrup.

In contrast, less lactate was consumed (based on DOC removal and HPLC data). With emulsified vegetable oil, the lack of significant accumulation of organic acids and lower levels of sulfate reduction suggested that it, too, was poorly utilized. Most of the DOC removal with vegetable oil was likely due to adsorption to the soil. Use of vegetable oil in remediation of chlorinated ethenes typically results in a decrease in the aqueous phase concentration due to partitioning (75). However, such an effect was not apparent in this study for the halomethanes or 1,1-DCE, even with repeated additions of vegetable oil.

The only apparent effect of the emulsified vegetable oil was on the product distribution

41 from [ 14 C]CT, i.e., pNSR was generally higher in the treatments with oil added (Table 2-

3). Although treatments with lactate + B 12 and emulsified vegetable oil + B 12 were not

tested in this study, the lack of evidence for significant metabolism of these substrates

suggests they are not likely to be effective with halomethanes under the conditions

evaluated.

In previous studies of anaerobic transformation of halomethanes, the effectiveness

of sulfate-reducing cultures grown on lactate and acetate (36) and acetogenic cultures

growing on fructose has been demonstrated (47). Due to the high background

concentration of sulfate at the industrial site that was a focus of this study, use of a sulfate

reducing culture for bioaugmentation was evaluated first. The initial response to

bioaugmentation with this culture was promising. However, significant amounts of CS 2

began to accumulate after several weeks. In the high and medium concentration

microcosms, the rate of CFC-11 transformation slowed considerably and sulfate

reduction was replaced by fermentation of the lactate to propionate (59). To address

these concerns, a second sulfate-reducing culture was evaluated using ethanol as a

substrate and addition of ferrous chloride to mitigate CS 2 formation by lowering the

sulfide concentration. Based on sulfate consumption and accumulation of stoichiometric

levels of acetate from ethanol, it was apparent that the culture was active. However, there

was no improvement in the rate of CFC-11 or CF transformation. One factor

contributing to the ineffectiveness of the SRB bioaugmentation cultures may have been

the toxicity of the halomethanes, especially the high concentration of CF. Attempts to

acclimate the ethanol-grown SRB culture to increasing amounts of CF (along with B 12 )

42 were unsuccessful. In contrast, the fermentative enrichment grown on corn syrup (along

with B 12 ) readily acclimated to CF, up to 500 mg/L. Subsequent additions of this culture to the microcosms yielded high rates of CF transformation. The ability of acetogenic cultures to biotransform halomethanes has been attributed to enzymes associated with the acetyl-CoA pathway (27).

Regardless of the treatments tested, halomethane transformation occurred in a consistent order, i.e., CT was consumed first, followed by CFC-11 and then CF. This is in agreement with thermodynamic expectations for the predominant transformation pathways observed in this study, i.e., more free energy is gained by transformation of CT versus CFC-11 and more from CFC-11 than CF via reductive hydrolysis and substitution or net hydrolysis (Table 2-6). The free energy change for reductive dechlorination of CT and CFC-11 are similar, but this was not a significant pathway.

The in situ conditions at the industrial site offered an interesting opportunity to observe the microbial community response to pH adjustment and biostimulation. The areas of the site with low and medium concentrations of halomethanes have acidic pH levels, while the high concentration area is near neutral pH. DGGE results revealed major changes in community structure for the low and medium concentration microcosms. In contrast, there was comparatively little shift in the structure of the high concentration microcosms. These results suggest that pH was a more significant factor than the concentration of halomethanes during the response to biostimulation. Lei and

VanderGheynst (62) also observed a significant effect of pH adjustment on microbial structure during composting. Apparently microbes in the high concentration area at the

43 industrial site have acclimated to the presence of high levels of halomethanes, based on a lack of significant change in response to biostimulation. Furthermore, there was more diversity of microbes in the as-is high concentration treatment compared to the medium and low concentration microcosms, also an apparent consequence of exposure to different pH levels over many decades.

Use of bioaugmentation to remove 1,1-DCE was not attempted until the halomethanes were removed. Chlororespiration of 1,1-DCE requires the presence of

Dehalococcoides , which are strongly inhibited by low concentrations of CF during reductive dechlorination of tetrachloroethene, trichloroethene, cis -dichloroethene, and

VC (22, 73). Although the effect of CF on reduction of 1,1-DCE has not been reported, inhibition by CF seems highly likely, due to the need for Dehalococcoides to carry out the reaction. Following removal of halomethanes from the high and medium concentration microcosms, bioaugmentation with a 1,1-DCE respiring culture was effective. This confirmed the potential to use bioremediation for complete removal of high concentrations of halomethanes, followed by removal of 1,1-DCE.

44 2.6 Tables and Figures

Table 2-1 Groundwater pH adjustment

Halomethane Groundwater Na 2CO 3 added Precipitate formed concentration pH (mg/L) (mg/L) a Initial Adjusted

High 6.6 7.5 116 0 Medium 3.3 7.3 1,330 480 Low 3.5 7.3 1,730 790 a Based on measurement of suspended solids with a glass fiber filter.

45 Table 2-2 Target concentrations of halogenated contaminants in the microcosms Concentration (mg/L) a Compound High Medium Low CF 500 28 1.8 CT 8.8 10 6.5 CFC-11 26 2.9 0.75 1,1-DCE 9.1 0.11 0 a The designation of “high” concentration is based on CF and CFC-11; CT concentrations were relatively similar in each of the plumes.

46

Table 2-3 Products from transformation of [ 14 C]CT Halomethane concentrations Treatment % of [ 14 C]CT added a CH 4 CO CM DCM CS 2 CF CT other CO 2 sNSR pNSR UA loss b Low bioaugmentation 0.16 0 0.40 2.5 14 0.94 0.11 2.6 20 15 16 0.65 28

corn syrup + B12 0.40 0.09 0.36 1.5 0.28 0.76 0.84 0.38 36 18 15 4.1 21 corn syrup 0.01 0.54 0.25 1.3 5.1 13 6.2 0.71 6.2 6.0 25 2.3 34 vegetable oil 0.05 0.69 0.29 0.98 0.38 15 19 0.67 2.1 2.0 22 2.1 35 lactate 0 0 0.20 1.1 5.1 8.2 23 2.7 4.9 5.4 10 8.0 32 as-is 1.4 0.05 0.27 3.1 1.1 3.9 39 1.4 5.5 2.6 9.8 3.7 28 AC 0 0 0.71 0.92 0.17 2.6 52 3.1 1.1 0.40 2.6 8.5 28 Medium bioaugmentation 0.07 0 0.04 0 5.6 0.35 0.02 0.36 18 12 8.5 0 54 corn syrup + B12 0.46 0.02 0.44 0.14 1.6 0.20 0.03 0.32 29 13 11 6.8 37 corn syrup 0.02 0.01 0.10 0.21 2.8 6.4 0.19 2.6 8.8 7.2 17 0 55

47 vegetable oil 0 0 0.24 1.3 0.11 10 12 0.73 1.2 2.7 41 1.6 28 lactate 0.01 0.47 0.07 0.23 0.01 4.9 10 3.5 6.8 7.4 13 5.7 47 as-is 0.51 4.9 1.8 0.16 0.03 6.3 25 1.1 6.0 4.3 20 0 30 AC 0 0 0.24 0.47 0.12 0.84 60 1.4 0.89 0.51 2.2 1.9 31 High bioaugmentation 0.99 0 0.12 0.0 2.1 0.0 0.02 0.05 9.1 31 9.2 12 35 corn syrup + B12 1.4 35 0.64 0.0 0.36 0.0 0.50 0.33 6.7 18 7.7 0.0 30 corn syrup 0 0 0.02 0.0 1.1 2.4 0.05 0.61 6.7 16 18 9.2 47 vegetable oil 0 0 0 0.0 0.0 2.3 0.04 0.01 9.1 3.5 49 7.2 29 lactate 0.09 2.3 0.46 0.86 0.0 3.5 0 0.19 11 11 23 13 35 as-is 0 0 0.07 0 0.27 7.0 11 0.65 24 6.4 18 11 22 AC 0 0 0.06 0 0 0.60 52 0.21 1.7 0.32 0.93 0.68 44 a UA=unaccounted for, i.e., percentage of remaining 14 C that cannot be assigned to a particular compound or category. b numbers in bold represent the main products formed.

Table 2-4 Products from transformation of [ 14 C]CF Halomethane concentrations Treatment % of [ 14 C]CF added a CH 4 CO CM DCM CS 2 CF CT other CO 2 sNSR pNSR UA loss b Low bioaugmentation 0.08 0 0.40 24 4.9 0.91 0.06 0.71 11 15 7.7 1.0 35

corn syrup + B 12 0.42 34 1.3 1.8 0.15 0.86 0.04 0.48 13 14 7.5 0.41 27 corn syrup 0 0 0.13 1.5 0.23 53 0.54 0.43 1.7 11 9.5 0.87 22 vegetable oil 0 0 0.09 1.4 0.12 46 0.63 0.53 2.2 7.6 7.5 1.3 33 lactate 0 0 0.20 1.2 0.10 48 0.63 0.34 2.2 10 6.3 0.75 30 as-is 0 0 0.17 2.3 0.18 59 1.2 0.50 1.5 7.6 8.7 5.8 13 AC 0.08 0.10 0.28 2.8 0.16 60 1.2 0.41 3.6 6.1 5.6 7.4 12 Medium bioaugmentation 0.15 0 0.04 0.09 0.44 0.05 0.02 0.31 24 19 2.2 0 54

corn syrup + B 12 0.23 0.06 0.05 0.04 0.06 0.25 0.01 0.11 31 14 4.0 0 50 corn syrup 0 0 0.01 0.03 0.04 16 0.05 0.14 3.0 11 1.4 4.5 64

48 vegetable oil 0.49 19 0.54 2.8 0 7.3 0 1.75 3.1 11 2.9 0 51 lactate 0 0 0.06 0.03 0.07 19 0.03 0.11 3.9 9.9 1.6 3.58 62 as-is 1.5 14 1.7 0.09 0.44 35 0.70 0.75 4.2 10 3.6 0 28 AC 0 0 0 0.20 0.08 59 0.67 0.50 3.7 8.3 4.8 7.7 15 High bioaugmentation 1.3 0 0.06 0.11 0.15 0.05 0.0 0.22 13 32 5.6 5.1 42

corn syrup + B 12 0.26 37 0.30 0.35 1.2 0.0 0.0 0.39 1.8 13 1.0 0.0 44 corn syrup 0.0 0.0 0.0 0.0 0.13 15 0.0 0.14 1.6 13 1.8 1.2 67 vegetable oil 0.06 0.32 0.0 0.07 0.07 12 0.0 0.20 3.0 9.3 3.0 5.8 66 lactate 0.0 0.0 0.0 0.0 0.17 13 0.0 0.19 0.9 14 3.7 0.5 68 as-is 0.14 2.6 0.04 0.60 0.10 58 0.22 0.26 4.5 11 3.5 1.1 18 AC 0.06 0 0 0.59 0.14 64 0.98 0.24 3.6 7.8 1.9 4.4 17 a UA=unaccounted for, i.e., percentage of remaining 14 C that cannot be assigned to a particular compound or category. b numbers in bold represent the main products formed.

Table 2-5 DOC and sulfate removal for different treatment strategies

Halomethane mg DOC mg DOC concentration DOC (mg/L) added/mmol removed/mmol Sulfate (mg/L) ranges Treatment added removed halomethanes halomethanes initial removed Low bioaugmentation 3,020 340 15,000 1,690 3,680 1,530 corn syrup + B 12 1,140 239 5,660 1,190 522 corn syrup 1,110 323 5,500 1,600 651 vegetable oil 772 649 3,830 3,220 543 lactate 1,110 8 5,500 40 163 Medium bioaugmentation 3,440 846 5,010 1,230 2,740 2,680 corn syrup + B 12 1,620 542 2,360 787 1,140 corn syrup 1,470 505 2,140 733 904 vegetable oil 1,020 821 1,490 1,190 0 49 lactate 1,470 292 2,140 424 548 High bioaugmentation 4,000 279 564 38 443 443 corn syrup + B 12 2,250 265 317 36 57 corn syrup 2,060 404 291 55 443 vegetable oil 1,750 1,160 247 157 59 lactate 1,750 29 247 4 15

Table 2-6 Reactions involved in the transformation of halomethanes and associated Gibbs free energy changesa Reactions G°' Reductive dechlorination + - - CCl 4 + H + 2e → CHCl 3 + Cl -102.2 + - - CCl 3F + H + 2e → CHCl 2F + Cl -103.2 + - - CHCl 3+ H + 2e → CH 2Cl 2 + Cl -84.5 Reductive hydrolysis - + - CCl 4 + 2e + H 2O → CO + 2H + 4Cl -424.7 - + - - CCl 3F + 2e + H 2O → CO + 2H + 3Cl + F -415.4 - + - CHCl 3 + 2e + H 2O → HCHO + H + 3Cl -244.9 Substitution or net hydrolysis - + - CCl 4 + 2HS → CS 2 + 2H + 4Cl -501.2 - - - CCl 3F + 2HS → CS 2 + 2H+ + 3Cl + F -474.7 + - CHCl 3 + H 2O → CO + 3H + 3Cl -339.6 a 25 °C, reactants and products at 1M or 1 atm except for H+ at pH 7.0; free energies of formation were obtained from published data (16, 61, 68, 85).

50

300 20

250 a 16 200 12

150

8 100

(µmol/bottle) 4 2 50

0 0 0 200 400 600 800 1000

300 16

C1 C2 C3 C4 250 b 12

CF, CFC-11 CF, (µmol/bottle) 200

150 8

100

4 50 CT,DCM, HCFC-21,1,1-DCE, ethene, CS

0 0

0 200 400 600 800

Time (days) CF CFC-11 CT DCM HCFC-21 CS2 1,1-DCE Ethene

Figure 2-1 Representative performance of microcosms with high concentrations of halomethanes, treated by biostimulation ( a) and bioaugmentation ( b); arrow= addition of electron donor ( ↓↓↓=corn syrup, ↓↓↓= lactate, ↓↓↓= ethanol); =addition of culture (C1= lactate SRB, C2= ethanol SRB, C3=corn syrup fermentative, and C4=1,1-DCE respiring culture).

51

25 0.4

a 20 0.3 C4 15 0.2 10 0.1

(µmol/bottle) 5

2

0 0.0 0 100 200 300 400 500 600

15 0.3

C1 C2 b

10 C3 C4 0.2 1,1-DCE, ethene (µmol/bottle) 1,1-DCE,

5 0.1 CT, CF, CFC-11, DCM, HCFC-21, CS

0 0.0 0 100 200 300 400 500 600 700 800 Time (days)

CF CFC-11 CT DCM

HCFC-21 CS2 1,1-DCE Ethene

Figure 2-2 Representative performance of microcosms with medium concentrations of halomethanes, treated by biostimulation (a) and bioaugmentation (b); arrow= addition of electron donor ( ↓↓↓=corn syrup, ↓↓↓= lactate, ↓↓↓= ethanol; =addition of culture (C1=SRB lactate, C2=SRB ethanol, C3=corn syrup fermentative, and C4=1,1-DCE respiring culture).

52

0.4 CT low

med 0.3 high

0.2

0.1

0.0

) -1 0.05 CFC-11

0.04 0.03

0.02

0.01

0.00 Bioaug. + Lactate Firstorder rate constant (d 0.03 CF

0.02

0.01

0.00

as-is

Lactate corn syrup corn

Bioaug. +B12 Bioaug. corn syrupB12 + corn

oil veg. emulsified Figure 2-3 Comparison of first-order rate constants for CT (a), CFC-11 (b), and CF (c) with different treatments, for high, medium and low concentrations. Error bars are one standard deviation of six microcosms.

53

12 3 4 5 6 7 8 9

a b e f c g d h i

Figure 2-4 DGGE results for biostimulation microcosms. Lanes 1-3 are for microcosms with low concentrations of halomethanes, 4-6 for medium concentrations, and 7-9 for high concentrations; lanes 1, 4 and 7 are for the as-is treatment; lane 2, 5 and 8 are for biostimulation with corn syrup; and land 3, 6, and 9 are for biostimulation with corn syrup + B 12 . Bands that were identified are labeled a-i.

54

3. ANAEROBIC BIOTRANSFORMATION OF HIGH CONCENTRATIONS OF

CHLOROFORM BY AN ENRICHMENT CULTURE AND TWO ISOLATES

3.1 Abstract

Bioremediation of high concentrations of chloroform (CF) in groundwater remains highly challenging because of its extremely high toxicity and inhibitory effect on most anaerobic prokaryotes. The overall objective of this research is to develop cultures that can readily transform hundreds of mg/L of CF and is practical for field application.

DHM-1, a natural aquifer derived fermentative enrichment culture grown on corn syrup, was demonstrated to be able to transform over 500 mg/L CF in the presence of catalytic amounts of vitamin B 12 at a rate as high as 22 mg/L/d in a mineral salts medium.

Production of non-toxic end products, i.e., CO, CO 2, and organic acids indicates

hydrolytic reactions as the dominating pathway. DHM-1 is facultative, anaerobic and can

grow on corn syrup while transforming high concentrations of CF, which makes it a

promising culture for bioaugmentation. The two isolates from DHM-1 are both likely

Pantoea spp., and both possess an equal or even slightly better CF transforming

capability than DHM-1. Development of DHM-1 and the two isolates suggests that

bioaugmentation of source zones containing high concentrations of CF may be a feasible

remediation strategy.

3.2 Introduction

In the 2007 CERCLA Priority List of Hazardous Substances, CF ranks eleventh

overall, the third highest among chlorinated organics, after vinyl chloride and

polychlorinated biphenyls (8). This is a reflection of how frequently CF is detected in

55

groundwater and its high level of toxicity. When present at hazardous waste sites, CF is

often a focal point for evaluating the feasibility of bioremediation, since it is very toxic to

most obligate anaerobic prokaryotes. For example, only 1 mg/L of CF completely

inhibited tetrachloroethene (PCE) dechlorination by a mixed chlororespiring anaerobic

culture (73). Inhibition of reductive dechlorination of chloroethenes by CF is a general

problem for sites co-contaminated with CF, and can only be overcome by first removing

the CF (73).

In spite of major recent advances in bioremediation for chlorinated organic

compounds, treatment of CF, especially at high concentrations (e.g., >100 mg/L), still

remains challenging. Although aerobic biotransformation of CF is possible (e.g.,

cometabolism by a butane-grown strain (30)), CF is even more difficult to cometabolize

than trichloroethene (88). Biotransformation of CF by mixed or pure cultures under

methanogenic (9, 43) and sulfate reducing (42) conditions has been reported, however,

only at low mg/L CF concentrations.

Corrinoids such as vitamin B 12 (i.e., cyanocobalamin) are effective catalysts for

increasing the rate of halomethane biotransformation under anaerobic conditions.

Addition of vitamin B 12 also shifts the pathway away from reductive dechlorination and towards hydrolytic and substitutive reactions forming CO, CO 2 and organic acids (12, 47,

48). With catalytic amounts of B 12 added, an enrichment culture grown on dichloromethane (DCM) as the sole substrate (12) and a lactate-grown sulfate-reducing enrichment culture (36) were able to biotransform up to 260 mg/L and 270 mg/L of CF, respectively. However, use of the DCM culture is not feasible for in situ bioremediation

56

because it requires DCM as a growth substrate and only grows well at 35 oC. A major disadvantage of the lactate-grown culture is the accumulation of DCM during CF transformation.

The microcosm study described in Chapter 2 demonstrated that bioaugmentation is a technically feasible remediation strategy for high concentrations of halomethanes. Of the three enrichment cultures tested, the fermentative one that grows on corn syrup showed the most promise for biotransforming CF to environmentally benign products.

The objectives of this study were 1) to further characterize the fermentative culture in terms of its maximum rate of CF transformation, its dependence on B12 for transformation of CF, and its ability to grow on corn syrup in the presence of CF; 2) to identify the transformation products from CF; and 3) to isolate and identify the microbes in the enrichment culture that are responsible for CF biotransformation.

3.3 Materials and methods

3.3.1 Chemicals, radioisotopes and culture media

CF (99.7%, Shelton Scientific) and DCM (99.9%, AlliedSignal) were obtained as neat liquids. CF was added to serum bottles as either a water saturated solution (to provide up to 15 mg/L) or as a neat liquid (to provide up to 500 mg/L). Chloromethane

(CM; 99%, Praxair) and methane (99.99%, Matheson) were obtained as neat gases. Corn syrup (regular type, Sweetener Products Company) was used as the primary substrate.

The chemical oxygen demand (COD) for corn syrup was calculated based on its presumed composition, i.e., C 6H12 O6. Cyanocobalamin (i.e., vitamin B 12 , USP grade) was obtained from Research Organics, Inc. All other reagents were ACS grade or higher.

57

Neat [ 14 C]CF (0.5 mCi/mmol) was purchased from American Radiolabeled Chemicals,

Inc. and a stock solution was prepared in distilled deionized water (approximately 10 7 disintegrations per minute (dpm) per mL).

A previously described (32) mineral salts medium (MSM) was used, with the following modifications: NaCl and KH 2PO 4 were not added; the concentration of

K2HPO 4 and NaHCO 3 was changed to 0.41 g/L and 4.0 g/L, respectively; and the headspace consisted of 30% CO 2 and 70% N 2 instead of helium. Sodium sulfide (0.5 mM) was added to the MSM inside an anaerobic chamber. Pure cultures were grown in filter sterilized MSM and stock solutions of corn syrup, sodium sulfide, and B 12 (0.45

µm). Tryptic soy agar (TSA) was obtained from MO BIO Laboratories, Inc.

