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dissertation entitled

BIOTRANSFORMATION OF NON-VOLATILE
ORGANOFLUORINE COMPOUNDS

presented by

Blake Douglas Key

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Environmental Eng.

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MSU to An Affirmative AotloNEquol Opportunity Institution
mm:

BIOTRANSFORMATION OF NON-VOLATILE
ORGANOFLUORINE COMPOUNDS

By

Blake Douglas Key

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Civil and Environmental Engineering

1996

ABSTRACT

BIOTRANSFORMATION OF NON-VOLATILE
ORGANOFLUORINE COMPOUNDS

By

Blake Douglas Key

This research addresses the potential for biotransformation of non-volatile fluorinated
organics, with a focus on sulfonates and carboxylates. Biodegradation of the following
model sulfonates was evaluated: difluoromethane sulfonate (DFMS, CHFQSO3Na),
trifluoromethane sulfonate (TFMS; CF3SO3Na), 2,2,2-trifluoroethane sulfonic acid
(TES; CF3CH2803H), perfluorooctane sulfonate (PFOSA; C3F17SO3K), and
lH,lH,2H,2H-perfluorooctane sulfonic acid (H-PFOSA; C5F13C2H4SO3H). Those
compounds with hydrogen at the alpha-carbon degraded under aerobic and sulfur-
limiting conditions. DFMS , TF3, and H-PFOSA were transformed by a variety of
bacteria, including Pseudomonads, Bacillus subtilis, and Escherichia coli. However, E.
coli was not capable of transforming H-PFOSA. DFMS was completely defluorinated
with a stoichiometric yield of fluoride. TFMS and H-PFOSA were only partially
defluon’nated with equimolar fluoride release from TES and 1-1.4 moles fluoride
release from H-PFOSA. Eight volatile and fluorinated byproducts of H-PFOSA were

detected by mass spectrometry and atomic emission detection.

Oxygen was required for growth on and transformation of DFMS. Complete shut down
of transformation occurred with the removal of glucose or ammonium. Inhibition
studies with other sulfur sources suggest that