3.3.2 Development of an enrichment culture acclimated to CF

An enrichment culture was started in the absence of CF by combining soil (20 g) from an uncontaminated area of the industrial site as inoculum, MSM (100 mL) and corn syrup (40 mM) as the primary substrate. The bottles were prepared in an anaerobic chamber containing an atmosphere of approximately 98% N 2 and 2% H 2. After 1-2 weeks of incubation, the protein level reached 200 µg/mL and a 1% transfer was made to

MSM and corn syrup. When protein again reached 200 µg/mL, the culture was concentrated by centrifugation (5500 g) and resuspended in MSM (200 mL) plus corn syrup in a 250 mL bottle sealed with a Teflon-lined rubber septum and screw cap.

Acclimation to CF began by adding 2.8 µmol per bottle. Taking into account partitioning to the headspace (see below), the aqueous phase concentration was 1.5 mg/L. The initial dose of vitamin B 12 was 10 µM. As CF was transformed, increasing amounts of CF were

58

added. B 12 was added along with CF to maintain a 3% molar ratio of B 12 to cumulative

CF added. Periodic additions of corn syrup were made when there was a cessation of a decrease in pH, i.e., a decrease in pH indicated that fermentation was occurring due to accumulation of organic acids (predominantly formate, acetate and propionate), so when the pH stopped decreasing, more corn syrup was added (approximately 960 mg COD/L per dose). When the pH decreased to 6.7-7.0, it was raised to 7.5-7.7 with 8 M NaOH.

After consuming a single dose of 500 mg/L of CF, a 1% transfer of the enrichment culture was made to MSM plus corn syrup (960 mg COD/L ), B 12 (136 µM ) and CF (36 µL neat = 452 µmol per bottle, or an aqueous phase concentration of 500 mg/L) in 160 mL serum bottles with 100 mL of liquid phase. When the CF remaining was below 0.5 mg/L, further enrichment of the culture was achieved by sequential 1-5% transfers along with the same initial conditions for CF, B 12 and corn syrup. The amount of corn syrup needed gradually decreased so that by the 13 th transfer of the culture, only one dose (960 mg COD/L ) was made at the start. The enrichment culture obtained was designated DHM-1.

3.3.3 Pure culture isolation and acclimation to CF

Samples of DHM-1 were streaked on TSA and incubated (35 °C) aerobically for

24 h. Well isolated colonies were returned to MSM + corn syrup + CF + B 12 and incubated anaerobically. When CF transformation was complete, the process was repeated (i.e., restreaking, aerobic growth on TSA, and return of isolated colonies to liquid culture under anaerobic conditions). Samples from the resulting bottles were again restreaked and grown on TSA for up to 48 h. At this point, two morphologies were

59

identified; one was a larger white (slightly yellow) colony (1-2 mm) that grew more

quickly (i.e., within 24 h), the other a smaller yellow colony ( ≤ 1 mm) that grew more slowly (i.e., within 48 h). Samples of both colonies were restreaked on TSA four to seven times before returning the isolates to MSM with CF. The purity of the isolates was confirmed based on microscopic observation and sequencing of their 16S rRNA genes

(see below).

Once the isolates were obtained, they were exposed to CF again, as follows.

Single colonies from plates were used to inoculate MSM. Corn syrup was added, the headspace was purged with a mixture of 70% N 2 and 30% CO 2, and the cultures were allowed to grow for 24-48 h on a shaker table in the absence of CF or B 12 . Neat CF and

B12 were then added and the bottles were incubated for 2-3 days on a shaker table to

ensure that the neat CF dissolved. The bottles were then maintained as described in the

section on CF biotransformation. In subsequent transfers of the cultures to MSM (5%

v/v), growth on corn syrup prior to CF addition was no longer considered necessary, i.e.,

the CF, B 12 and corn syrup were added at the same time as inoculation. The purity of the

cultures was routinely checked by streaking on TSA.

3.3.4 CF biotransformation

CF biotransformation experiments were performed in 160 mL serum bottles,

which were prepared in an anaerobic chamber. After dispensing MSM (93 mL) and

inoculating the bottles (5 mL of DHM-1 or the isolates), they were removed from the

chamber. Corn syrup and B 12 were added (yielding a total liquid volume of 100 mL) to

provide initial concentrations of 960 mg COD/L and 0.136 mM (i.e., 3 mole percent of

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the total CF), respectively. The headspace was then purged for 1 min with a gas mixture

(30% CO 2/70% N 2) and the bottles were sealed with Teflon-lined red rubber septa and

aluminum crimp caps. CF was then added; the amount was confirmed gravimetrically.

The bottles were incubated for 2-3 days on a shaker table to ensure that the neat CF

dissolved. They were then transferred to the anaerobic chamber, where they were

incubated quiescently, in an inverted position and shielded from light. The bottles were

removed weekly to monitor and adjust pH as needed (to stay in the range of 6.7-7.7), add

corn syrup as needed, and remove headspace samples for gas chromatographic analysis.

They were shaken vigorously prior to headspace sampling to ensure that the aqueous and

headspace phases were in equilibrium.

One of the CF biotransformation experiments was performed using [ 14 C]CF along with CF, thereby allowing for measurement of the 14 C products formed. In order to minimize addition of any 14 C-labeled impurities, aliquots of the [ 14 C]CF stock solution

(60 µL) were injected onto a gas chromatograph (GC) containing a stainless steel column

(3.175 mm × 2.44 m) packed with 1% SP-1000 on 60/80 Carbopack B (Supelco). N2 was used as the carrier gas (28.5 mL/min at 150 °C). The outlet of the column was connected to stainless steel tubing (1.59 mm) that exited the GC oven and terminated with a needle, which was inserted into the microcosm headspace during the interval when CF eluted

(6.35-6.85 min). The total 14 C added was approximately 0.45 Ci/bottle.

3.3.5 Analytical methods and 14 C distribution

CF, DCM, CM, and methane were monitored by gas chromatographic analysis of

0.5 mL headspace samples, as previously described (35). A Hewlett Packard 5890 Series

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II GC equipped with a flame ionization detector was used. A response factor for each

compound was measured to relate the total mass present in a bottle to the GC peak area

(38). Aqueous phase concentrations were calculated based on Henry’s law constants at

23°C and the volume of headspace (60 mL) and liquid (100 mL) per bottle (35). The

detection limits are approximately 10, 9, and 1 µg/L for CF, DCM and CM, respectively.

The total amount of 14 C in the bottles was quantified by counting samples of the

headspace (0.5-mL) and liquid (0.1-mL) in liquid scintillation cocktail (LSC).

Distribution of the 14 C-labeled compounds (DCM, CM, CO and methane) was determined by a combination of headspace and aqueous-phase analyses (33, 35). Volatile compounds (DCM, CM, CO and methane) were analyzed with a GC/combustion technique. A 0.5-mL headspace sample was injected onto the Carbopack column and the well-separated compounds were routed to a catalytic combustion tube (containing CuO), where the compounds were oxidized at 800°C to CO 2. Each fraction was then trapped in

3 mL of 0.5 M NaOH and counted in 15 mL LSC. A second headspace sample was injected onto a stainless steel column (2.44-m by 3.175-mm) packed with Carbosieve SII

(Supelco) to separate methane, CO and CO 2, which were not adequately separated by the

Carbopack column.

14 14 CO 2 and C-labeled nonvolatile compounds were measured after analysis of the

14 C-labeled volatile compounds was completed. After raising the pH of the liquid to drive the CO 2 in headspace into the liquid phase (by adding 0.75 mL of 10 M NaOH), samples of the liquid phase (10 mL) were transferred to a test tube that was connected to a second test tube containing 10 mL 0.5 M NaOH. Nitrogen gas was sparged into the first tube and

62

over into the second. The pH in the first tube was then lowered by injecting 0.25 mL of 6

14 M HCl, to facilitate stripping of CO 2, which was trapped in the second tube. C activity

remaining in the first tube was referred to as nonstrippable residue (NSR). The percent

distribution of 14 C, including losses, was calculated as described elsewhere (19).

The distribution of 14 C in NSR was evaluated by injecting filtered samples (200

µL) onto a WATERS high performance liquid chromatograph (HPLC) using 0.01 N

H2SO 4 as the eluant and an Aminex® HPX-87H column (300 mm x 7.8 mm) for

separation of organic acids. Eluent samples were collected in LSC according to the

elution times for lactate, formate, acetate, propionate, ethanol, isobutyrate and butyrate

(based on the retention times of authentic material).

3.3.6 Denaturing gradient gel electrophoresis (DGGE) and protein analysis

DNA was extracted from cultures using an UltraClean® Microbial DNA isolation

kit (MO BIO Laboratories, Inc.). A segment of the 16S rRNA gene of the bacteria

domain was amplified with primer 1055F and 1406R with a 40 base GC clamp (18). A

touchdown PCR program (18) was adapted as follows: an initial denaturation step at 94

°C for 5 min; 10 cycles at 94 °C for 1 min, 65 °C for 1 min (touch down -1°C/cycle), 72

°C for 3 min; 20 more cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min, with a

final extension step at 72 °C for 10 min. The amplified rDNA was analyzed by DGGE

(74). Bands of DNA excised from the DGGE gel were soaked in 1 × SB buffer for a week, re-amplified by PCR, purified with a QIAquick  PCR purification kit (QIAGEN

Sciences), then submitted to MWG-Biotech, Inc. (Huntsville, AL) for sequencing.

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Species were identified by sequence alignment via Basic Local Alignment Search Tool

(BLAST).

Protein was measured with a BCA™ protein assay kit (Pierce Chemical Company)

following the manufacturer’s enhanced protocol after lysing the cells (15).

3.3.7 Identification of isolates

DNA was isolated from the two pure cultures using the same kit described above.

DNA was quantified spectrophotometrically and 30 µg was used as a template. The

small subunit 16S rRNA gene was amplified using primers 8F and 1492R (26, 84) using

the following PCR conditions: one cycle at 95 °C, 3 min; 55 °C, 1 min; 72 °C, 2 min; 35 cycles at 95 °C, 45 sec; 55 °C, 45 sec; 72 °C, 2 min; and one cycle of 72 °C for 15 min.

The resulting PCR product was cloned using the TOPO-TA cloning system (Invitrogen,

Inc, Carlsbad, CA). Recombinants were selected for using 50 g/mL kanamycin and 20

g/mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Recombinant plasmids were isolated using the alkaline mini-preparation and subsequently digested with EcoRI. Plasmids with full length inserts were submitted to the Clemson University

Genomics Institute for DNA sequence analysis. 16S rRNA gene sequences of the two isolates were identified via BLAST (2) and deposited in the GenBank database under accession numbers FJ745299 and FJ745300.

3.4 Results

3.4.1 Development of DHM-1

An enrichment culture capable of transforming CF was acclimated to increasing concentrations (Figure 3-1a). CF transformation rates increased throughout the

64

enrichment process and reached an average rate of 6.6 mg/L/day with the final dose of

500 mg/L. Accumulation of reductive dechlorination products, i.e., DCM and CM never exceeded 2 and 1 mg/L, respectively, and no methane was produced. With a subsequent

1% transfer, the resulting culture biotransformed 500 mg/L of CF at an average rate of 17 mg/L/d (Figure 3-1b). This rate increased somewhat through repeated transfers, reaching a maximum of 22 mg/L/d. Formation of DCM and CM continued to be negligible.

3.4.2 Product distribution from [ 14 C]CF

The principle products from [14 C]CF transformation by DHM-1 were CO

(67±2%), NSR (15±0.1%) and CO 2 (2.7±0.7%). All of the other potential products, including DCM, CM, and methane, were negligible (consistent with GC headspace analyses). HPLC fractionation revealed that the NSR consisted mainly of formate

(36±3%) and propionate (13±1%). The total loss of 14 C-labeled material during the 26 day incubation period was 13% (±0.8 %).

3.4.3 Growth of DHM-1 while transforming CF

Significant growth of DHM-1 (based on an increase in protein concentration) occurred in the presence of high concentrations of CF (Figure 3-2a). The initial protein concentration in all treatments was approximately 5 µg/mL. As expected, corn syrup was required for the growth of DHM-1; no significant growth occurred without it (i.e., in the treatments with the culture and MSM plus “CF only”; “B12 only”; and the “blank,” i.e.,

MSM only). Among the four treatments with corn syrup added, protein levels increased to approximately 100 µg/mL. The presence of CF did not have an inhibitory effect on protein yield when B 12 was present and CF was transformed (Figure 3-2b, “CF+B 12 +CS”).

65

However, the presence of CF without B 12 did have a modest inhibitory effect on protein yield (Figure 3-2a, “CF+CS”) and CF transformation was minor (Figure 3-2b, “CF+CS”).

Addition of B 12 with corn syrup (without CF) did not increase the protein yield above corn syrup alone (also without CF) (Figure 3-2a, “B 12 +CS” versus “CS”). No significant transformation of CF occurred in the live treatment with only CF added (Figure 3-2b,

“CF only”) or in the control with MSM and B 12 but no DHM-1 (Figure 3-2b, “MC+B 12 ”).

These results indicate that CF removal was a biotic process, requiring the presence of

DHM-1, corn syrup and B 12 .

Based on monitoring of protein concentration over time, most of the growth of

DHM-1 occurred when CF levels were in the 400 to 500 mg/L range (Figure 3-3). Only after growth reached a plateau (in this case with only one dose of 960 mg COD/L corn syrup on day 0) did most of the CF undergo transformation. A similar growth pattern for

DHM-1 was observed with only corn syrup added (i.e., without CF and B 12 ) (Appendix

B.1). Since the protein concentration was 60 µg/mL for most of the time period when CF was transformed, the average overall transformation rate of 22 mg/L/d can be expressed as 0.37 mg CF/mg protein/d.

3.4.4 Importance of sulfide to CF transformation

The contribution of sulfide to CF transformation by the DHM-1 enrichment culture was examined by replacing sulfide in the MSM with an equimolar amount of sodium sulfate (0.5 mM) (Appendix B.2). Without sulfide present, the color of the MSM was blue (based on the resazurin), indicating the E h was above -110 mV. Approximately

12 h after inoculation, the color of the medium became clear (based on the color change

66

in the resazurin) and turbidity in the bottles indicated growth had occurred. Nevertheless,

the rate of CF transformation was considerably slower than controls with the standard

MSM containing sulfide, i.e., approximately 5.5 mg/L/d versus 22 mg/L/d. In an effort to

restore the rate of CF transformation, the bottles were reinoculuated with the same

amount of DHM-1 on days 18 and 26 and corn syrup was added on day 18. The rate of

CF transformation did not improve. On day 48 more corn syrup was added and on day

50, sulfide was added so that the aqueous concentration was the same as in MSM, i.e., 0.5 mM. The rate of CF transformation increased significantly to 16 mg/L/d, similar to the performance of DHM-1 in sulfide-reduced MSM.

3.4.5 DGGE Analysis of DHM-1

During development of the DHM-1 culture, DNA from the fourth transfer was extracted and analyzed by DGGE. A total of six bands were resolved (Figure 3-4, lane 1).

Sequencing of bands B, C, D and E were most similar to Enterobacter spp. (95-99%), while band F was most similar to a Propionibacteriaceae sp. (98%). The latter was no

longer detectable by the sixth transfer (Figure 3-4, lane 2). The intensity of Band A,

which was not sequenced, decreased to a nearly undetectable level in the tenth transfer

version of the culture. The four bands that remained were all associated with

Enterobacter spp., indicating that they are the ones responsible for CF biotransformation.

3.4.6 Isolate identification and CF transformation

The two strains isolated from DHM-1 are Gram negative, motile rods. The faster

growing isolate was designated strain DHM-1B and the slower growing one was

designated strain DHM-1T. Based on their 16S rRNA gene sequences, the closest match

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for both strains is to Pantoea (Enterobacter) spp. Strains DHM-1B and DHM-1T align most closely with Pantoea sp. Nj-40 (100%) and Enterobacter spp. (up to 99%), respectively. When evaluated by DGGE, the isolates exhibited multiple bands (Figure 3-

4, lanes 4 and 5). The presence of multiple bands for pure cultures during DGGE has been reported previously (6).

Strains DHM-1B and DHM-1T retained the ability to transform high concentrations of CF. After inoculating them in MSM, there was a lag of 10 to 20 days before the onset of CF transformation. However, with subsequent transfers (5% v/v), the rate of CF transformation was similar and occasionally faster than the DHM-1 enrichment culture (Figure 3-5), reaching as high as 26 mg/L/d. As with DHM-1, transformation of 500 mg/L of CF by both isolates yielded negligible amounts of DCM and CM (i.e., less than 1.5 mg/L and 0.03 mg/L, respectively) and no methane. In addition, growth of both strains was not inhibited by the presence and/or transformation of CF, as was shown with DHM-1 (Figure 3-2a).

3.5 Discussion

DHM-1 is the first enrichment culture reported that is capable of biotransforming high concentrations of CF, e.g., at least as high as 500 mg/L. The two Pantoea spp. isolated from DHM-1 are the first strains reported that possess this dechlorination capability. Growth on corn syrup is required along with the presence of a catalytic level of vitamin B 12 , which appears to direct the transformation pathway away from reductive dechlorination and towards net hydrolysis to CO, oxidation to CO 2, and incorporation into organic acids. A similar role for B 12 was reported by Hashsham and Freedman for

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transformation of high concentrations of CT by fructose-grown A. woodii (47). Becker and Freedman (12) also observed the need for B 12 during transformation of CF by a

DCM-grown culture, yielding similar end products as DHM-1.

The ability of the DHM-1 enrichment culture and strains DHM-1B and DHM-1T to grow on corn syrup in spite of the presence of high concentrations of CF is especially noteworthy, given the toxicity of CF to most anaerobic microbes. With cometabolic processes, it is common for relatively low concentrations of the nongrowth substrates to inhibit growth on the primary substrate. For example, CF was found to be inhibitory to the growth of an anaerobic culture that cometabolizes CF (12). Less than 10 µM 1,1-

DCE and cis -DCE, or 100 µM of CF, DCM, vinyl chloride, and trichloroethylene is inhibitory to the growth of most types of methanotrophs (3, 45, 52). This is partly why cometabolic processes are more difficult to engineer than metabolic processes, i.e., growth on the primary substrate is often not feasible in the presence of the non-growth substrate, due to competitive inhibition of a shared enzyme (e.g., an oxygenase) and/or product toxicity. The ability of the cultures obtained during this study offers a potentially significant advantage for in situ application, i.e., only a low initial dose of culture may be needed since the microbes are capable of growth on corn syrup in the presence of high concentrations of CF.

Pantoea (or Enterobacter) is not a genus commonly associated with biodegradation of halogenated compounds. Nevertheless, a few strains with this ability have been reported, including some Enterobacter strains that dechlorinate polychlorinated biphenyls (1), DDT (13), toxaphene (60) and tetrachloroethene (80). In

69

addition, it appears that tolerance of high concentrations of halogenated compounds may

be a characteristic of Enterobacter species. For example, strain MS-1, which is closely

related to E. agglomerans , is able to grow in the presence of tetrachloroethene at

concentrations above saturation (2-10 mM) (80). Likewise, strain D-1, which is closely

related E. colacae , is able to grow in the presence of toxaphene at concentrations above

saturation (3-96 mg/L) (60).

Enterobacter are facultative microbes. This makes the DHM-1 enrichment

culture and the two isolates easier to handle than obligate anaerobic cultures, especially

for field application. The ability of the isolates to transform CF was retained even after

repeated streaking on TSA and incubation under aerobic conditions during the culture

isolation process. The cultures also retained their CF-transformation ability after at least

24 h exposure to air and then returning them to anaerobic MSM (Appendix B.3). It may,

therefore, be possible to apply DHM-1 in the field even before the in-situ redox

conditions are sufficiently reducing to achieve CF transformation. Fermentative growth

of DHM-1 on corn syrup causes the redox level of the surrounding environment to

decrease below -110 mV within several days, thereby creating conditions that are

favorable for CF transformation. This is similar to the ability of strain MS-1 to grow in

the presence of PCE under conditions that are too oxidized to support reductive

dechlorination of PCE; however, once the redox conditions become more reducing,

chlororespiration of PCE to cis -1,2-dichloroethene by Strain MS-1 commences (80).

Egli et al. (27) have noted that anaerobic dechlorination of chlorinated methanes is often associated with microbes that possess the acetyl-CoA pathway, which involves

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coenzyme B 12 as a carrier for one carbon methyl groups. The presumed benefit of adding supplemental B12 is to mitigate the toxic effects of the chlorinated compounds on coenzyme B 12 and carbon monoxide dehydrogenase (47). However, Enterobacter spp. ferment sugars via a mixed acid pathway that does not involve the acetyl-CoA pathway and has no dependence on B 12 (53, 69), so it is less clear what role B 12 plays in facilitating CF cometabolism by these microbes. However, some genera of

Enterobacteriaceae use a B 12 -dependent dehydratase to mitigate toxicity during metabolism of 1,2-propanediol and 1,2-ethanediol (87). The reactions take place in microcompartments that provide protection from DNA and cellular damage by a reactive metabolic intermediate (76). B12 -dependent reductive dehalogenases have been purified from several anaerobes mediating reductive dechlorination of chloroethenes (10).

However, transformation of CF by DHM-1 and strains DHM-1T and DHM-1B proceeds mainly via net hydrolysis to CO, not via reductive dechlorination to DCM, CM, or methane. It is not yet known if a B 12 -dependent dehalogenase homologous to reductive dehalogenases is involved in the hydrolytic pathway for CF transformation, or, more generally, what role B 12 plays in CF transformation by the isolates obtained in this study.

In addition to catalytic levels of B 12 , transformation of CF by DHM-1 appears to require sulfide. The MSM used in this study contains 0.5 mM; whether or not lower or higher concentrations affect the rate of CF transformation is not yet known. The role of sulfide (0.5 mM) in CT transformation by Pseudomonas stutzeri KC via a bacterial transition metal chelator has been demonstrated, with the undesirable effect of increased production of CS 2 (65). CS 2 formation during transformation of CF by DHM-1 is not a

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concern. However, maintaining a low level of sulfide in groundwater poses some

practical challenges for in situ remediation, including a potentially high demand for sulfides by the aquifer soil. Substitution of sulfide with another reluctant such as dithionate is worthy of further investigation, including the effect on the transformation rate of CF by DHM-1.

The appearance of multiple bands on the DGGE gel for DHM-1B and DHM-1T likely represent multiple copies of the 16S rRNA gene. Enterobacter are known to possess multiple copies of the rRNA operon. For example, two species reportedly have seven copies of operon (54).

It is not yet known what properties of DHM-1 and strains DHM-1B and DHM-1T enable them to grow on corn syrup in the presence of such high concentrations of CF as well as transform the CF. One possibility is the presence of microcompartments, the presence of which in several types of fermentative microbes has been linked to B 12 -

dependent degradation of 1,2-propanediol (76). The microcompartments in this

environment protect the cells from damage from a toxic metabolic intermediate.

Alterations to membrane fluidity may also play a role in the ability of the cultures in this

study to tolerate high concentrations of CF. Alterations in membrane structure,

particularly in fatty-acids composition, phospholipid headgroups, and protein content, is a

principal adaptive mechanism used by bacteria to resist the toxic effect of solvents (50).

The use of cultures in bioaugmentation is restricted to those that do not contain

human pathogens. Some species of Enterobacter (also known as Pantoea ) are known to behave as pathogens (17, 78). However, the pathogenicity of DHM-1, DHM-1T and

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DHM-1B is not yet known. If pathogenicity is not a concern, then these microbes show

significant promise for in situ bioaugmentation at sites with high concentrations of

halomethanes, based on their ability to grow in the presence of CF using an inexpensive

substrate (i.e., corn syrup) and transform CF with the aid of catalytic levels of B 12 . The predominant transformation products formed (CO, CO 2, and organic acids) are environmentally benign.

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3.6 Figures

600 a 15 500 10 5 400 0

300 0 5 10 15 20 25

CF(mg/L) 200

100 0 0 25 50 75 100 125 150 175 200 225 600 b 500 400

300 CF(mg/L) (mg/L) 200

100

0 0 5 10 15 20 25 30 35

Time (days)

Figure 3-1 Increase in the rate of CF transformation during initial development of the DHM-1 enrichment culture (a) and following a transfer (1% v/v) in MSM (b). Results in panel b are the average of duplicate bottles; error bars represent the data range.

74

120 a 100

80

g/mL) 60

40

( Protein 20 0

S S y k CS CS C n 2+C 2 only Bla 12+ CF+ B1 CF onl B1 CF+B 700 b

600

500

400 CF+B12+CS

300 CF+CS CF only CF(mg/L) 200 MC+B12 100

0 0 5 10 15 20 25 30 35 40

Time (days)

Figure 3-2 Effect of CF on growth of DHM-1 based on protein yields after 35-39 days of incubation (a); and associated CF transformation in the three live treatments and media control with B 12 but no culture (MC+B 12 ) (b); blank=live culture + MSM only; CS=corn syrup; error bars are the standard deviation for duplicate measurements from duplicate bottles. Corn syrup was added on days 0 and 22.

75

600 70

60 500 50 400 CF+B12+CS

protein 40 g/mL) 300 30

CF(mg/L) 200 20 Protein ( Protein 100 10

0 0 0 5 10 15 20 25

Time (days)

Figure 3-3 Growth of DHM-1 while transforming CF. The average of duplicate bottles are shown. Error bars are the standard deviation for duplicate measurements from duplicate bottles. Corn syrup was added only on day 0.

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1 2 3 4 5

Figure 3-4 Variation of microbial structure of DHM-1 along with transfers and comparison among the mixed culture and its isolates by DGGE. Lane 1, 2, and 3 are the fourth, sixth, and tenth transfers of DHM-1, respectively; lane 4 is strain DHM- 1B, lane 5 is strain DHM-1T.

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600 Pantoea DHM-1B

Pantoea DHM-1T 500

400

300

CF(mg/L) 200 100

0 0 5 10 15 20 25 30 Time (days)

Figure 3-5 CF transformation by the two Pantoea strains. Formation of DCM and CM was minor (< 1% of CF transformed). Results are the average of duplicate bottles; error bars represent the data range.

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4. EVALUATION OF CAPACITY OF DHM-1 FOR TRANSFORMING HIGH

CONCENTRATIONS OF HALOMETHANES IN MEDIA AND MICROCOSM

AND COMPARISON TO A COMMERCIAL CULTURE

4.1 Abstract

Following the development and characterization of DHM-1, a natural aquifer derived enrichment culture capable of transforming over 500 mg/L chloroform (CF), the capacity of DHM-1 for biotransforming high concentrations of halogenated methanes was explored. With addition of catalytic amounts of vitamin B 12 , DHM-1 can readily transform at least 2000 mg/L CF. Furthermore, DHM-1 is active on transformation of carbon tetrachloride (CT) and trichlorofluoromethane (CFC-11), too. Complete transformation of 11 mg/L CT, 24 mg/L CFC-11 and 504 mg/L CF was achieved in less than four months by DHM-1 in mineral salts medium. CO, CO 2 and organic acids are the prevailing end products of transformation of CT and CF, indicating hydrolytic reactions instead of reductive dechlorination are the dominant pathways. This study also demonstrated the capability of SDC-9, a commercially available mixed culture, to transform high concentrations of halomethanes. DHM-1, has an advantage over SDC-9 in terms of the rate of CF transformation, while SDC-9 exhibited a higher rate on CFC-11.

A microcosm study further demonstrated the use of these two cultures for bioaugmentation to enhance in situ bioremediation of groundwater contaminated by high

concentrations of halomethanes.

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4.2 Introduction

In spite of major recent advances in bioremediation of groundwater, efficient in situ bioremediation strategies to treat groundwater contaminated with high concentrations of halogenated methanes such as CT, CF, and CFC-11 are still lacking. On the 2007

CERCLA Priority List of Hazardous Substances, CF ranks eleventh, the third highest among chlorinated organics, after vinyl chloride and polychlorinated biphenyls (8). This is a reflection of how often CF is detected in groundwater and its high level of toxicity.

Although not ranked as highly, CT is also on the list, and CFC-11 is often a co- contaminant with CT and CF. Among these compounds, CF is often the focal point for evaluating the feasibility of bioremediation because of its high toxicity to most obligate anaerobic prokaryotes. For instance, tetrachloroethene dechlorination by a mixed dechlorinating anaerobic culture was completely inhibited by only 1 mg/L of CF (73).

Inhibition of reductive dechlorination of chloroethenes by CF is a major concern for sites co-contaminated with CF, and can only be overcome by removing the CF first (73).

Although bioaugmentation is rapidly maturing as a technology for treatment of chlorinated ethenes (28), there is comparably little information for halogenated methanes.

Information was found for only one commercially available culture, SDC-9, that is being marketed for its ability to transform CT and CF (28). However, the characteristics of

SDC-9 in terms of transformation rates and products formed during halomethane transformation have not been reported.

The results presented in Chapter 2 demonstrated that bioaugmentation is a technically feasible strategy for sites contaminated with high concentrations of

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halomethanes. Of the three enrichment cultures described in Chapter 2, the fermentative culture that grows on corn syrup exhibited the most promise, especially in terms of its ability to cometabolize CF. Corn syrup was the most effective substrate tested and addition of catalytic levels of vitamin B 12 was essential for sustaining a rapid rate of transformation to environmentally benign products.

In Chapter 3, the characteristics of the DHM-1 enrichment culture were further explored, based mainly on its ability to cometabolize CF. Two isolates (strains DHM-1B and DHM-1T) obtained from DHM-1 were also evaluated in terms of their ability to cometabolize CF. The objectives of this study were 1) to further characterize DHM-1 in terms of its ability to cometabolize CT and CFC-11, alone and in mixtures that included

CF, and the tolerance of DHM-1 to concentrations of CF up to 4,000 mg/L; and 2) to compare DHM-1 to SDC-9 with respect to their transformation rates for CT, CFC-11 and

CF; the distribution of products formed using [ 14 C]CT and [ 14 C]CF; and the performance of these cultures in microcosms. The microcosms were similar to the ones described in

Chapter 2, although the focus was on bioaugmentation and the effect of various amendments on performance of DHM-1 and SDC-9.

4.3 Materials and methods

4.3.1 Chemicals, radioisotopes, enrichment cultures and culture media

CT (99.9%, Sigma-Aldrich), CF (99.7%, Shelton Scientific), dichloromethane

(DCM; 99.9%, AlliedSignal), CFC-11 (99%, Aldrich), and CS 2 (100%, J.T.Baker) were obtained as neat liquids. Chloromethane (CM; 99%, Praxair), dichlorofluoromethane

(HCFC-21; 99%, SynQuest Labs), chlorofluoromethane (HCFC-31; 99%, SynQuest

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Labs), and methane (99.99%, Matheson) were obtained as neat gases. Neat 14 C-labeled

CT (1.0 mCi/mmol) and CF (0.5 mCi/mmol) were purchased from American

Radiolabeled Chemicals, Inc. Stock solutions of [ 14 C]CT and [ 14 C]CF were prepared in

distilled deionized water at concentrations of approximately 10 7 disintegrations per

minute (dpm) per mL. Sodium lactate (60% syrup, EM Science) and corn syrup (regular

type, Sweetener Products Company) were used as electron donors. The chemical oxygen

demand (COD) for corn syrup was calculated based on its presumed composition, i.e.,

C6H12 O6. Cyanocobalamin (i.e., vitamin B 12 , USP grade) was obtained from Research

Organics, Inc.

Two types of enrichment cultures were used in this study, DHM-1 and SDC-9.

The development of DHM-1 is described in Chapter 3, including its ability to biotransform up to 500 mg/L of CF to nontoxic products. Corn syrup serves as the growth substrate. SDC-9 is a commercially available culture marketed for use in bioremediation of chlorinated ethenes and ethanes.

The mineral salts medium (MSM) used to grow DHM-1 is the same as that described in Chapter 3. The revised anaerobic mineral medium (RAMM) used for SDC-

9 was prepared as previously described (81), with the following changes. Yeast extract was added (0.05 g/L) and the concentration of sodium sulfide was decreased to 0.12 g/L

Na 2S⋅9H 2O (i.e., 0.5 mM sulfide), so that the MSM and RAMM had the same concentrations of both of these components.

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4.3.2 Transformation experiments with the enrichment cultures

Transformation experiments employing DHM-1 and SDC-9 were performed with

the individual halomethanes (approximately 15 mg/L CT, 26 mg/L CFC-11 and 500

mg/L CF). The same initial protein concentrations were used for both cultures

(approximately 5 µg/mL). Treatments with and without vitamin B 12 (3% molar ratio)

were prepared in 160 mL serum bottles with 100 mL of MSM or RAMM and 60 mL of

headspace, in an anaerobic chamber (Coy laboratory Products, Inc.) containing an

atmosphere of approximately 98% N 2 and 2% H 2. The initial dose of substrate provided was approximately 960 mg/L as COD. After purging the headspace with 30% CO 2/70%

N2 for 1 min, the bottles were sealed with 20-mm Teflon-faced red rubber septa and aluminum crimp caps. Halomethanes were then added using neat compounds. Media controls (no culture present) with and without B 12 for each halomethane were prepared in

MSM and RAMM.

In the bottles that received CT and CF, [ 14 C]CT and [ 14 C]CF were also added to provide an initial 14 C activity of 10 6 dpm/bottle. The labeled compounds were added prior to adding the neat compounds. The gas chromatographic method described in

Chapter 3 was used to add the labeled compounds, to minimize the introduction of 14 C impurities in aqueous stocks.

The bottles were quiescently incubated in an inverted position in boxes at room temperature (22 °C) in the anaerobic chamber. pH was monitored weekly and maintained between 6.7-7.7. A second dose of substrate was added when the pH stopped decreasing, signifying a cessation in substrate utilization, as described in Chapter 3.

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The ability of the DHM-1 culture to transform two-component mixtures of the halomethanes was examined, as well as transformation when the three halomethanes were present at the same time. For the two-component experiments, a single dose of B 12 was provided at the start (3% molar ratio of the total halomethanes added). When all of the halomethanes were present, the first dose of B 12 was made based on the molar amount of CT (i.e., 3 mole percent). When CT transformation was nearly complete, a second dose of B 12 was made based on the molar amount of CFC-11 added, and when CFC-11 was nearly consumed, a third dose was added based on the CF added. Along with the second dose of B 12 , a second addition of DHM-1 was made (adding another 5 µg/mL of culture), since preliminary tests suggested that the culture’s activity on CFC-11 and CF was inhibited after completing transformation of CT, presumably because the CT transformation pathway may involve some inhibitory intermediates, e.g., thiophosgene

(65). In addition to media controls with B 12 present, water controls (WC) and autoclaved controls (AC) were prepared as part of the experiment with all three halomethanes present. WC consisted of 100 mL of distilled deionized water; AC consisted of bottles that were inoculated with DHM-1 in MSM and then autoclaved for 1 h, after which the neat halomethanes were added.

4.3.3 Microcosm evaluation of bioaugmentation with DHM-1 and SDC-9

The effectiveness of the two enrichment cultures for use in bioaugmentation was compared in a microcosm study. In addition to bioaugmentation, the other treatments included as-is conditions (no substrate or B 12 added), biostimulation with corn syrup, and biostimulation with corn syrup + B 12 . All treatments were prepared in triplicate. Soil

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from the high concentration area of the industrial site described in Chapter 2 was used.

Five types of liquid were used, depending on the type of treatment (Table 4-1). All were

prepared with a sample of municipal tap water obtained from the industrial site. Use of

buffered municipal water was designed to simulate extraction of groundwater and

injection of municipal water, to adjust the in situ pH without accumulating precipitates

(column B in Table 4-1). To determine if bioaugmentation requires the presence of the

chemicals found in the MSM and RAMM, the respective media were prepared using the

municipal water (column F D for DHM-1 and F S for SDC-9 in Table 4-1). However, the

municipal water was not autoclaved or deoxygenated prior to adding the chemicals, as

was done with the media. In addition, sulfide for F D and F S treatments was not added until the first bioaugmentation event. Considering the potential difficulties of adding sulfide in situ , two other types of media were prepared using municipal water with an equimolar amount of sulfate replacing the sulfide (column FD' for DHM-1 and F S' for

SDC-9 in Table 4-1). The intent was for sulfate to be microbially reduced to sulfide in the microcosms. Resazurin was added (1 mg/L) to all bottles to serve as a redox indicator.

The microcosms were prepared in 160 mL serum bottles with 20 g of soil and 50 mL of liquid (Table 4-1) inside an anaerobic chamber. After purging the headspace with high purity N 2 for 1 min, the microcosms were sealed with 20-mm Teflon-faced red rubber septa and aluminum crimp caps. The microcosms were then placed on a shaker table for one hour to facilitate obtaining equilibrium between the soil and liquid phase.

Liquid samples (0.2 mL) were removed to measure sulfate and confirm that the initial pH

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was between 7.3 and 7.6. CT, CFC-11, and CT were then added using neat liquids to

achieve initial concentrations of 10, 26, and 500 mg/L, respectively. Due to the high

vapor pressure of CFC-11, it was necessary to cool the neat material and the syringe used

to add it to 4ºC. Finally, an initial dose of corn syrup or lactate was made (720 mg/L

COD) to the appropriate treatments (Table 4-1). The microcosms were quiescently

stored in an inverted position in boxes at room temperature in the anaerobic chamber.

Vitamin B 12 was added using the same dosing strategy described above for the experiment with DHM-1 in MSM with CT, CFC-11, and CF present at the same time.

However, the first dose of B 12 (0.31 µmol/bottle) was not made until the liquid reached

an E h of less than -110 mV, based on the color change of the resazurin. The second dose

(2.42 µmol/bottle) was made when consumption of CT was nearly complete and the third

dose (7.94 µmol/bottle) was made when CFC-11 was nearly gone.

The first bioaugmentation dose was made at the same time or shortly after the first

addition of B 12 , i.e., after low redox conditions had developed. The volume of

enrichment culture added was adjusted so that the resulting protein concentration in the

microcosms was 5 µg/mL (i.e., the same concentration used to inoculate the cultures in

media alone). A second dose of bioaugmentation culture was made once CT was nearly

consumed. Subsequent additions were made in an attempt to achieve the same rates in

the microcosms that were observed with DHM-1 in MSM.

pH was monitored approximately once per month and was maintained in the

range of 6.7-7.5. Additional amounts of corn syrup or lactate (720 mg COD/L per dose)

were provided when the pH stopped decreasing, as described in Chapter 3.

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4.3.4 Analytical methods and 14 C distribution

The amount of volatile organic compounds (CT, CFC-11, CF, DCM, CM, HCFC-

21, HCFC-31, methane and CS 2) present in serum bottles was determined based on

analysis of headspace samples using gas chromatography. For the microcosm

experiments, the same method described in Chapter 2 was used and for the

transformation experiments, the same method described in Chapter 3 was used.

Compounds were separated and quantified using a Hewlett Packard 5890 Series II gas

chromatograph (GC) equipped with a flame ionization detector and a 2.44 m × u3.175 mm column packed with 1% SP-1000 on 60/80 Carbopack B (Supelco). A GC response factor for each volatile organic compound was measured to relate the total mass of the compound to the GC peak area (38). Previously reported values for Hc at 23°C was used

(7, 38, 77). The detection limit of this method was lower than the maximum contaminant level for each of the chlorinated compounds.

Sulfate was measured on a Dionex AS50 ion chromatography system equipped with a CD25 Conductivity detector and a Dionex guard column (AG9-HC, 4 mm x 50 mm) followed by an IonPac ® AS9-HC anion-exchange column (4 mm x 250 mm).

Eluant (9 mM Na 2CO 3) was delivered at 1.0 mL per min. Protein concentration was measured with a BCA TM protein assay kit (Pierce Chemical Company) by following the manufacturer’s enhanced protocol after lysing the cells (15).

The amount of 14 C activity and its distribution were quantified using the same methods described in Chapter 3. The total 14 C activity remaining in bottles was measured

by counting 0.5 mL headspace and 0.1 mL liquid phase sample. The distribution of 14 C

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was determined through headspace and liquid phase analyses. Volatile 14 C-labeled compounds were analyzed with headspace samples and a GC-combustion technique (35).

14 14 CO 2 and C-labeled nonvolatile compounds were determined through liquid phase analyses (33).

4.3.5 Sulfide demand test and dissolved sulfide measurement

Dissolved sulfide was analyzed following the methylene blue colorimetric assay in Standard Methods (40). To investigate the sulfide demand of the soil used to prepare the microcosms, bottles in duplicate were prepared with 20 g of soil and 50 mL municipal water (1 mg/L resazurin added) inside the anaerobic chamber. After purging the headspace with high purity N 2 for 1 min, sulfide was added (2, 4, 8, 12, and 16 mM).

The bottles were incubated in the anaerobic chamber and the remaining dissolved sulfide was measured after 7 and 21 days of incubation.

4.4 Results

4.4.1 Biotransformation of CT, CFC-11 and CF by DHM-1

When provided with corn syrup and B 12 , DHM-1 readily transformed the three halomethanes, although the culture was developed to transform CF and was not previously acclimated to CT or CFC-11. Transformation of approximately 16 mg/L CT

(i.e., 18 µmol/bottle) was completed in 14 days (Figure 4-1a). Up to 1.5 mg/L (2.9

µmol/bottle) of CS 2 accumulated, accounting for 16% of the CT transformed, indicating the substitutive pathway was involved in CT transformation. Formation of reductive dechlorination products (i.e., CF, DCM and CM) was negligible. Over a 14 day incubation period, transformation of CT was not observed in the live control (i.e., DHM-

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1 with corn syrup but without B 12 ), although some removal (20%) occurred in the media

+ B 12 control. No transformation of CT occurred in a control with media without B 12

( Appendix C.1), indicating that B 12 contributed to abiotic transformation of CT.

Biotransformation of CFC-11 started after a lag phase of approximately 10 days

(Figure 4-1b). Then, 24 mg/L (56 µmol/bottle) of CFC-11 was completely transformed

by day 48. Approximately equal amounts of HCFC-21 and CS 2 (i.e., approximately 3

µmol/bottle each) accumulated, accounting for 11% of the total CFC-11 transformed,

while formation of HCFC-31 was negligible. There was only a minor amount of CFC-11

transformation in the live control (i.e., DHM-1 with corn syrup but without B 12 ) and

media + B 12 control, proving the CFC-11 transformation was biotic and required a

catalytic amount of B 12 . No transformation of CFC-11 occurred in a control with media without B 12 (Appendix C.1).

DHM-1 transformed 513 mg/L CF (i.e., 464 µmol/bottle) in as short as 23 days including a 5-day lag phase (Figure 4-1c), or an average rate of 22 mg/L/d. This is consistent with previously reported rates for DHM-1 (Chapter 3). Only 0.8 mg/L DCM

(1 µmol/bottle) and 0.9 mg/L of CS 2 (1.8 µmol/bottle) accumulated, accounting for 0.6% of the CF transformed. In the live control (i.e., DHM-1 with corn syrup but without B 12 ) and media + B 12 control, there was no transformation of CF. No transformation of CF

occurred in a control with media without B 12 (Appendix C.1),

In the vicinity of a source zone of CF that contains nonaqueous phase liquid, the

aqueous phase concentration of CF will be higher than 500 mg/L. Experiments were

conducted to determine the ability of DHM-1 to transform higher concentrations. As

89

shown in Figure 4-2, an initial concentration of CF of approximately 1000 mg/L (i.e., 900

µmol/bottle) was transformed by DHM-1 in 85 days and 2000 mg/L (i.e., 1800

µmol/bottle) was consumed in 180 days. Accumulation of DCM and CS 2 was negligible, i.e., less than 0.5 and 1.0 µmol/bottle, respectively. WC results proved that diffusive loss of CF over the incubation period was minor. The activity of DHM-1 on CF ceased when the initial CF concentration was 4000 mg/L (i.e., 3.6 mmol/bottle), which is approximately 50% of saturation of CF in water. In spite of these high initial concentrations of CF, growth of DHM-1 on corn syrup was not adversely affected. An increase of 94, 102 and 133 µg/mL of protein on approximately 1920 mg COD/L corn syrup was detected at the end of the incubation period (Figure 4-2), for initial CF concentrations of 1000, 2000 and 4000 mg/L, respectively. This level of growth is similar to previously reported values for DHM-1 in the presence and absence of CF at

500 mg/L (Chapter 3). Although growth of DHM-1 in the presence of CT and CFC-11 was not measured in terms of protein levels, an increase in the turbidity of the culture was observed, suggesting that DHM-1 also grows on corn syrup in the presence of CT and

CFC-11.

4.4.2 Biotransformation of halomethane mixtures by DHM-1

In the presence of 12 mg/L CT (13 µmol/bottle) and 512 mg/L CF (463

µmol/bottle), DHM-1 biotransformed both halomethanes simultaneously (Figure 4-3a), with no apparent effect of CT on CF or vice versa (i.e., comparing Figure 4-1 and Figure

4-3). Transformation of CT and CF was complete in 10 and 33 days, respectively. A minor amount of CS 2 (5.2 µmol/bottle) accumulated, accounting for 1% of the sum of CT

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and CF transformed. Formation of DCM (0.6 µmol/bottle) was negligible (Figure 4-3a).

With a mixture of 23 mg/L CFC-11 (55 µmol/bottle) and 509 mg/L CF (461 µmol/bottle),

DHM-1 biotransformed the CFC-11 in 29 days, which was somewhat faster than being transformed individually (Figure 4-1b). This may indicate that DHM-1 functions better on CFC-11 when CF is present. In contrast, transformation of CF was inhibited by the presence of CFC-11; CF transformation did not begin until the concentration of CFC-11 dropped to 6 mg/L (14 µmol/bottle) on day 18, then finished by day 48 (Figure 4-3b).

Minor amounts of CS 2, DCM and HCFC-21 accumulated (4.2, 0.5 and 1.8 µmol/bottle, respectively) in comparison to the amount of halomethanes removed (Figure 4-3b). With a mixture of 16 mg/L CT (17 µmol/bottle) and 24 mg/L CFC-11 (57 µmol/bottle), CT was completely transformed in 10 days, and following a lag phase of approximately 10 days, CFC-11 was consumed by day 47. These patterns are similar to what occurred with the individual compounds (Figure 4-1), indicating no apparent interaction between CT and CFC-11. The combination of CT and CFC-11 resulted in more CS 2 accumulation

(5.8 µmol/bottle) than the other two-component mixtures, accounting for 7.7% of the CT and CFC-11 transformed. Formation of HCFC-21 (3.3 µmol/bottle) was also slightly higher than in the mixture of CFC-11 and CF, while formation of DCM was negligible

(Figure 4-3c).

When CT, CFC-11, and CF were added at the same time (11, 24, and 500 mg/L, respectively), the pattern of transformation was similar to what was observed in the two- component mixtures. CT (12 µmol/bottle) transformation was completed first, followed by CFC-11 (57 µmol/bottle) and then CF (456 µmol/bottle) (Figure 4-4). This same

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order of transformation was observed in a microcosm study with the same mixture of halomethanes (Chapter 2). One difference between the live bottles shown in Figure 4-4 and Figure 4-3 is the pattern of B 12 addition. With the two-component mixtures, B 12 was added only at the start. With the three-component mixture, the same molar ratio of B 12 was added, although the additions were timed to coincide when transformation of each compound began. CT was removed to below detection by day 10. Transformation of

CFC-11 accelerated after day 8 and proceeded at a nearly linear rate through day 75. As was observed in the CFC-11 + CF mixture (Figure 4-3b), a high rate of CF transformation did not start until transformation of CFC-11 was nearly complete on day

65 (2.5 mg/L, 6 µmol/bottle). Then, the remaining 462 mg/L CF (418 µmol/bottle) was removed by day 112 at a rate comparable to those for single (Figure 4-1c) and two- component mixtures (Figure 4-3b). Only 2 µmol/bottle CS 2 accumulated, accounting for

0.4% of the halomethanes transformed. Formation of reductive dechlorination products

(DCM, CM, HCFC-21, HCFC-31) was negligible.

In the controls with all three halomethanes present, nearly complete transformation of CT occurred by day 113 in the media control and AC, versus no significant losses from the WC (Figure 4-4a). Approximately 33% and 11% of CFC-11 was removed in the autoclaved and media controls, respectively, while loss of CFC-11 in

WC was minor (Figure 4-4b). Losses of CF in all three controls were minor (1.5-3.8%).

These results demonstrate that transformation of CF was exclusively a biotic process, while abiotic processes contributed to transformation of CT and CFC-11. However, transformation of CT and CFC-11 was considerably faster in the presence of live cells,

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and DHM-1 was able to achieve complete transformation of a mixture of CT, CFC-11

and CF at high initial concentrations in less than four months in MSM.

4.4.3 Transformation rates

The commercially available bioaugmentation culture SDC-9 also exhibited an

ability to transform CT (15 mg/L), CFC-11 (25 mg/L), and CF (500 mg/L). The trends

observed for DHM-1 in Figure 4-1 were similar for SDC-9, although there were some

differences in the rates. Both cultures showed the highest transformation rate on CF, i.e.,

over an order of magnitude higher than for CT and CFC-11 (Table 4-2). The average and

maximum rates for CF transformation by DHM-1 were approximately two times greater

than for SDC-9. SDC-9, however, transformed CFC-11 at a higher rate than DHM-1.

The average CT transformation rate for DHM-1 was slightly higher than for SDC-9

although their maximum rates were not statistically different (Student’s t-test, α=0.025).

The live control and media control for SDC-9 were different from those used for

DHM-1, due to the differences in media composition between RAMM and MSM.

Nevertheless, the SDC-9 controls behaved similarly. Over the same length of incubation time as the live treatment with SDC-9 (i.e., 21, 33, and 41 days for transformation of CT,

CFC-11, and CF, respectively), less than 10% transformation of CFC-11 and CF occurred in the live control, with 15% loss of CT. In the media with B 12 control, losses of CFC-11

and CF were minor, while CT decreased by 34%. This was somewhat higher than the

comparable control for DHM-1 (Figure 4-1a), although the incubation time for SDC-9

was 7 days longer. In media controls with no B 12 added, losses of CT, CFC-11 and CF

were insignificant.

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4.4.4 14 C product distribution

14 14 14 CO and CO 2 were the dominant products from transformation of [ C]CT and

14 [ C]CF (Table 4-3). The sum of CO and CO 2 accounted for approximately 70% of CT and CF transformation by both cultures. CO 2 predominated from CT transformation (i.e., over 50%) while CO was predominant from CF transformation, especially by DHM-1.

Nonstrippable residue (NSR) was the third most significant product. Only a minor amount of CS 2 accumulated from CT transformation, and relatively more CS 2 was generated by SDC-9 than DHM-1 (i.e., 10% vs. 5.2%), consistent with GC results. High performance liquid chromatographic fractionation of the NSR (recovery efficiencies were

75 ±7.5% and 59 ±3.2% for the NSR formed by DHM-1 and SDC-9, respectively) indicated that the main products were formate (35%) and propionate (18%) for DHM-1; and formate (21%), acetate (11%), and propionate (9%) for SDC-9.

4.4.5 Microcosm evaluation of bioaugmentation

Redox conditions conducive to halomethane transformation (i.e., E h<-110 mV) were established within one week in all treatments that received corn syrup and media- augmented water (i.e., F D’ or F D in Table 4-1), while it took 15 days for the microcosms prepared with buffered water (i.e., B), 25 days for the lactate-amended SDC-9 microcosms that received media-augmented water (i.e., F S’ or F S), and 50 days for microcosms with only buffered water (i.e., B). This contrast between corn syrup and lactate is consistent with that observed in the previously reported microcosm study for the same site (Chapter 2). As a result, the first dose of bioaugmentation of DHM-1 was implemented on day 15, while bioaugmentation of SDC-9 was not done until day 51.

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The initial sulfate concentration in microcosms was 500-600 mg/L, approximately

one third of which was from the site municipal water, with the balance from the soil.

Significant decreases in sulfate were observed by day 60 (up to complete removal) in all

treatments receiving corn syrup or lactate, especially in the media augmented tap waters.

This indicated the presence of indigenous sulfate reducing bacteria, even though high

concentrations of halomethanes were present.

Microcosm results are shown in Figure 4-5; each data point is the average of

triplicate bottles. The transformation rates observed are slower than what was reported

for both cultures in MSM or RAMM. For CT (Figure 4-5a, d, and g), transformation

rates grouped in the following order, from fastest to slowest: 1) the biostimulation

treatment with corn syrup + B 12 in sulfate-amended media (F D’); all of the treatments

with DHM-1 added; and the two SDC-9 treatments with amended media (FS’ and F S); 2) the biostimulation treatment with corn syrup + B 12 in buffered water (B); 3) the treatment

with SDC-9 added to buffered water (B); and 4) the biostimulation treatments with corn

syrup (no B 12 ) in sulfate-amended media (F D’) and the one with corn syrup (no B 12 ) in buffered water (B). These results suggest that addition of B 12 is essential for obtaining high rates of CT transformation, while bioaugmentation is not. The presence of media rather than simply buffered tap water was beneficial.

Activity on CFC-11 was not initiated until removal of CT was nearly complete.

However, significant levels of CFC-11 transformation were initially restricted to the two treatments receiving SDC-9 in media-amended water (FS’ and FS) (Figure 4-5h).

Transformation of CFC-11 in the DHM-1 treatments with media-amended water (F D’ and

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FD) also started after CT was removed, but at a slower rate (Figure 4-5e). Addition of sulfide (0.5 mM on day 134) appears to have improved the rate of CFC-11 removal with

DHM-1. On day 162, the dissolved sulfide concentration was close to the detection limit, indicating that most of the sulfide was consumed. Based on this result, a separate experiment was performed to measure the extent of sulfide consumption by reaction with the soil. In serum bottles that received soil and municipal water, various initial amounts of sulfide were added and the amount remaining was measured after varying periods of incubation. As shown in Figure 4-6, soil from the industrial site exerted a significant demand on sulfide. To achieve a soluble sulfide level of 0.5 mM after 21 days of incubation required a dose of 0.85 mg S 2-/g soil, equivalent to an initial sulfide dose of 8 mM. The importance of sulfide to maintaining high rates of CF transformation by DHM-

1 were reported in Chapter 3, although similar experiments have not yet been performed with CT and CFC-11. In the treatments with sulfate-amended media (F D’, F S’ and B), additional sulfate was added, equimolar to the amount of sulfide added to the treatments with sulfide-amended water (F D and F S).

As with CT, the transformation rates observed through day 205 for CFC-11 in the microcosms are slower than in MSM and RAMM. SDC-9 bioaugmented treatments were faster than those with DHM-1, consistent with its faster rate on CFC-11 observed in media (Table 4-2). Unlike CT, high rates of CFC-11 biotransformation requires bioaugmentation. Faster rates of CFC-11 transformation were also dependent on the presence of B 12 and media-amended water. Although transformation of CF was not observed as of day 205, activity is expected as soon as the CFC-11 is consumed.

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4.5 Discussion

This study demonstrated the ability of the DHM-1 enrichment culture to

biotransform high concentrations of mixtures of halomethanes. Furthermore, growth of

DHM-1 on corn syrup was not affected by the presence of CF at concentrations up to

4,000 mg/L, although transformation activity ceased at this level. It appears that tolerance

to high concentrations of halogenated solvents is characteristic of Enterobacter species.

For example, one strain closely related to Enterobacter agglomerans (strain MS-1) can grow in the presence of up to 10 mM tetrachloroethene (80). This characteristic of DHM-

1 may further broaden its applicability in source zone remediation for sites highly contaminated with CF. For example, physical remediation strategies that target the nonaqueous phase liquid may precede biotic transformation of residual CF at concentrations up to 2,000 mg/L, which DHM-1 is capable of transforming.

In comparison to SDC-9, a commercially available bioaugmentation culture,

DHM-1 is advantageous with respect to its higher rate of CF transformation. However,

SDC-9 exhibits a faster transformation rate with CFC-11. The choice of culture for use in bioaugmentation must, therefore, take into account site specific conditions, e.g., the relative amounts of CFC-11 and CF. With DHM-1, acclimation to CFC-11 may yield higher rates of transformation, as was observed when the culture was exposed to increasing levels of CF during successive transfers in MSM.

In the presence of B 12 , both bioaugmentation cultures transformed CT and CF to environmentally benign products. Over 50% of CT was transformed to CO 2. This is comparable to the recovery of CO 2 from CT transformation by a DCM-grown culture (48)

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and fructose-grown Acetobacterium woodii (47), also in the presence of B 12 . As shown in Figure 1-1, conversion of CT to CO 2 may occur via net hydrolysis of CT, hydrolysis of

CS 2 formed from CT via a substitutive reaction, or oxidation of CO generated through reductive hydrolysis of CT (47). Accumulation of CS 2 from CT transformation is unwanted because of its toxicity. Formation of CS 2 depends on a number of factors, including the presence of sulfide, coenzymes such as B 12 , or catalysts such as biotite or pyrite. DHM-1 converted approximately one half as much CT to CS 2 as SDC-9 (Table 4-

3), while the percentage formed by SDC-9 (10%) was similar to previous reports for a

DCM-grown culture (i.e., 11%) (48) and fructose-grown Acetobacterium woodii (i.e., 13-

18%) (47). The highest reported output of CS 2 from CT is for a lactate-grown sulfate- reducing enrichment culture (73%) (36), most likely related to a higher concentration of sulfide in the media.

For SDC-9 and DHM-1, CO was a major product from CF (55-67%) and, to a lesser extent, from CT (14-15%). Reductive hydrolysis of CT to CO and net hydrolysis of CF to CO are shown in Figure 1-1. B12 plays a significant role in favoring conversion of CT and CF to CO (12, 47). Unlike CS 2, CO is not a major concern from a toxicity standpoint. Many anaerobes that use the acetyl-CoA pathway can grow on CO and others can oxidize CO to CO 2 via CO dehydrogenase (66). Both SDC-9 and DHM-1 convert significant amounts of CT and CF to organic acids, which are also environmentally acceptable endpoints.

During the microcosm study reported in Chapter 2, the fermentative enrichment culture was not used until nearly all of the CT and CFC-11 were consumed, so its

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principal function was to accelerate the rate of CF transformation. During this

microcosm study, the intent was to use DHM-1 from the outset, to accelerate the removal

of CT and CFC-11 as well as CF. Based on the results through day 205, bioaugmentation

did not provide a significant benefit in terms of CT removal; i.e., biostimulation with

corn syrup + B 12 was equally fast in terms of CT transformation. The CT transformation rates for both enrichment cultures in the microcosms were lower than what was achieved with DHM-1 in MSM and SDC-9 in RAMM (Table 4-2). This suggested that the soil exhibited some type of inhibitory influence on CT transformation. Sulfide may be playing a key role. The importance of sulfide in maintaining a high rate of CF transformation by DHM-1 was described in Chapter 3. Although a similar evaluation has not yet been performed with CT, other studies have demonstrated the importance of sulfide in CT transformation (65). The soil used in the microcosm study exerted a high demand for sulfide, so it appears unlikely that a residual level was available during CT transformation. The soil demand for sulfide observed in this study was similar to a sandy soil used in another microcosm study (i.e., approximately 0.4-0.6 mg S 2-/g soil) which investigated the use of calcium polysulfide to abiotically reduce hexavalent chromium

(37). To compensate for the soil sulfide demand, consideration should be given to increased addition of sulfide or sulfate, with the intent of fostering in situ sulfate

reduction. If a sufficient level of sulfide can be achieved, bioaugmentation to accelerate

CT removal may then become worthwhile. The higher concentration of sulfide and

sulfate in the microcosms with MSM and RAMM-modified tap water were not sufficient

to overcome the sulfide demand of the soil. It should also be noted that there is a risk

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from overdosing with sulfide, in the form of increased conversion of CT and CFC-11 to

CS 2. Getting the sulfide level correct in situ will be challenging.

Unlike the CT results, bioaugmentation of the microcosms has yielded higher transformation rates for CFC-11 in comparison to biostimulation with corn syrup + B 12 .

Nevertheless, CFC-11 transformation rates are slower in the microcosms when compared to the results for DHM-1 in MSM and SDC-9 in RAMM (Table 4-2). It is not yet clear if sulfide is a limiting factor for CFC-11 transformation. Monitoring of sulfide is recommended so that once CFC-11 is consumed, sulfide levels close to 0.5 mM should be maintained to facilitate a high rate of CF biotransformation.

Although monitoring of the microcosms is still in progress, the information collected through day 205 from the experiments conducted in media indicates that both

DHM-1 and SDC-9 are good candidates for bioaugmentation of sites contaminated with high concentrations of halomethanes, especially CFC-11 and CF. Since industrial sites that have high concentrations of CF may have a different mix of co-contaminants than the ones evaluated in this study, additional research is needed to evaluate the tolerance of these cultures to a wide range of organic and inorganic compounds.

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4.6 Tables and figures

Table 4-1 Experimental design for the microcosm study Treatment Type of water used a b,f c,f d,f e,f B FD' FD FS' FS As-is √ √ Corn syrup √ √ Corn syrup + B 12 √ √ Corn syrup + B 12 + DHM-1 √ √ √ Lactate + B 12 + SDC-9 √ √ √ a B=buffered tap water, i.e., tap water + 4 g/L NaHCO 3 + sulfate.

b FD'=DHM-1 media (i.e., modified MSM) prepared with tap water and sulfate replacing sulfide. c FD=DHM-1 media (i.e., modified MSM) prepared with tap water + sulfide.

d FS'=SDC-9 media (i.e., modified RAMM) prepared with tap water and sulfate replacing sulfide. e FS= SDC-9 media (i.e., modified RAMM) prepared with tap water + sulfide. f One half the amount of phosphate was used versus the amounts found in MSM and RAMM, to reduce the possibility of precipitate formation during delivery into groundwater; the amount of NaHCO 3 in RAMM was increased to 2.4 g/L, to reach neutral pH; the same amount of sulfide (0.5 mM) and yeast extract (50 mg/L) was added to the modified MSM and RAMM.

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Table 4-2 Halomethane transformation rates (mg/L/d) halomethane DHM-1 SDC-9 Average CT 1.3±0.01 a 0.82±0.06 CFC-11 0.54±0.01 0.67±0.12 CF 22±0.13 13±0.08 Maximum CT 1.3±0.18 b 1.1±0.04 CFC-11 1.1±0.05 2.3±0.30 CF 33±0.42 17±0.86 a Data range for duplicate bottles. b Standard error of the best fit line for duplicate bottles.

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Table 4-3 Comparison of end product distribution of CT and CF transformation by DHM-1 and SDC-9

Halomethane Culture % of [ 14 C]CT or [ 14 C]CF added a CH 4 CO CM DCM CS 2 CF CT other CO 2 NSR UA Loss CT DHM-1 0.1 14 b 0.3 0 5.2 2.2 0 0.1 51 27 0 0 SDC-9 0.9 15 1.9 0 10 4.0 0.4 0.4 51 16 0 0 CF DHM-1 0.5 67 0 0 0 0.9 0.2 0.8 2.7 15 0 13 SDC-9 0.4 55 1.7 0 0 0 0 0.3 9.9 16 0 17 a UA=unaccounted for, i.e., percentage of remaining 14 C that cannot be assigned to a particular compound or category. b Numbers in bold represent the main products formed. 103

CT (DHM-1 + B12) CT (LC) CT (MC+B12) 20 CF CS2 15

10

CT(mg/L) (mg/L) 5

0 0 2 4 6 8 10 12 14 16

CFC-11 (DHM-1 + B12) CFC-11 (LC) CFC-11 (MC+B12) 50 HCFC-21 CS2 40

30 20 10

CFC-11(mg/L) 0 0 10 20 30 40 50 CF (DHM-1 + B12) CF (LC) CF (MC+B12) 700 DCM CS2 600 500 400 300 200 CF(mg/L) 100 0 0 5 10 15 20 25 30

Time (days)

Figure 4-1 Transformation of CT (a), CFC-11 (b) and CF (c) by DHM-1 in MSM with corn syrup and B 12 added. LC=live control, MC+B 12 =media control with B 12 . Daughter products formed only in the DHM-1 + B 12 treatments. ↓↓↓=addition of corn syrup; B 12 was added only at t=0. Error bars are the data range for duplicate bottles.

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4000 Live 3500 WC

3000 2500

CF(mg/L) 2000

1500 1000 500

0 0 25 50 75 100 125 150 175 200

Time (days)

Figure 4-2 CF transformation by DHM-1 in MSM with corn syrup and B 12 added (at t=0), at varying initial CF concentrations. Arrows indicate addition of corn syrup. Error bars represent the data range for duplicate bottles.

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600 15 CF 500 CT DCM 400 CS2 10

300

,DCM (mg/L) a 2 CF (mg/L) 200 5

100 CT,CS

0 0 0 10 20 30 40 50 600 40 500 CF 30 400 CFC-11 HCFC-21 300 20

CF CF (mg/L) (mg/L) 200 b 10 100 CFC-11,HCFC-21 (mg/L) (mg/L) CFC-11,HCFC-21 0 0 0 10 20 30 40 50 40

CT 30 (mg/L) (mg/L) 2 CFC-11 HCFC-21 20 CS2

10 c

Halomethanes,CS 0 0 10 20 30 40 50 Time (days)

Figure 4-3 Performance of DHM-1 in transforming mixtures of CT + CF (a), CFC- 11 + CF (b) and CT + CFC-11 (c) in MSM and B 12 added (t=0). Arrows indicate addition of corn syrup.

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30 WC MC+B12 AC 20 10 a

CT (mg/L) (mg/L) 0

40 WC MC+B12 AC 30 20 10 b 0 CFC-11 (mg/L) CFC-11 0 20 40 60 80 100 120 800 WC MC+B12 AC 600 400 200 c (mg/L) (mg/L) CF 0 0 20 40 60 80 100 120

800 40

700 35 CF 600 CT 30 (mg/L)

500 CFC-11 25 2 CS2 400 20 300 15 (mg/L) CF d 200 10

100 5 CT,CFC-11,CS 0 0 0 20 40 60 80 100 120 Time (days)

Figure 4-4 Behavior of a mixture of halomethanes in controls (a, b and c) and live bottles with DHM-1 and corn syrup + B12 in MSM (d). ↓↓↓ = addition of corn syrup; = addition of B 12 ; = reinoculation with DHM-1; WC=water controls, MC+B 12 = media control with B 12 , and AC=autoclaved control with B 12 . Error bars are the data range for duplicate bottles.

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buffered tap water buffered tap water + B12 buffered tap water + B12 media with sulfate buffered tap water + B12 media with sulfate + B12 media with sulfate + B12 media with sulfate + B12 media with sulfide + B12 media with sulfide + B12 12 Biostimulation 12 Bioaugmentation with DHM-1 12 Bioaugmentation with SDC-9 10 10 10 8 8 8 6 6 6 4 4 4 (mg/L) (mg/L) CT CT 2 a 2 2 d 0 0 0 g 50 50 50 40 40 40

30 30 30

20 20 108 20 10 10 10 b e (mg/L) (mg/L) CFC-11 CFC-11 0 0 0 h

600 600 600 500 500 500 400 400 400 300 300 300 200 200 200 CF (mg/L) (mg/L) CF CF 100 100 100 c 0 0 f 0 i 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 Time (days) Time (days) Time (days)

Figure 4-5 Performance of biostimulation (a-c) and bioaugmentation with DHM-1 (d-f) and SDC-9 (g-i) in microcosms. ↓↓↓ = addition of electron donor; =addition of B 12 ; =reinoculation; = addition of sulfide.

6 day 7

5 day 21

4

3

2

(mM) sulfide 1 0.5 mM 0 0.0 0.5 1.0 1.5 2.0 2- Sulfide added (mg S /g soil)

Figure 4-6 Sulfide demand of the soil used in the microcosms. The dashed line at 0.5 mM represents the target concentration for microcosms.

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5. CONCLUSIONS

This study is the first to demonstrate that biostimulation and bioaugmentation are technically feasible remediation strategies for treatment of groundwater contaminated with high concentrations of halomethanes, e.g., approximately 10 mg/L CT, 20-30 mg/L of CFC-11, and up to 2000 of mg/L of CF. Use of a catalytic amount of vitamin B 12 is required to achieve reasonable transformation rates and direct the transformation pathways towards environmentally benign products. Corn syrup was the most effective primary substrate tested. A principal outcome of this dissertation was the development of a fermentative enrichment culture, DHM-1, that is able to grow on corn syrup in the presence of high concentrations of CF and transform it, along with CT and CFC-11, at high rates to mainly CO, CO 2, and organic acids.

The specific conclusions from this study are:

1. Based on a microcosm study using soil and groundwater from an industrial site in

California contaminated with high concentrations of CT, CFC-11 and CF, corn

syrup was a more effective primary substrate for biostimulation than lactate and

emulsified vegetable oil. Use of corn syrup shortened the time required to

establish the low redox conditions needed for rapid anaerobic transformation of

halomethanes and also yielded higher rates of halomethane transformation.

2. The effectiveness of corn syrup was magnified considerably by addition of

cyanocobalamin (vitamin B 12 ) at a 1.3% molar ratio to the initial concentration of

halomethanes. Under this condition, transformation of approximately 500 mg/L

CF, 26 mg/L CFC-11 and 10 mg/L CT was completed in microcosms in

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approximately 1000 days. The predominant end products were CO, CO 2 and NSR,

demonstrating the importance of hydrolytic transformation processes rather than

reductive dechlorination.

3. Three bioaugmentation cultures were initially tested for their ability to increase

the rates of halomethane transformation. All of the cultures were developed with

soil from an industrial site that was a focus of this research. The lactate-grown

SRB enrichment was initially beneficial for CT and CFC-11 transformation, but

gradually lost its ability to respire lactate and also resulted in an undesirably high

level of CS 2 as a product. An ethanol-grown SRB enrichment was ineffective in

enhancing the rate of CFC-11 or CF transformation. A corn syrup-grown

fermentative culture exhibited considerable promise in transforming residual

levels of CFC-11 and high concentrations of CF; this culture became the focus of

further development.

4. Through repeated transfers in MSM (with corn syrup as the substrate and B 12

added) and acclimation to high concentrations of CF, the enrichment culture

DHM-1 was developed. A patent application covering DHM-1 has been filed.

The culture is dominated by Pantoea (also known as Enterobacter ) spp. and

shows promise for use in bioaugmentation of sites contaminated with high

concentrations of CF. Distinctive characteristics of DHM-1 include a) the ability

to transform CF concentrations as high as 2000 mg/L to mainly CO, CO 2, and

organic acids at a maximum rate of 22 mg/L/d; b) the ability to grow on corn

syrup in the presence of CF at concentrations at least as high as 4000 mg/L; and c)

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the ability to alternate growth between under aerobic and fermentative conditions

without losing the ability to anaerobically biotransform CF, making it relatively

easy to handle for in situ applications (i.e., in comparison to obligate anaerobes).

5. High rates of CF transformation by DHM-1 require the presence of 0.5 mM

sulfide.

6. Two Pantoea spp. were isolated from the DHM-1 enrichment culture: Strain

DHM-1B, which grows comparatively fast and strain DHM-1T, which grows

more slowly. Both isolates grow fermentatively on corn syrup and in the presence

of B 12 are able to cometabolize 500 mg/L of CF at rates similar to the DHM-1

enrichment culture.

7. DHM-1 cometabolizes CT and CFC-11 in addition to CF. When the

halomethanes are present together (500 mg/L CF, 26 mg/L CFC-11 and 10 mg/L

CT) in MSM, DHM-1 completes biotransformation of the mixture in less than

four months, using corn syrup as its growth substrate and B 12 as an amendment.

8. In comparison to SDC-9, a commercially available bioaugmentation culture,

DHM-1 transforms CF at a higher rate while SDC-9 transforms CFC-11 more

quickly; they exhibit similar rates of transformation with CT. Like DHM-1, SDC-

9 converts CT and CF mainly to CO 2, CO and organic acids.

9. DHM-1 and SDC-9 were evaluated in a second microcosm study with soil from

the same industrial site. For CT removal, biostimulation with corn syrup + B 12

was equally effective in comparison to bioaugmentation. However, after CT was

consumed, bioaugmented treatments significantly increased the rate of CFC-11

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transformation. SDC-9 exhibited a higher rate on CFC-11 than DHM-1, and

addition of chemicals (in particular, sulfide) found in MSM and RAMM to tap

water increased the rate of transformation over buffered tap water.

10. Based on its ability to grow in the presence of CF at concentrations as high as

4000 mg/L and to biotransform CF as concentrations as high as 2000 mg/L,

DHM-1 shows promise for in situ remediation of source zones containing

nonaqueous phase CF. Nevertheless, site specific conditions, including the mix of

contaminants present and their concentrations, will likely dictate what type of

bioaugmentation culture will most likely to be successful.

113

6. APPENDICES

114

A. Supplementary results of Chapter 2

A.1 GC headspace results of individual microcosms in Chapter 2

Nomenclature for the microcosms

The format used to name the microcosms is: X – Y – Z - #, where

• X =

o CT if [ 14 C]CT was added

o CF if [ 14 C]CF was added

• Y =

o H for the high concentration microcosm;

o M for the medium concentration microcosm;

o L for the low concentration microcosm;

• Z =

o AI for as-is conditions (i.e., no amendments except pH adjustment);

o LA for biostimulation with lactate;

o EM for biostimulation with emulsified vegetable oil;

o CS for biostimulation with corn syrup;

o CS-B12 for biostimulation with corn syrup + B 12 ;

o LA-SRB for bioaugmentation with enrichment cultures of SRB, although

other cultures were also applied later

o AC for autoclaved control; and

• # = bottle number 1, 2, or 3; or ave (average of the triplicate, applicable only to AI

and AC)

115

250 CF CFC-11 16 CT-H-LA-SRB-1 CT DCM 1,1-DCE CHCl2F 14 200 CS2 F,

12 2 10 mol/bot) 150 , CHCl , 8 2

100 6 4 50 CF, CFC-11 ( 1,1-DCE (µmol/bottle) 2 CT,DCM, CS

0 0 0 100 200 300 400 500 600 700 800 900 1,000

Time (days)

250 CF CFC-11 20 CT-H-LA-SRB-2 CT DCM 1,1-DCE CHCl2F CS2 200 F, 15 2

150 mol/bot) , CHCl 2

10 mol/bottle) 100

5 50 1,1-DCE ( CT,CS DCM, CF, CFC-11 (

0 0 0 100 200 300 400 500 600 700 800 900 1,000 1,100

Time (days)

250 CT-H-LA-SRB-3 CF 16 CFC-11 CT 14 200 DCM F,

1,1-DCE 12 2

10 mol/bot) 150 , CHCl , 2

8 mol/bottle) 100 6

4 CF, CFC-11 ( 1,1-DCE ( 50 CT,DCM, CS 2

0 0 0 100 200 300 400 500 600 700 800 900 1,000 Time (days)

116

250 18 CF-H-LA-SRB-1 CF CFC-11 16 CT

200 DCM F, 14 2 1,1-DCE CHCl2F 12 , CHCl

150 2 10 8 100 6 CF, CFC-11 (µmol/bot) CT, DCM, CS 50 4 1,1-DCE (mmol/bottle) 2 0 0 0 100 200 300 400 500 600 700 800 900 1,000 Time (days)

250 CF 18 CF-H-LA-SRB-2 CFC-11 16 CT DCM

200 F,

14 2 1,1-DCE CHCl2F 12 CS2

150 CHCl , 10 2 8 100 6 CT,DCM, CS 1,1-DCE (mmol/bottle) CF, CFC-11 (µmol/bot) 50 4 2 0 0 0 100 200 300 400 500 600 700 800 900 1,000 1,100 Time (days)

250 CF CFC-11 18 CF-H-LA-SRB-3 CT DCM 16 1,1-DCE CHCl2F

200 CS2 F,

14 2 12 , CHCl

150 2 10 8 100 6 CT, DCM, CS 1,1-DCE (mmol/bottle) CF,CFC-11 (µmol/bot) 50 4 2 0 0 0 100 200 300 400 500 600 700 800 900 1,000 Time (days)

117

14 CF CFC-11 0.30 CT-M-LA-SRB-1 CT DCM 12 CHCl2F CS2 0.25 1,1-DCE 10 2 0.20 8 F, CS 2 0.15 6 CHCl (µmol/bottle) 0.10 4 CT,CF,CFC-11, DCM, 1,1-DCE (µmol/bottle)

2 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

14 0.30 CT-M-LA-SRB-2 CF CFC-11 CT DCM 12 CHCl2F CS2 0.25 1,1-DCE 10

2 0.20 8 F,CS

2 0.15 6 CHCl

(µmol/bottle) 0.10 4 CT,CF,DCM, CFC-11, 1,1-DCE (µmol/bottle) 2 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

14 0.30 CF CFC-11 CT-M-LA-SRB-3 CT DCM 12 CHCl2F CS2 0.25 1,1-DCE 10

2 0.20 8 F, CS

2 0.15 6 CHCl (µmol/bottle) 0.10 4 1,1-DCE(µmol/bottle) CT,CF,CFC-11, DCM,

2 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

118

16 0.30 CF-M-LA-SRB-1 CT CF DCM CFC-11 14 CHCl2F CS2 0.25 1,1-DCE 12 0.20 2 10 F, CS

2 8 0.15

6 CHCl

(µmol/bottle) 0.10 1,1-DCE(µmol/bottle) 4 CT,CF,CFC-11, DCM, 0.05 2

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

16 0.30 CF-M-LA-SRB-2 CT CF 14 DCM 0.25 12 CFC-11 CHCl2F

2 0.20 10 CS2 1,1-DCE F,CS

2 8 0.15

6 CHCl

(µmol/bottle) 0.10 4 1,1-DCE (µmol/bottle) CT,CF, DCM, CFC-11, 0.05 2

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

16 0.25 CF-M-LA-SRB-3 CT CF 14 DCM CFC-11 0.20 12 CHCl2F

2 CS2 10 1,1-DCE 0.15 F, CS 2 8 0.10 CHCl 6 (µmol/bottle) 1,1-DCE (µmol/bottle)

CT,CF, DCM, CFC-11, 4 0.05 2

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

119

8.0 CT CF CT-L-LA-SRB-1 DCM CFC-11

F, 7.0

2 CHCl2F CS2 6.0 CH2ClF

5.0

4.0

(µmol/bottle) 3.0 2

CS 2.0

CT,CF,DCM, CFC-11, CHCl 1.0

0.0 0 50 100 150 200 250 300 350 Time (days)

7.0 CT CT-L-LA-SRB-2 CF F,

2 6.0 DCM CFC-11 5.0 CHCl2F CS2 4.0

3.0 (µmol/bottle) 2 2.0 CS

1.0 CT,CF,DCM, CFC-11, CHCl 0.0 0 50 100 150 200 250 300 350 Time (days)

7.0 CT

F, CT-L-LA-SRB-3 2 6.0 CF DCM 5.0 CFC-11 CHCl2F 4.0 CS2

3.0 (µmol/bottle) 2

CS 2.0

CT,CF, DCM, CFC-11, CHCl 1.0

0.0 0 50 100 150 200 250 300 350

Time (days)

120

7.0 CF-L-LA-SRB-1 CT CF

F, 6.0 DCM 2 CFC-11 5.0 CHCl2F CS2 4.0

3.0 (µmol/bottle) 2 2.0 CS

1.0 CT,CF, DCM, CFC-11, CHCl 0.0 0 50 100 150 200 250 300 Time (days)

7.0 CT CF CF-L-LA-SRB-2

F, DCM 2 6.0 CFC-11 CHCl2F 5.0 CS2

4.0

3.0 (µmol/bottle) 2

CS 2.0

CT,CF,DCM, CFC-11, CHCl 1.0

0.0 0 50 100 150 200 250 300 Time (days)

7.0 CF-L-LA-SRB-3 CF DCM F, 2 6.0 CFC-11 CHCl2F 5.0 CS2

4.0

3.0 (µmol/bottle) 2

CS 2.0

CT,CF,CFC-11, DCM, CHCl 1.0

0.0 0 50 100 150 200 250 300 Time (days)

121

350 20 CT-H-AI-ave CF CFC-11 18 300 CT 1,1-DCE 16 250 14 mol/bot) µ mol/bot) 12

µ 200 10 150 8

100 6 CT,1,1-DCE(

CF, CFC-11( 4 50 2 0 0 0 200 400 600 800 1000 1200 Time (days)

20 CT-M-AI-ave CF 0.35 CFC-11 CT 0.30 1,1-DCE 15 0.25

0.20 mol/bottle) 10 µ

mol/bot) 0.15 µ (

CT,CF, CFC-11 0.10

5 1,1-DCE( 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900

Time (days)

8.0 CT-L-AI-ave CF 7.0 CFC-11 CT 6.0 mol/bot) µ 5.0 4.0 3.0 2.0

CT, CF, CFC-11( 1.0 0.0 0 100 200 300 400 500 600 700

Time (days)

122

300 18 CF-H-AI-ave CF CFC-11 CT 16 250 1,1-DCE 14 200 12 10 150 8 100 6 CT, 1,1-DCE (µmol/bot) CF, CFC-11 (µmol/bot) 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

25 0.35 CF CF-M-AI-ave CFC-11 0.30 20 CT 1,1-DCE 0.25

15 0.20

10 0.15 (µmol/bottle) CT,CF, CFC-11 0.10 1,1-DCE(µmol/bottle) 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

8 CF-L-AI-ave CF 7 CFC-11 6 CT

mol/bottle) 5 µ 4

3

2

CT,CF, CFC-11 ( 1

0 0 100 200 300 400 500 Time (days)

123

350 20 CT-H-CS-1 CF CFC-11 18 300 CT 1,1-DCE 16 250 14 mol/bot) mol/bot)

µ 12 µ 200 10 150 8 100 6 CF, CFC-11 ( CT, 1,1-DCE ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

350 18 CT-H-CS-2 CF CFC-11 16 300 CT 14 250 1,1-DCE mol/bot)

mol/bot) 12 µ µ 200 10

150 8 6 100 CT,1,1-DCE( CF, CFC-11 ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

350 20 CT-H-CS-3 CF CFC-11 18 300 CT 16 1,1-DCE 250 14 mol/bot) mol/bot) µ µ 200 12 10 150 8 100 6 CT,1,1-DCE( CF, CFC-11 ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

124

300 18 CF CF-H-CS-1 CFC-11 16 250 CT 1,1-DCE 14 200 12 mol/bot) mol/bot) µ µ 10 150 8 100 6 CT, 1,1-DCE ( CF, CFC-11( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

300 18 CF-H-CS-2 CF CFC-11 16 250 CT 1,1-DCE 14 200 12 mol/bot) mol/bot) µ µ 10 150 8 100 6 CF,CFC-11 ( 4 CT, 1,1-DCE ( 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

300 CF-H-CS-3 CF 18 CFC-11 CT 16 250 1,1-DCE 14

200 12 mol/bottle) mol/bottle) µ

µ 10 150 8

100 6 CT, 1,1-DCE (

CF, CFC-11 ( 4 50 2

0 0 0 200 400 600 800 1000 1200 1400 Time (days)

125

25 0.45 CT-M-CS-1 CF CFC-11 0.40 CT 20 1,1-DCE 0.35 0.30 15 0.25 mol/bottle) 0.20 µ mol/bot)

µ 10 ( 0.15 CT,CF,CFC-11

5 0.10 1,1-DCE ( 0.05 0 0.00 0 100 200 300 400 500 600 700 Time (days)

25 CF 0.35 CT-M-CS-2 CFC-11 CT 0.30 20 1,1-DCE 0.25 15 0.20 mol/bottle) µ

mol/bot) 0.15 10 µ (

CT,CF,CFC-11 0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 Time (days)

25 0.35 CT-M-CS-3 CF CFC-11 CT 0.30 20 1,1-DCE 0.25 15

0.20 mol/bottle) µ

mol/bottle) 0.15

µ 10 ( CT,CF,CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 200 400 600 800 1000 1200 Time (days)

126

25 0.30 CF-M-CS-1 CF CFC-11 0.25 20 CT 1,1-DCE 0.20 15 mol/bottle)

0.15 µ mol/bottle)

µ 10 ( CT,CF, CFC-11 0.10 1,1-DCE(

5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

25 0.30 CF-M-CS-2 CF CFC-11 CT 0.25 20 1,1-DCE

0.20 15 mol/bottle)

0.15 µ

mol/bottle) 10 µ (

CT, CF, CFC-11 0.10

5 1,1-DCE ( 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

25 CF 0.30 CF-M-CS-3 CFC-11 CT 0.25 20 1,1-DCE

0.20 15 mol/bottle) 0.15 µ mol/bottle)

µ 10 ( CT, CF, CFC-11 0.10 1,1-DCE (

5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

127

7.0 CT-L-CS-1 CF CFC-11 6.0 CT 5.0 mol/bottle) µ 4.0

3.0

2.0

CT,CF, CFC-11 ( 1.0

0.0 0 50 100 150 200 250 300 350 400 Time (days)

7.0 CT-L-CS-2 CF 6.0 CT CFC-11 5.0 mol/bottle) µ 4.0

3.0

2.0

CT,CF, CFC-11 ( 1.0

0.0 0 100 200 300 400 500 600 700 800 Time (days)

7.0 CT-L-CS-3 CF 6.0 CFC-11 CT 5.0 mol/bottle)

µ 4.0

3.0

2.0

1.0 CT,CF, CFC-11 (

0.0 0 100 200 300 400 500 600 700 800 Time (days)

128

6.0 CF-L-CS-1 CF CFC-11 5.0 CT

4.0 mol/bottle) µ

3.0

2.0

CT, CF,CFC-11 ( 1.0

0.0 0 100 200 300 400 500 Time (days)

6.0 CF-L-CS-2 CF 5.0 CFC-11 CT 4.0 mol/bottle) µ 3.0

2.0

1.0 CT, CF, CFC-11 (

0.0 0 100 200 300 400 500 Time (days)

6.0 CF-L-CS-3 CF 5.0 CFC-11 CT 4.0 mol/bottle)

µ 3.0

2.0

1.0 CT, CF,CFC-11 ( 0.0 0 100 200 300 400 500 Time (days)

129

350 CT-H-CS-B12-1 CF 20 CFC-11 18 300 DCM 16 250 CHCl2F 14 CT 12 200 1,1-DCE 10 150 8 100 6 CF, CFC-11 (µmol/bot) CT, 1,1-DCE (µmol/bot) 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

300 18 CT-H-CS-B12-2 CF CFC-11 16 250 CT 14 1,1-DCE 200 12 10 150 8

100 6 CF, CFC-11 (µmol/bot)

4 CT, 1,1-DCE (µmol/bot) 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

350 20 CT-H-CS-B12-3 CF 18 300 CFC-11 16 CT 250 14 1,1-DCE 12 200 10 150 8

100 6 CF, CFC-11 (µmol/bot) CT, 1,1-DCE (µmol/bot) 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

130

350 18 CF-H-CS-B12-1 CF 16 300 CFC-11 CT 14 250 1,1-DCE 12 mol/bot) mol/bot) µ µ 200 10

150 8 6

100 CT, 1,1-DCE ( CF, CFC-11 ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

300 CF-H-CS-B12-2 CF 20 CFC-11 CT 18 250 1,1-DCE 16 14

200 mol/bot) mol/bot) µ µ 12 150 10 8 100 6 CT,1,1-DCE ( CF, CFC-11 ( 4 50 2 0 0 0 200 400 600Time (days) 800 1000 1200 1400

300 20 CF-H-CS-B12-3 CF CFC-11 18 CT 250 1,1-DCE 16 14 200 mol/bot) mol/bot) 12 µ µ 150 10 8 100 6 CT,1,1-DCE( CF, CFC-11 ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

131

25 0.40 CT-M-CS-B12-1 CF CFC-11 0.35 20 DCM CHCl2F 0.30 CT 15 1,1-DCE 0.25 0.20 F (µmol/bottle) 2 10 0.15 1,1-DCE (µmol/bottle) CT,CF,DCM, CFC-11, CHCl 0.10 5 0.05

0 0.00 0 100 200 300 400 500 600 700 Time (days)

25 0.40 CT-M-CS-B12-2 CF CFC-11 0.35 20 DCM CHCl2F 0.30 CT 0.25 15 1,1-DCE 0.20

F(µmol/bottle) 10 2 0.15 1,1-DCE (µmol/bottle)

CHCl 0.10 CT,CF,CFC-11, DCM, 5 0.05

0 0.00 0 100 200 300 400 500 600 700 Time (days)

25 0.40 CT-M-CS-B12-3 CF CFC-11 0.35 20 DCM CHCl2F 0.30 CT 15 0.25 1,1-DCE 0.20

10 0.15 F(µmol/bottle) 2

0.10 1,1-DCE (µmol/bottle) CT,CF,DCM,CFC-11, CHCl 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 1000 1100 Time (days)

132

25 0.35 CF-M-CS-B12-1 CF CFC-11 0.30 20 CT DCM 0.25 CH2ClF 15 1,1-DCE

0.20 mol/bottle) µ

mol/bottle) 10 0.15 µ ( CT, CF,CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

25 CF 0.30 CF-M-CS-B12-2 CT CFC-11 0.25 20 DCM CHCl2F 1,1-DCE 0.20 15 mol/bottle)

0.15 µ 10 mol/bottle)

µ 0.10 ( CT, CF,CFC-11 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

25 CF 0.25 CF-M-CS-B12-3 CFC-11 CT 20 DCM 0.20 CHCl2F 1,1-DCE 15 0.15 mol/bottle) µ

mol/bottle) 10 0.10 µ ( CT, CF, CFC-11

5 0.05 1,1-DCE (

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

133

7.0 CT-L-CS-B12-1 CF 6.0 CFC-11 DCM 5.0 CHCl2F CT 4.0

3.0

2.0 CT,CF, CFC-11 (µmol/bottle) 1.0

0.0 0 50 100 150 200 250 300 350 400 Time (days)

6.0 CT-L-CS-B12-2 CF CFC-11 5.0 DCM 4.0 CHCl2F CT 3.0

2.0

1.0 CT, CF,CFC-11 (µmol/bottle)

0.0 0 100 200 300 400 500 600 700 800 Time (days)

6.0 CT-L-CS-B12-3 CF CFC-11 5.0 DCM CHCl2F 4.0 CT

3.0

2.0

1.0 CT,CF, CFC-11 (µmol/bottle)

0.0 0 100 200 300 400 500 600 700 800 Time (days)

134

6.0 CF-L-CS-B12-1 CF CFC-11 5.0 CT DCM 4.0 mol/bottle) CHCl2F µ 3.0

2.0

1.0 CT, CF, CFC-11(

0.0 0 50 100 150 200 250 300 350 400 450 500 Time (days)

6.0 CF-L-CS-B12-2 CF CFC-11 5.0 CT DCM 4.0 CHCl2F mol/bottle) µ 3.0

2.0

1.0 CT, CF, CFC-11(

0.0 0 100 200 300 400 500 Time (days)

6.0 CF-L-CS-B12-3 CF CFC-11 5.0 CT DCM CHCl2F 4.0 mol/bottle) µ 3.0

2.0

1.0 CT, CF,CFC-11 (

0.0 0 100 200 300 400 500 Time (days)

135

350 20 CT-H-LA-1 CF 18 300 CFC-11 CT 16 250 1,1-DCE 14

200 12 10 150 8

100 6 CF, CFC-11 (µmol/bot)

4 CT, 1,1-DCE (µmol/bot) 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

350 18 CT-H-LA-2 CF 16 300 CFC-11 CT 14 250 1,1-DCE 12

200 10

150 8 6 100 CT, 1,1-DCE (µmol/bot) CF, CFC-11(µmol/bot) 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

350 18 CT-H-LA-3 CF 16 300 CFC-11 CT 14 250 1,1-DCE 12 200 10

150 8 6 100 CT, 1,1-DCE (µmol/bot)

CF, CFC-11 (µmol/bot) 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

136

300 18 CF-H-LA-1 CF CFC-11 16 250 CT 14 1,1-DCE 200 12 mol/bot) mol/bot) µ

µ 10 150 8 100 6

4 CT,1,1-DCE( CF, CFC-11 ( 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

300 20 CF-H-LA-2 CF CFC-11 18 250 CT 16 1,1-DCE 14 200 mol/bot) mol/bot)

12 µ µ 150 10 8 100 6 CT, 1,1-DCE ( CF, CFC-11( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

300 20 CF-H-LA-3 CF CFC-11 18 250 CT 16 1,1-DCE 14 200 mol/bot)

mol/bot) 12 µ µ 150 10 8 100 6 CT, 1,1-DCE ( CF, CFC-11 ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

137

25 CT-M-LA-1 CF 0.40 CFC-11 0.35 CT 20 1,1-DCE 0.30

15 0.25 0.20 10

(µmol/bottle) 0.15 CT,CF, CFC-11 0.10 5 1,1-DCE (µmol/bottle) 0.05

0 0.00 0 100 200 300 400 500 600 700 Time (days)

25 CF 0.35 CT-M-LA-2 CFC-11 CT 0.30 20 1,1-DCE 0.25

15 0.20

10 0.15 (µmol/bottle) CT,CF, CFC-11

0.10 1,1-DCE (µmol/bottle) 5 0.05

0 0.00 0 100 200 300 400 500 600 700 Time (days)

25 0.35 CT-M-LA-3 CF CFC-11 0.30 20 CT 1,1-DCE 0.25

15

0.20 mol/bottle) µ

mol/bottle) 10 0.15 µ ( CT,CF, CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 200 400 600 800 1000 1200 Time (days)

138

25 0.40 CF-M-LA-1 CF CFC-11 CT 0.35 20 1,1-DCE 0.30

15 0.25 mol/bottle)

0.20 µ

mol/bottle) 10

µ 0.15 ( CT, CF, CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

25 0.35 CF-M-LA-2 CF CFC-11 CT 0.30 20 1,1-DCE 0.25 15 0.20 mol/bottle) 0.15 µ 10 mol/bottle) µ (

CT, CF, CFC-11 0.10

5 1,1-DCE ( 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

25 CF 0.35 CF-M-LA-3 CFC-11 CT 0.30 20 1,1-DCE 0.25

15 mol/bottle) 0.20 µ

mol/bottle) 0.15

µ 10 ( CT, CF,CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

139

8.0 CT-L-LA-1 CF 7.0 CFC-11 CT 6.0 mol/bottle)

µ 5.0

4.0

3.0

2.0

CT, CF, CFC-11( 1.0

0.0 0 50 100 150 200 250 300 350 400 Time (days)

8.0 CT-L-LA-2 7.0 CF 6.0 CFC-11 CT mol/bottle)

µ 5.0

4.0

3.0

2.0

CT, CF, CFC-11( 1.0

0.0 0 100 200 300 400 500 600 700 800 Time (days)

8.0 CT-L-LA-3 CF 7.0 CFC-11 6.0 CT mol/bottle) 5.0 µ

4.0

3.0

2.0 CT, CF,CFC-11 ( 1.0

0.0 0 100 200 300 400 500 600 700 800 Time (days)

140

6.0 CF-L-LA-1 CF CFC-11 5.0 CT

4.0 mol/bottle) µ 3.0

2.0

1.0 CT, CF, CFC-11 (

0.0 0 50 100 150 200 250 300 350 400 450 500 Time (days)

6.0 CF-L-LA-2 CF CFC-11 5.0 CT

4.0 mol/bottle) µ 3.0

2.0

1.0 CT, CF, CFC-11 (

0.0 0 50 100 150 200 250 300 350 400 450 500 Time (days)

6.0 CF-L-LA-3 CF 5.0 CFC-11 CT

4.0 mol/bottle) µ 3.0

2.0

1.0 CT,CF, CFC-11 (

0.0 0 50 100 150 200 250 300 350 400 450 500 Time (days)

141

350 20 CT-H-EM-1 CF CFC-11 18 300 CT 1,1-DCE 16 250 14 mol/bot) mol/bot) µ 200 12 µ 10 150 8 100 6 CF, CFC-11( 4 CT,1,1-DCE( 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

350 20 CT-H-EM-2 CF CFC-11 18 300 CT 16 1,1-DCE 250 14 mol/bot)

mol/bot) 12 µ

µ 200 10 150 8 100 6 CT, 1,1-DCE (

CF, CFC-11 ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

300 20 CT-H-EM-3 CF CFC-11 18 250 CT 16 1,1-DCE

14 mol/bot)

mol/bot) 200 µ µ 12 150 10 8 100

6 CT, 1,1-DCE ( CF, CFC-11 ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

142

300 18 CF-H-EM-1 CF CFC-11 16 250 CT 14 1,1-DCE 200 12 mol/bot) mol/bot) µ µ 10 150 8

100 6

CF,CFC-11 ( 4 CT, 1,1-DCE ( 50 2

0 0 0 200 400 600 800 1000 1200 1400 Time (days)

300 18 CF-H-EM-2 CF CFC-11 16 250 CT 14 1,1-DCE

200 12 mol/bot) mol/bot) µ µ 10 150 8

100 6 CT, 1,1-DCE( CF, CFC-11 ( 4 50 2

0 0 0 200 400 600 800 1000 1200 1400 Time (days)

350 CF 18 CF-H-EM-3 CFC-11 16 300 CT 1,1-DCE 14 250 12 mol/bot) mol/bot) µ µ 200 10

150 8 6 100 CT, 1,1-DCE ( CF, CFC-11 ( 4 50 2 0 0 0 200 400 600 800 1000 1200 1400 Time (days)

143

25 0.40 CT-M-EM-1 CF CFC-11 0.35 20 CT 1,1-DCE 0.30

15 0.25 mol/bottle) 0.20 µ

mol/bottle) 10 µ 0.15 ( CT,CF, CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 Time (days)

25 0.40 CT-M-EM-2 CF CFC-11 0.35 20 CT 1,1-DCE 0.30

15 0.25 mol/bottle)

0.20 µ

mol/bottle) 10 0.15 µ ( CT,CF,CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 Time (days)

25 0.35 CT-M-EM-3 CF CFC-11 0.30 20 CT 1,1-DCE 0.25

15 0.20 mol/bottle) µ

mol/bottle) 0.15 µ 10 ( CT,CF, CFC-11 0.10 1,1-DCE( 5 0.05

0 0.00 0 200 400 600 800 1000 1200 Time (days)

144

25 0.35 CF-M-EM-1 CF CFC-11 0.30 20 CT 1,1-DCE 0.25

15 mol/bottle)

0.20 µ

mol/bottle) 0.15

µ 10 ( CT, CF,CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

25 CF 0.35 CF-M-EM-2 CFC-11 CT 0.30 20 1,1-DCE 0.25

15

0.20 mol/bottle) µ

mol/bottle) 0.15

µ 10 ( CT,CF, CFC-11

0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

25 0.30 CF-M-EM-3 CF CFC-11 0.25 20 CT 1,1-DCE 0.20 15 mol/bottle)

0.15 µ mol/bottle)

µ 10 ( CT,CF, CFC-11 0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

145

7.0 CT-L-EM-1 CF 6.0 CFC-11 CT 5.0 mol/bottle)

µ 4.0

3.0

2.0

CT,CF, CFC-11 ( 1.0

0.0 0 50 100 150 200 250 300 350 400 Time (days)

6.0 CT-L-EM-2 CF CFC-11 5.0 CT

4.0 mol/bottle) µ 3.0

2.0

1.0 CT, CF, CFC-11(

0.0 0 100 200 300 400 500 600 700 800 Time (days)

7.0 CT-L-EM-3 CF CFC-11 6.0 CT 5.0

4.0

3.0

2.0

1.0 CT, CF, CFC-11 (umol/bottle)

0.0 0 100 200 300 400 500 600 700 Time (days)

146

7.0 CF-L-EM-1 CF 6.0 CFC-11 CT 5.0 mol/bottle) µ 4.0

3.0

2.0

CT, CF,CFC-11 ( 1.0

0.0 0 50 100 150 200 250 300 350 400 450 500 Time (days)

7.0 CF-L-EM-2 CF 6.0 CFC-11 CT 5.0 mol/bottle) µ 4.0

3.0

2.0

CT, CF, CFC-11 ( 1.0

0.0 0 50 100 150 200 250 300 350 400 450 500 Time (days)

7.0 CF-L-EM-3 CF 6.0 CFC-11 CT 5.0 mol/bottle) µ 4.0

3.0

2.0

CT, CF,CFC-11 ( 1.0

0.0 0 100 200 300 400 500 Time (days)

147

300 CF 18 CT-H-AC-ave CFC-11 16 250 CT 1,1-DCE 14 200 12 mol/bot) mol/bot) µ

µ 10 150 8 100 6

4 CT,1,1-DCE( CF, CFC-11 ( 50 2 0 0 0 200 400 600 800 1000 1200 Time (days)

25 CF 0.35 CT-M-AC-ave CFC-11 CT 1,1-DCE 0.30 20 0.25

15 0.20 mol/bottle) µ

mol/bottle) 10 0.15 µ ( CT,CF, CFC-11 0.10 1,1-DCE ( 5 0.05

0 0.00 0 100 200 300 400 500 600 700 Time (days)

8.0 CT-L-AC-ave CF 7.0 CFC-11 CT 6.0

5.0 mol/bottle) µ 4.0

3.0

2.0

CT, CF, CFC-11( 1.0

0.0 0 100 200 300 400 500 600 700 Time (days)

148

300 20 CF-H-AC-ave CF CFC-11 250 CT 1,1-DCE 15

200 mol/bot) µ mol/bot) µ 150 10

100

5 CT, 1,1-DCE ( CF, CFC-11 ( 50

0 0 0 200 400 600 800 1000 1200 1400 Time (days)

25 CF 0.35 CF-M-AC-ave CFC-11 CT 0.30 20 1,1-DCE 0.25 15 0.20 mol/bottle) µ 0.15 10 mol/bottle) µ (

CT, CF, CFC-11 0.10 5 1,1-DCE ( 0.05

0 0.00 0 100 200 300 400 500 600 700 800 900 Time (days)

7.0 CF-L-AC-ave CF CFC-11 6.0 CT

5.0 mol/bottle)

µ 4.0

3.0

2.0

CT, CF,CFC-11 ( 1.0

0.0 0 100 200 300 400 500 Time (days)

Figure 6-1 GC headspace results of all individual microcosms in the microcosm study in Chapter 2

149

A.2 Data used to determine first order rate constants

H-SRB on CT

0 -1 150 200 250 300 -2 -3 -4 -5 y = -0.1479x + 32.293 R2 = 0.7084

ln(C/Co) -6 -7 -8 -9 -10 Time (days)

H-SRB on CFC-11

0 150 250 350 450 550 650 750 -2

-4 y = -0.0168x + 4.6265 -6 R2 = 0.7038 ln(C/Co) -8

-10

-12 Time (days)

H-SRB on CF

0 150 250 350 450 550 650 750 850

-2

-4 y = -0.0129x + 5.1771 R2 = 0.3618 -6 ln(C/Co) -8

-10

-12 Time (days)

150

M-SRB on CT

0 -1 150 160 170 180 190 200 210 -2 -3 -4 -5 y = -0.142x + 21.164

ln(C/Co) -6 2 -7 R = 0.8141 -8 -9 -10 Time (days)

M-SRB on CFC-11

0 150 250 350 450 550 -1

-2

-3 ln(C/Co) -4

-5 y = -0.0143x + 2.3011 R2 = 0.7361 -6 Time (days)

M-SRB on CF

0 -1 150 250 350 450 550 650 750 850 -2 -3 -4

-5 y = -0.0147x + 4.3577 ln(C/Co) -6 R2 = 0.8163 -7 -8 -9 Time (days)

151

L-SRB on CT

0 95 105 115 125 135 145 -2

-4 y = -0.3169x + 29.904 2 -6 R = 0.8713 ln(C/Co) -8

-10

-12 Time (days)

L-SRB on CFC-11

0 90 140 190 240 290 -1

-2 y = -0.0381x + 3.5317 -3 R2 = 0.7352 -4

-5 ln(C/Co) -6

-7

-8

-9 Time (days)

L-SRB on CF

0 120 140 160 180 200 220 240 260 280 300 320 -1

-2

-3 y = -0.0189x + 2.8139 ln(C/Co) -4 R2 = 0.2916

-5

-6 Time (days)

152

H-CS+B12 on CT

0 -1 150 170 190 210 230 250 -2 -3 y = -0.0399x + 1.6841 -4 R2 = 0.6277 -5

ln(C/Co) -6 -7 -8 -9 -10 Time (days)

H-CS+B12 on CFC-11

0 150 350 550 750 950 1150 1350 -2

-4

-6 ln(C/Co) -8 y = -0.006x + 1.6601 -10 R2 = 0.6195

-12 Time (days)

H-CS+B12 on CF

0 -1 150 350 550 750 950 1150 1350 -2 -3 -4 -5 y = -0.006x + 2.6529 -6 ln(C/Co) 2 -7 R = 0.5419 -8 -9 -10 Time (days)

153

M-CS+B12 on CT

0 -1 150 160 170 180 190 200 210 220 -2 -3 -4 y = -0.0494x + 3.037 R2 = 0.585 -5 ln(C/Co) -6 -7 -8 -9 Time (days)

M-CS+B12 on CFC-11

0 -0.5 150 250 350 450 550 650 750 850 -1 -1.5 -2 -2.5 y = -0.0076x + 1.305 R2 = 0.8687 ln(C/Co) -3 -3.5 -4 -4.5 -5 Time (days)

M-CS+B12 on CF

0 -1 150 350 550 750 950 1150 -2 -3 -4 -5

ln(C/Co) -6 -7 -8 y = -0.0129x + 3.6461 -9 R2 = 0.597 -10 Time (days)

154

L-CS+B12 on CT

0 -1 20 70 120 170 220 -2 -3 -4 y = -0.0341x + 1.388 2 -5 R = 0.2969 ln(C/Co) -6 -7 -8 -9 Time (days)

L-CS+B12 on CFC-11

0 80 180 280 380 480 580 -1 -2 -3

-4

ln(C/Co) -5 -6

-7 y = -0.0116x + 1.0215 2 -8 R = 0.3269 Time (days)

L-CS+B12 on CF

0 120 220 320 420 520 620 720 820 -1

-2

-3

-4 ln(C/Co)

-5 y = -0.0069x + 0.8636 -6 R2 = 0.398 -7 Time (days)

155

H-CS on CT

0 -1 20 220 420 620 820 1020 -2 -3 -4 -5

ln(C/Co) -6 -7 y = -0.012x + 1.4074 -8 R2 = 0.8812 -9 -10 Time (days)

H-CS on CFC-11

0 80 280 480 680 880 1080 1280 1480

-0.2 ln(C/Co) -0.4 y = -0.00050x + 0.12144 R2 = 0.95364

-0.6 Time (days)

H-CS on CF

0 120 320 520 720 920 1120 1320

-0.5

-1 ln(C/Co)

-1.5 y = -0.0016x + 0.3687 R2 = 0.9696 -2 Time (days)

156

M-CS on CT

0 -1 20 120 220 320 420 520 620 720 -2 -3 -4 -5

ln(C/Co) -6 -7 y = -0.0152x + 2.6686 -8 R2 = 0.6933 -9 -10 Time (days)

M-CS on CFC-11

0 80 280 480 680 880 1080

-0.5 ln(C/Co) y = -0.0006x + 0.0394 R2 = 0.4811

-1 Time (days)

M-CS on CF

0 120 320 520 720 920 1120

-0.5 ln(C/Co) -1 y = -0.0016x + 0.4701 R2 = 0.8854

-1.5 Time (days)

157

L-CS on CT

0 -1 20 120 220 320 420 520 620 -2 -3 -4 -5 ln(C/Co) -6 y = -0.017x + 2.5833 -7 R2 = 0.4396 -8 -9 Time (days)

L-CS on CFC-11

0 80 180 280 380 480 580 680 780

-0.2

-0.4 ln(C/Co)

-0.6 y = -0.0008x + 0.1304 R2 = 0.7751 -0.8 Time (days)

L-CS on CF

0.7

y = -0.0015x + 1.234 0.2 R2 = 0.5235 ln(C/Co)

0 100 200 300 400 500 600 700 800

-0.3 Time (days)

158

H-LA on CT

0 -1 20 220 420 620 820 1020 1220 -2 -3 -4 y = -0.0087x + 0.6446 -5 R2 = 0.7704

ln(C/Co) -6 -7 -8 -9 -10 Time (days)

H-LA on CFC-11

0 80 280 480 680 880 1080 1280 1480

-0.5 ln(C/Co)

y = -0.0005x + 0.137 R2 = 0.9409

-1 Time (days)

H-LA on CF

0 120 320 520 720 920 1120 1320

-0.5

-1 ln(C/Co)

-1.5 y = -0.0014x + 0.2043 R2 = 0.6449

-2 Time (days)

159

M-LA on CT

0 -1 20 220 420 620 820 1020 1220 -2 -3 -4 -5 ln(C/Co) -6 y = -0.0077x + 1.3764 -7 R2 = 0.4653 -8 -9 Time (days)

M-LA on CFC-11

0 80 280 480 680 880 1080

-0.2 ln(C/Co) y = -0.0005x + 0.1485 R2 = 0.901

-0.4 Time (days)

M-LA on CF

0 120 320 520 720 920 1120 -0.5

-1

-1.5 y = -0.0016x + 0.3983 ln(C/Co) -2 R2 = 0.7532

-2.5

-3 Time (days)

160

L-LA on CT

0 20 220 420 620 820 -0.5 -1

-1.5 -2 y = -0.0026x + 0.4982 R2 = 0.5053 ln(C/Co) -2.5 -3 -3.5

-4 Time (days)

L-LA on CFC-11

0 80 180 280 380 480 580 680 780

-0.2 ln(C/Co)

y = -0.0004x + 0.0783 R2 = 0.6692 -0.4 Time (days)

L-LA on CF

1 y = -0.0006x + 0.3382 R2 = 0.1329

0.5 ln(C/Co) 0 120 220 320 420 520 620 720 820

-0.5 Time (days)

161

H-EM on CT

0 -1 20 220 420 620 820 1020 -2 -3 -4 -5 ln(C/Co) -6 -7 y = -0.0089x + 0.4561 R2 = 0.7921 -8 -9 Time (days)

H-EM on CFC-11

0 80 280 480 680 880 1080 1280 1480

-0.5 ln(C/Co)

y = -0.0006x + 0.1047 R2 = 0.8013 -1 Time (days)

H-EM on CF

0 120 320 520 720 920 1120 1320 -0.5

-1

ln(C/Co) -1.5 y = -0.0019x + 0.3637 -2 R2 = 0.9728

-2.5 Time (days)

162

M-EM on CT

0 20 220 420 620 820 1020 1220 -0.5

-1

-1.5

-2 ln(C/Co) y = -0.0033x + 0.3401 -2.5 R2 = 0.9134 -3

-3.5 Time (days)

M-EM on CFC-11

0 80 280 480 680 880 1080

-0.5 ln(C/Co)

y = -0.0005x + 0.0986 R2 = 0.683

-1 Time (days)

M-EM on CF

0 120 320 520 720 920 1120 -0.5

-1

ln(C/Co) -1.5

-2 y = -0.0022x + 0.4644 R2 = 0.8618 -2.5 Time (days)

163

L-EM on CT

0 20 220 420 620 820 -0.5

-1

ln(C/Co) -1.5

-2 y = -0.003x + 0.2643 R2 = 0.866 -2.5 Time (days)

L-EM on CFC-11

0 80 180 280 380 480 580 680 780

-0.2 ln(C/Co)

y = -0.0005x + 0.0925 R2 = 0.4699 -0.4 Time (days)

L-EM on CF

1

y = -0.0014x + 0.74 0.5 R2 = 0.6206 ln(C/Co) 0 120 220 320 420 520 620 720 820

-0.5 Time (days)

164

H-AI on CT

0 20 220 420 620 820 1020 1220 1420 -0.5

-1

ln(C/Co) -1.5

-2 y = -0.0016x - 0.0175 R2 = 0.7409 -2.5 Time (days)

H-AI on CFC-11

y = -0.0002x + 0.1249 0.2 R2 = 0.7203 ln(C/Co)

80 280 480 680 880 1080 1280 1480

-0.3 Time (days)

H-AI on CF

0.5

0 120 320 520 720 920 1120 1320 ln(C/Co) -0.5 y = -0.0003x + 0.1496 R2 = 0.553

-1 Time (days)

165

M-AI on CT 0 20 220 420 620 820 1020 1220

-0.5 ln(C/Co) -1 y = -0.0012x + 0.1408 R2 = 0.921

-1.5 Time (days)

M-AI on CFC-11

0.5

y = -0.0001x + 0.072 0.3 R2 = 0.3554

0.1 ln(C/Co)

-0.1 80 280 480 680 880 1080

-0.3 Time (days)

M-AI on CF 0.3

120 320 520 720 920 1120 -0.2 ln(C/Co) -0.7 y = -0.0008x + 0.3077 R2 = 0.4482

-1.2 Time (days)

166

L-AI on CT 0 20 220 420 620 820

-0.5

-1 y = -0.0019x + 0.3422 ln(C/Co) R2 = 0.5081 -1.5

-2 Time (days)

L-AI on CFC-11

0.5

0.0 80 180 280 380 480 580 680 780 ln(C/Co) y = -0.0004x + 0.1185 R2 = 0.4029

-0.5 Time (days)

L-AI on CF

0.5

0.0 120 220 320 420 520 620 720 820 ln(C/Co) -0.5 y = -0.0008x + 0.2237 R2 = 0.4502

-1.0 Time (days)

Figure 6-2 Data used to determine first order rate constants for the microcosm study in Chapter 2

167

A.3 Sample data used to determine the first order transformation rate constants

Principle

The starting point of first order transformation of each parent contaminant (i.e., the end point of lag phase) in each individual microcosm, was determined as the point where a steady trend of removal initiated, not necessarily the maximum transformation phase initiated. However, for 1,1-DCE in bioaugmentation microcosms to which a chlororespiring culture was bioaugmented, results after bioaugmentation was used for determining the rate constants to emphasize the effectiveness of bioaugmentation.

Examples

Example 1: biostimulation microcosm CF-H-CS-B12-1 (determined first order reaction phase was highlighted in each figure) CF (CF-H-B12-1) CT (CF-H-B12-1) 2.0 1.0 0.0 0.0 -1.0 0 500 1000 1500 0 50 100 150 200 -2.0 -2.0 -3.0 -4.0 y = -0.0033x + 1.4018 y = -0.0769x + 6.2819 -4.0 R2 = 0.5175 -6.0 R2 = 0.918 -5.0 -8.0 -6.0 -7.0 -10.0 CFC-11 (CF-H-B12-1) 1,1-DCE (CF-H-B12-1) 0.5 0.2 0.0 0.0 0 500 1000 1500 -0.2 0 500 1000 1500 -0.5 -0.4 -1.0 -0.6 -1.5 -0.8 y = -0.0024x + 0.4399 y = -0.0008x - 0.0782 -2.0 -1.0 R2 = 0.9811 R2 = 0.9483 -2.5 -1.2

Figure 6-3 Sample data of first order transformation following a lag phase for a biostimulation microcosm with corn syrup plus B 12 (CF-H-B12 -1)

168

Example 2: bioaugmentation microcosm CF-H-CS-B12-1

CT (CF-H-SRB-1) CF (CF-H-SRB-1) 2.0 2.0 0.0 0.0 0 200 400 600 800 -2.0 0 200 400 600 800 -2.0 -4.0 -4.0 y = -0.211x + 48.546 y = -0.0167x + 6.8798 -6.0 -6.0 R2 = 0.9149 R2 = 0.4045 -8.0 -8.0 -10.0 -10.0 -12.0 -12.0

CFC-11 (CF-H-SRB-1) 1,1-DCE (CF-H-SRB-1)

2.0 0.0 0 200 400 600 800 1000 0.0 -1.0 0 200 400 600 800 1000 -2.0 -2.0 -4.0 -3.0 y = -0.0363x + 26.429 y = -0.0169x + 4.8399 2 -6.0 R = 0.8529 R2 = 0.6567 -4.0 -8.0 -5.0 -10.0 -6.0 -12.0

Figure 6-4 Sample data of first order transformation following a lag phase for a bioaugmentation microcosm (CF-H-SRB-1)

169

A.4 Percent removal of halogenated compounds in the microcosm study in Chapter 2 Table 6-1 Percent removal of parent contaminants in the microcosm study in Chapter 2 Treatment % removal a CT CFC-11 CF 1,1-DCE High WC (>800 days) 17.6 ± 13.9% 7.5 ± 9.0% 15.3 ± 11.4% 21.1 ± 8.0% AC 18.8 ± 9.0% 7.0 ± 2.7% 15.4 ± 7.2% 23.5 ± 3.2 % as-is 80.5 ± 14.8% 6.7 ± 5.3% 14.4 ± 11.2% 26.4 ± 9.8% lac 100.0 ± 0.0% 37.3 ± 5.0% 82.8 ± 4.9% 71.1 ± 9.2% EM 99.5 ± 0.2% 45.1 ± 7.4% 84.4 ± 4.9% 72.7 ± 7.4% CS 99.8 ± 0.3% 38.1 ± 5.7% 80.2 ± 5.0% 68.3 ± 6.4% CS + B12 99.5 ± 0.2% 92.6 ± 5.7% 99.9 ± 0.1% 68.1 ± 1.9% lac + B12 + bioaug 99.8 ± 0.2% 100.0 ± 0.0% 100.0 ± 0.0% 99.3 ± 0.5% Medium AC 13.7 ± 8.2% 2.8 ± 3.1% 3.5 ± 5.2% 18.1 ± 10.5% as-is 54.0 ± 11.1% 2.1 ± 2.6% 27.3 ± 27.7% 71.5 ± 10.2% lac 89.5 ± 16.8% 21.2 ± 7.2% 57.5 ± 17.5% 53.6 ± 8.8% EM 87.4 ± 7.9% 25.2 ± 7.7% 71.5 ± 17.4% 57.3 ± 8.6% CS 99.9 ± 0.1% 34.7 ± 9.9% 57.1 ± 10.4% 66.3 ± 8.9% CS + B12 99.9 ± 0.1% 98.2 ± 0.3% 99.9 ± 0.1% 90.5 ± 16.0% lac + B12 + bioaug 99.9 ± 0.1% 99.4 ± 0.1% 99.6 ± 0.4% 99.3 ± 1.3% Low AC 4.8 ± 5.7% 0.5 ± 1.1 % 17.6 ± 20.4 % as-is 26.3 ± 28.6% 4.4 ± 9.1% 14.5 ± 15.2% lac 51.2 ± 25.4% 11.0 ± 7.3% 4.0 ± 8.4% EM 64.9 ± 17.0% 12.8 ± 8.9% 7.2 ± 7.9% CS 88.2 ± 17.7% 21.0 ± 14.5% 0.0 ± 0.0% CS + B12 100.0 ± 0.0% 99.5 ± 0.4% 98.5 ± 1.1% lac + B12 + bioaug 100.0 ± 0.0% 100.0 ± 0.0 % 98.3 ± 1.9 % a Average of triplicate microcosms ± standard deviation.

170

A.5 Characteristics of the soils used to prepare the microcosms. a Table 6-2 Characteristics of the soils used to prepare the microcosms High Medium Low (RPB-1) (RPB-2) (RPB-3) Moisture (%) 24.4 19.7 22.7 Organic Matter (%) 0.155 0.176 0.245 Soil pH 5.6 3.9 3.7 Buffer pH 7.8 7.7 7.5 Carbon (%) 0.075 0.043 0.041 Nitrogen (%) 0.021 0.026 0.025 C/N Ratio 3.6 1.7 1.6 Nitrate-N (ppm) 2 3 4 Phosphorus (ppm) b 256 169 236 Potassium (ppm) b 31 30 32 Calcium (ppm) b 654 1,954 1,265 Magnesium (ppm) b 113 28 105 Sodium (ppm) b 355 36 62 Sulfur (ppm) b 33 1,252 815 Boron (ppm) b 0.1 0 0.1 Zinc (ppm) b 1.2 0.4 0.6 Manganese (ppm) b 20 4 4 Copper (ppm) b 0.4 0.2 0.3 Iron (ppm) 35 165 87 Aluminum (ppm) 347 211 466 Bulk Density (kg/m 3) 1,818 2,000 1,905 a High, medium and low refer to the halomethane concentrations; abbreviations in parentheses refer to the soil core used. b Results converted from lb/acre to ppm by dividing by 2 (according to the analysis report from Agricultural Service Laboratory, Clemson University).

171

A.6 Characteristics of the precipitates formed by pH adjustment Table 6-3 Composition of the precipitates (%) AS-MWO-4 AS-MWO-10 (Medium) (Low) N 0.00 0.00 P 0.06 0.04 K 0.03 0.02 Ca 5.41 9.27 Mg 0.08 0.56 S 1.28 1.09 Cl 0.00 0.00 Zn 0.0459 0.0346 Cu 0.0023 0.0048 Mn 0.0521 0.152 Fe 15.6 5.51 Na 0.764 1.27 B 0.00 0.00 Al 11.0 11.6 nitrate 0.00 0.00 Color of precipitate brown orange/brownish

18 AS-MWO-4 16 AS-MWO-10

14

12

10

8 Percent

6

4

2

0 N P K Ca Mg S Cl Zn Cu Mn Fe Na B Al nitrate Compound

Figure 6-5 Analysis of the precipitate generated during pH adjustment of groundwater

172

A.7 Organic acids analysis for the microcosms in Chapter 2 at the end of incubation Table 6-4 Organic acids analysis for the microcosms in Chapter 2 at the end of incubation Treatment % of COD added Total lactate Formate Acetate Propionate Butyrate CF -L- lac + B12 + bioaug 85.7% 0.0% 0.0% 44.3% 41.4% 0.0% CS + B12 21.9% 0.0% 2.8% 19.1% 0.0% 0.0% CS 34.8% 0.0% 1.0% 26.4% 4.5% 2.9% emul oil 3.2% 2.3% 0.1% 0.6% 0.1% 0.0% lactate 86.5% 86.5% 0.0% 0.0% 0.0% 0.0% CT -L- lac + B12 + bioaug 90.4% 0.0% 0.0% 47.6% 42.8% 0.0% CS + B12 60.1% 0.0% 0.1% 50.9% 4.6% 4.5% CS 41.4% 0.5% 1.4% 34.6% 4.9% 0.0% emul oil 0.8% 0.0% 0.0% 0.8% 0.0% 0.0% lactate 88.6% 80.0% 0.0% 8.7% 0.0% 0.0% CF -M- lac + B12 + bioaug 63.9% 0.0% 0.0% 39.9% 23.9% 0.0% CS + B12 56.4% 0.0% 0.0% 56.4% 0.0% 0.0% CS 51.8% 0.0% 0.1% 46.5% 5.2% 0.0% emul oil 1.4% 0.7% 0.0% 0.1% 0.5% 0.0% lactate 60.0% 2.7% 0.0% 49.7% 7.6% 0.0% CT -M- lac + B12 + bioaug 65.1% 0.0% 0.0% 41.4% 23.7% 0.0% CS + B12 47.1% 0.0% 0.0% 45.5% 1.6% 0.0% CS 49.3% 0.0% 0.1% 46.0% 3.2% 0.0% emul oil 1.6% 1.3% 0.1% 0.2% 0.0% 0.0% lactate 84.2% 77.9% 0.0% 4.4% 1.8% 0.0% CF -H- lac + B12 + bioaug 68.9% 0.0% 0.0% 31.0% 37.1% 0.8% CS + B12 46.3% 2.9% 6.1% 21.8% 14.7% 0.7% CS 57.2% 1.5% 5.0% 27.4% 2.2% 21.2% emul oil 1.0% 0.3% 0.0% 0.8% 0.0% 0.0% lactate 84.0% 41.0% 0.0% 10.7% 32.3% 0.0% CT -H- lac + B12 + bioaug 67.9% 0.0% 0.0% 24.4% 41.6% 1.9% CS + B12 50.5% 14.0% 4.6% 19.6% 8.4% 3.9% CS 50.0% 0.7% 5.3% 28.6% 5.1% 10.3% emul oil 3.8% 0.5% 0.0% 1.7% 1.6% 0.0% lactate 78.5% 75.4% 0.1% 0.6% 2.4% 0.0%

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A.8 Fate of lactate in bioaugmentation microcosms

20 a 18 16 14 12 10 8 Lactate 6 Acetate

Organic acids (mM) acids Organic Cumlative lactate added 4 Propionate 2 Cumlative lactate consumed 0 0 20 40 60 80 100 120 140 160 180 200 Time (days after bioaugmenting)

45 Lactate

40 Acetate b Cumlative Lactate 35 added Propionate 30 Cumlative Lactate 25 consumed

20

15

Organic acids (mM) acids Organic 10

5

0 0 50 100 150 200 250 Time (days after bioaugmenting)

Figure 6-6 Fate of lactate in the high (a) and medium (b) concentration bioaugmentation microcosms (in chapter 2)

174

B. Supplementary data for Chapter 3

B.1 Comparison of growth pattern of DHM-1 with and without CF

70

60

50

g/mL) 40

30

Protein( 20 DHM-1 + CS + B12 + CF 10 DHM-1 + CS only 0 0 5 10 15 20 25 Time (days)

Figure 6-7 Comparison of growth pattern of DHM-1 with and without CF

175

B.2 Effect of addition of sulfide on CF transformation by DHM-1 in MSM

600 reinoculation 500

400 0.5 mM sulfide 300

CF(mg/L) 200

100

0 0 10 20 30 40 50 60 70 Time (days)

Figure 6-8 Effect of sulfide (0.5 mM) on CF transformation by DHM-1 in MSM

176

B.3 CF-transformation ability of DHM-1 after 24 h exposure to air and then returning them to anaerobic MSM

700 600 500 400 "No air" 24 hr air 300 e.d. addition CF(mg/l) 200 100 0 0 10 20 30 40 50 60 Time (days)

Figure 6-9 CF-transformation ability of DHM-1 after 24 h exposure to air and then returning them to anaerobic MSM, in comparison with no air exposure

177

B.4 Sequences of partial 16S rRNA gene of the six bands of DHM-1 (Figure 3-4) cut off from DGGE

DGGE Sequence BLAST/Closest band ID (by MWG Biotech. 12/10/07) matches (from top to bottom) #1 CNNNTGGTAGTCGCACGAGCGCACCCTNATCCTTTGTTGCCAGCGGNTNNTGCCGGNAAN BLAST failed due to TCANAGAAAATNNCCGNGNTNAACTGCGAGAGAAAGNTGGGATTAACTCTCAATTCTTCT double sequences TGCCCNTTATATNNNGGGNTTCNCCCTTNTTNCANTGNNCNTATACAAAANNAGTCCAAC TCTNTANAGCATGAGCGACNTTNCTAAAATGCGTCTCTAATTTCGANTTGAGTCTGCAAC TCTCACTCCCTGAAATCTCGAATCTCCTATATAATCTCTAAATNNCAANTGACTACTGGT GAATACTATTCTTCCGGGCTTGTTGNACACCCCCCCTGGTGGNGNGGGCGNCGNGCCCCG NGCGCGGCGCGCCGCGCGNGA #2 GNTTGNTNTCCGCACGAGCGCACCCTTATCCTTTGTTGCCAGCGGTNCGGCCGGGAACTC Pantoea agglomerans ; AAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCC Enterobacter sp.

178 CTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGA GAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCA 99% TGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCC TTGTACACACCGCCCGTGGGGCGGGGGCGGCGGGCCGGGCGCGGGGCGCGGCGGGCGA

#3 CGGTTGGTAGTCCGCACGAGCGCACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAAC Enterobacter sp. TCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGG 99% CCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGC GAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTC CATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGG CCTTGTACACACCGCCCGTGGGGCGGGGGCGGCGGGCCGGGCGCGGGGCGCGGCGGGCGA AA #4 TTGGTAGTCCGCACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGGAACT Enterobacter , 95% CAAAGGANACTGCCAGTGATAAACTGGANGAAGGTGGGGATGACGTCAAGGTCATCATGG CCCTTACNAGTANGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGC GAGAGCAAGCGGACCTCATAAAGTGCGTCNTANTCCGGATTGGAGTCTGCAACTCNACTC CATGAAGTCGGAATCNCTAGTAATCNTAGATCANAATGCTACGGTGAATACNTTCCCGGG CCTTGTACACACCGCCCNTGGGGCGGGGGCGGCGGGCCGGGCGCGGGGCGCGNCGGGCGA GA

#5 NGGTTGGTAGTCCGCACGAGCGCACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAAC Pantoea agglomerans , TCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGG Enterobacter CCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGC GAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTC 99% CATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGG CCTTGTACACACCGCCCGTGGGGCGGGGGCGGCGGGCCGGGCGCGGGGCGCGGCGGGCGA A #6 TTNNTTNGGTAGTCCGCACGAGCGCAACCCTCGTCCTATGTTGCCAGCGGGTAATGCCGG Propionibacteriaceae GGTACTCATAGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCAT bacterium WN042 gene CATGCCCCTTATGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAAAGAGCTGCGAG CCTGTGAGGGTGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTC for 16S rRNA, etc. GACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTT 98% CCCGGGGCTTGTACACACCGCCCGTGGGGCGGGGGCGGCGGGCCGGGCGCGGGGCGCGGC GGGCGA

179

B.5 Complete sequences of 16S rRNA gene of the two Pantoea strains

Strain DHM-1B agagtttgatcctggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagcacaga gagcttgctctcgggtgacgagtggcggacgggtgagtaatgtctgggaaactgcctgatggggggggata actactggaaacggtagctaataccgcataacgtcgcaagaccaaggagggggaccttcgggcctcttgcc atcagatgtgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctgg tctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaa tattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatgaagaaggccttcgggttgtaaagta ctttcagcggggaggaaggtgttgcggttaataaccgcagcaattgacgttacccgcagaagaagcaccgg ctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcg cacgcaggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggca ggctagagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaatac cggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggatta gataccctggtagtccacgccgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggag ctaacgcgttaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggccc gcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccagag aacttagcagagatgctttggtgccttcgggaactctgagacaggtgctgcatggctgtcgtcagctcgtg ttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggttaggccggga actcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacga gtagggctacacacgtgctacaatggcgcatacaaagagaagcgacctcgcgagagcaagcggacctcata aagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagat cagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaa aagaagtaggtagcttaaccttcgggagggcgcttaccactttgtgattcatgactggggtgaagtcgtaa caaggtaacc

Strain DHM-1T

GCCCTTGGTTACCTTGTTACGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTT AAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTAT TCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCC GGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGC ACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTG GCAGTCTCCTTTGAGTTCCCGGCCTAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTA ACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAA GCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACAT GCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGT CGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGT GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGG GCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCT CTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTG ACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCT GCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGMYTAGGTTATTAACCTYAKCGCCT TCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTG CGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGC TGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTAATCCCA YCTGGGCACATCTGATAGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGC TACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGCC GGCAAAGTAGCAAGCTACTTTCCGCTGCCGCTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCT GAGCCAGGATCAAACTCTAAGGGC

180

B.6 Growth curves of the two Pantoea strains

0.7

0.6

0.5

0.4

0.3 OD600nm at

0.2 DHM-1B (+CF) DHM-1B (-CF) 0.1 DHM-1T (+CF) DHM-1T (-CF) 0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time (days)

Figure 6-10 Growth curves of the two DHM-1 isolates, i.e., DHM-1 B and DHM-1T, in MSM + corn syrup (960 mg COD/L) and B 12 (3% mole ratio), with and without CF (500 mg/L)

181

C. Supplementary data for Chapter 4

C.1 Media controls (without B 12 ) for CT, CFC-11 and CF

700 45

40 600 35 500 30

400 25

300 20 CF(mg/l) 15 200

CF 10 CT,CFC-11 (mg/L) 100 CT 5 CFC-11 0 0 0 10 20 30 40 50 60 Time (days)

Figure 6-11 Media controls (without B 12 ) for CT, CFC-11 and CF

182

C.2 Transformation of individual halomethane by SDC-9

CT (SDC-9+B12) CT (LC) CT (MC+B12) 20 CF CS2

15

10

(mg/L) CT 5

0 0 5 10 15 20 25 CFC-11 (SDC-9+B12) CFC-11 (LC) CFC-11 (MC+B12) HCFC-21 40 CS2

30

20

10

(mg/l) CFC-11 0 0 10 20 30 40 CF (SDC-9+B12) Time CF(days) (LC) CF (MC+B12) 600 DCM CS2 500 400 300 200 CF(mg/l) 100

0 0 10 20 30 40 50 60 Time (days) Figure 6-12 Transformation of individual halomethane by SDC-9 in RAMM, LC=live control, MC+B 12 =media control with B 12 . Daughter products formed only in the DHM-1 + B 12 treatments.

183

D. Protocols

D.1 PCR protocol using Eppendorf Master Mix

Sequence Reagent Amount ( l) 1 Autoclaved DDI water 26/23 28 (for NC) 2 Forward Primer 1 2 Reverse Primer 1 3 Master Mix 20 4 Template DNA 2/5 Note: 1. always include a negative control to show any contamination. Working in a clean hood is recommended. 2. Recommended template amount: 30-50 ng [from Dr. Kurtz]; 25-30 cycles [PCR Analysis, Chapter 16, Basic Techniques In Molecular Biology, Stefan Surzycki, 1999]

Votex, centrifuge @ 10K rpm for 0.5 min. Then ready for PCR using program TB-PCR1.

After PCR, run agarose gel at 80v for 1.5 hr . 1. Load: 1 l dye + 3 l PCR DNA sample; same amount for ladder. 2. Stain DNA on the gel by immersing in an ethidium bromide solution (about 5 l in 200 mL DDI water) in a plastic tray, shaking gently for about 10 min. 3. Pour the ethidium bromide solution and change to DDI water for rinsing. 4. Watch the gel under UV by BIO-RAD universal hood II and get a picture for the bands.

Gel Preparation Measure 70 mL 1X SB buffer into a conical flask, add 1.05g agarose into it (i.e. 1.5%), weigh it; put into microwave oven, carefully heat it until boiling, stop and check whether it’s become a clear solution, which may take about 1 min. Then slowly pour into gel chamber to avoid generating air bubble, put on the well maker, then cool about half a hour to solidify. Pack it with clean wrap and store in fridge.

184

D.2 DGGE protocol

1) Prepare 6% acrylamide solutions 0% 10% 50% 100% 40% acrylamid/bis (37.5:1) 15 mL 15 mL 15 mL 15 mL 20X SB 2 mL 2 mL 2 mL 2 mL Deionized formamide 4 20 mL 40 mL Urea 4.2 21 g 42 g DDI water To 100 mL To 100 mL To 100 mL To 100 mL a) Prepare the solutions according to the formulas in the table above. i) Be sure to work in the hood as much as possible. Acrylimde is a neurotoxic. Avoid breathing the vapors. ii) To prepare 100% solution, use a 150 or 250 conical flask (make a mark for 100 mL) first. After adding the first 4 items (total volume will be less than 100 mL), add a few mL DDI, stir and slightly heat for around 15 min, will completely dissolve. Then transfer to a 100 mL vol. flask topping to 100 mL with DDI. b) Degas the solutions for 30 minutes i) Place the solution in a 125 mL Erlenmeyer flask with a port ii) Place a stir bar in the flask and place a cork (stopper) in the flask iii) Place the flask on a stir plate, set to gently stir the solution (you don’t want a vortex to form) iv) Attach a vacuum hose to the port and apply vacuum pressure c) Store the solutions in amber bottles in the fridge for up to 1 month

2) Glass Plate Preparation a) ____ Rinse 5 X with DDI water b) ____ Air dry or use a lint-free tissue c) ____ Wipe the inner surface of the plates with 95% ethanol and dry with lint- free tissue d) ____ Apply gel-repel (CBS Scientific) to one side of the back glass plate (this will be the inside of the sandwich next to the gel)

3) Cast the Gel a) ____ Start with the back plate (the one with the rounded corners) b) ____ Apply the gasket starting at the bottom i) Make sure the rounded side of the gasket is on the inside (the side you applied gel repel to) and that the flat side is on the outside ii) Make sure the notches on the gasket align with the rounded corners of the glass plate iii) Work the gasket down around both sides c) ____ Place the gasketed plate on the lab bench with the tubing side up, and extend the bottom of the plate over the edge of the bench, approximately ¾ of an inch.

185

d) ____ Place the spacers along side the inside edges of the gasket i) Be sure the rounded scorner end of each spacer is facing the outside bottom of the plate, following the radius of the glass. e) ____ Place the notched plate on top of the bottom assembly, starting from the bottom edge and gently easing the plate down f) ____ Verify the gasket is smooth around the edges g) ____ Clamp along the bottom i) Make sure the glass rests against the body of both clamps h) ____ Lift the assembly and stand it on the base of the clamps i) ____ Clamp the sides of the assembly with two clamps per side i) Be sure the gasket is aligned between the plates, forming a seal j) ____ Blow the assembly with air, empty remained water inside the fitting and tubing. k) ____ Place the assembly upright on the lab bench near the stir plate to be used for the gradient caster l) ____ Tape the needle of the gradient caster to the top of the assembly, with the needle between the plates and centered horizontally m) ____ Make sure both valves on the gradient maker are closed n) ____ Place a small Teflon coated stir bar in cylinder 2 (C-2) o) ____ Make Solution A & Solution B (add TEMED and 10% APS to the acrylamide solutions) (Note: Prepare pipets with tips on, beaker, chemicals at hand to expedite this step .) i) Solution A is the lower % denaturant & Solution B is the higher % denaturant ii) Filter the acrylamide solutions (pour ~15 mL into a third beaker, suck 15 mL using a 20 mL syringe with a 18G1 needle) iii) Avoid introducing gas into the acrylamide solutions while filtering through a 0.45 um filter into a beaker, one beaker for each solution iv) Add 15 L TEMED v) Add 150 L 10% APS (Note: No need to mix them, handle step p immediately.) p) ____ Pour solution B (the denaturing acrylamide) into the cylinder (C-2) nearest the outlet q) ____ Open valve 1 (V-1) and allow approximately 1 mL of solution B to pass into C-1 r) ____ Close valve 1 (V-1) s) ____ Use a pipette to transfer all of the solution from C-1 back to C-2 t) ____ Open valve 2 (V-2) and allow some of solution be to flow into the glass plate assembly (Note: steps q-t are optional, can skip them. Handle steps p-w in a minute after step o!) u) ____ Add solution A to chamber C-1 v) ____ Turn on the stir plate w) ____ Open valve 1 (V-1) and then valve 2 (V-2) x) ____ Tilt the assemble in case there is air bubble i

186

y) ____ Once all of the acyrlamide solutions have fed into the glass plate assembly, insert the 16-well rectangular tooth comb z) ____ If the gel volume is not enough to fill the sandwich (to fill in around the comb), top-off with 0% denaturing acrylamide aa) ____ Allow the gel to polymerize for 2 hours

Starting protocol :

1. Take off the comb 2. Remove the clamps, put the assembly on the stand with the back plate facing outside, take the gasket out at the bottom 3. Fix the assembly and the stand using 4 bigger clamps 4. Transfer the assembly into the water bath, fill the top trough with water, remove air bubbles at the bottom using a syringe w/ a bent needle 5. Connect the influent tubing, control its flow by a adjustable clip to get continue dropping at the outlet and a suitable water level in the trough 6. When temperature reaches 60 oC, confirm there is no air bubble at the bottom, then load samples (around 5-15 µL depending on concentration of the PCR products; recommend 500 ng by H Schafer and G Muyzer in chapter 22, Method in Microbiology v30, v12, (2001)). 7. Connect the black cord, turn on 140, run 2.5 hrs 8. Watch the flow through the assembly from time to time.

Staining protocol :

1. Take the assembly out, remove the clamps and gasket 2. Put the gel with plates together in a shallow DDI water bath with the back plate facing up, and then remove it 3. Transfer the gel and front plate into staining bath (~200 mL), stain 10-20 min, then destain in DDI water bath for 5-10 min 4. Ready for viewing under UV.

187

D.3 Preparation of the medium for DHM-1

Table 6-5 Minimal salts medium for DHM-1 Constituent Concentration NH 4Cl 0.3 g/L KCl 0.3 g/L CaCl 2·2H 2O 0.15 g/L MgCl 2·6H 2O 0.4 g/L K2HPO 4 0.41 g/L NaHCO 3 4.0 g/L Resazurin 0.001 g/L Yeast extract 0.05 g/L a Na 2S·9H 2O 0.12 g/L Selenite solution b 1 mL/L Ferrous solution c 1 mL/L Trace metal solution d 1 mL/L a All compounds except sodium sulfide were added into autoclaved distilled deionized water, purged with 30% CO 2 and 70% N 2 for a few minutes until complete dissolution of mineral salts, and then placed in an anaerobic chamber where 0.12 g/L (i.e., 0.5 mM) sodium sulfide was added, media turned clear (indicating E h dropped below -110 mV) in hours. pH was approximately 7.5. b 3.0 mg Na 2SeO 3·5H 2O, 5.0 mg/L NaHCO 3. c 3.0 mg FeCl 2·4H 2O, 5.0 mg/L NaHCO 3. d FeCl 2·4H 2O (1.5 g/L), CoCl 2·6H 2O (0.190 g/L), MnCl 2·4H 2O (0.1 g/L), ZnCl 2 (0.07 g/L), H 3BO 4 (0.006 g/L), Na 2MoO 4·2H 2O (0.036 g/L), NiCl 2·6H 2O (0.024 g/L), and CuCl 2·2H 2O (0.002 g/L).

188

D.4 Preparation of RAMM for SDC-9

(Revised Anaerobic Minimal Media, modified from Shelton and Tiedje (81))

A. Buffer Solution (100x) Dissolve in 1 L dH 2O:

KH 2PO 4 27.0 g

K2HPO 4 35.0 g

B. Mineral Salts Solution (100x) Dissolve in 1 L dH 20:

NH 4Cl 53.0 g

CaCl 2-2H 2O 7.5 g

MgCl 2-6H 2O 10.0 g

FeCl 2-4H 2O 2.0 g

C. Trace Metals (1000x) Dissolve in 1 L dH 2O:

MnCl 2-4H 2O 5.00 g

H3BO 3 0.05 g

ZnCl 2 0.05 g

CuCl 2 0.03 g

NaMoO 4-2H 2O 0.01 g

CoCl 2-6H 2O 0.50 g

NiCl 2-6H 2O 0.05 g

Na 2SeO 3 0.05 g Add ~1 mL of 38% HCl to keep metals in suspension

D. Resazurin Solution (10,000x) Dissolve 0.1 g Resazurin in 10 mL dH 2O. 1x Media: 1) Mix a [1X] solution of A, B, C, and D in DDI water

2) Autoclave, then bring to boil (or almost boil) while sparging with high purity N2 3) Let cool while sparging

4) Add 1.2 g NaHCO 3 per liter of media, and 0.5 g/L yeast extract, then cap the bottle

5) Transfer into anaerobic chamber where 0.12 g/L Na 2S·9H 2O was added, cap the bottle, Media should turn clear in few hours, then may turn dark overnight. Store the media inside anaerobic chamber.

189

D.5 Transferring DHM-1

1) 5% transferred (i.e., add ~ 5 mL culture into ~ 93 mL fresh media (MSM)) 2) Add ~ 960 mg COD/L corn syrup (by ~0.266 mL 360 mg COD/mL stock a) and

1.84 mL B12 stock (1% weight/volume)

3) Purge the headspace with 30% CO 2/70% N 2 for 1 min 4) Spike 500 mg/l CF (by weight) by adding 36 µL neat CF 5) Equilibrate on shaker for 2-3 days before initial GC headspace analysis a assuming corn syrup is 100% composed of glucose

190

D.6 Dissolved sulfide measurement

Follow the methylene blue colorimetric assay in Standard Methods, except using 1/10 of the volumes of samples and reagents specified 1) Take 0.75 mL of each standard solution into a 2.0 mL microcentrifuge tube, using a replicate of one or two standards as tube B (as blank for spectrophotometer analysis)

2) Add 50 µL amine-H2SO 4 reagent, 15 µL FeCl 3 solution (tube A), while 50 µL

1+1 H2SO 4, 15 µL FeCl 3 solution (tube B) 3) Mix once by inverting gently, wait 3-5 min, develop blue color

4) Add 0.16 mL (NH 4)2PO 4, wait 3-15 min, produce white precipitate (suggest - centrifuge to settle the precipitate) 5) Measure absorption of the liquid phase samples at 664 nm using Beckman DU 640 spectrophotometer with 1 mL cuvette

191

E. Supplementary experimental results E.1 Amount of corn syrup added in each transfer of DHM-1 to transform 500 mg/L CF

Transfer Corn syrup added (# of dose, ~ 960 mg COD/L per dose) 1 2 2 2 3 3 4 3 5 2 6 2 7 2 8 2 9 2 10 2 11 2 12 2 13 1 14 1 15 1 16 1

192

E.2 Ability of a culture that grows anaerobically on DCM to use chlorofluoromethane

50 DCM-A-1 DCM 100,000 45 CH2ClF 90,000 40 80,000 35 70,000 30 60,000 25 50,000

mol/bottle) 20 40,000

15 30,000 (peak ClF area) 2

DCM( 10 20,000 CH 5 10,000 0 0 0 20 40 60 80 100 120 140

Time (days)

50 DCM-A-2 DCM 80,000 45 CH2ClF 70,000 40 60,000 35 30 50,000 25 40,000

mol/bottle) 20 30,000

(peak ClF area) 15 2 20,000

10 CH DCM( 5 10,000 0 0 0 20 40 60 80 100 120 140

Time (days)

193

40 DCM-B-1 DCM 90,000 35 CH2ClF 80,000 30 70,000 60,000 25 50,000 20

mol/bottle) 40,000 15 30,000 ClF (peak ClF area) 2 10 20,000 CH DCM( 5 10,000 0 0 0 20 40 60 80 100 120 Time (days)

DCM 50 DCM-B-2 80,000 45 CH2ClF 70,000 40 60,000 35 30 50,000 25 40,000 mol/bottle) 20 30,000 15 20,000 CH2ClF(peak area)

DCM( 10 5 10,000 0 0 0 20 40 60 80 100 120 Time (days)

Figure 6-13 Test of ability of a culture that grows anaerobically on DCM to use chlorofluoromethane. Where, DCM-A: CH 2ClF was added together with DCM after growth on DCM was established; DCM-B: only CH 2ClF was added after growth on DCM was established

194

E.3 Response factors of GC

Table 6-6 Response factors of GC for 160 mL serum bottles with 50 mL liquid phase and 99 mL headspace Compound GC RT RF Conversion Factor Date (min) (µmol/bottle/ R2 (µmol/btl to mg/L) Peak Area) Methane 0.50 3.981E-06 0.9903 0.000136 7/27/2005 Chloromethane 1.22 1.284E-05 0.9961 0.6224 6/25/2005

CH 2ClF 1.54 3.086E-05 0.9990 0.8689 6/25/2005

CHCl 2F 4.2 2.647E-05 0.9866 1.4406 6/25/2005 DCM 4.6 3.136E-05 0.9975 1.4649 7/27/2005

CS 2 5.4 7.490E-04 0.9949 0.5905 7/27/2005 CFC-11 5.68 1.587E-05 0.9965 0.3223 7/31/2008 CF 7.16 2.469E-05 0.9991 1.8881 7/27/2005 CT 8.6 8.780E-06 0.9989 0.9564 7/27/2005

Table 6-7 Response factors of GC for 160 mL serum bottles with 100 mL liquid phase and 60 mL headspace Compounds RF Conversion Factor Date

(µmol/bottle/ (µmol/btl to mg/L) Peak Area) Chloromethane 1.221E-05 0.4248 3/6/2007 a CHCl 2F 3.139E-05 0.9109 a CH 2ClF 5.703E-05 0.5830 DCM 5.315E-05 0.8101 1/11/2008 CS 2 7.223E-04 0.5150 1/11/2008 CFC-11 1.366E-05 0.4188 6/4/2007 CF 4.571E-05 1.1052 1/11/2008 CT 9.250E-06 0.9202 1/11/2008

a Estimated from the RFs from Table 6-6 using the equation: Cg = M/(V g+V L/H c) (i.e., using the equation to get the RFs for Cg/peak area, then convert C g to M using M=C g(V g+V L/H c)) because neat compounds of these two HCFCs were no longer available in the laboratory.

195

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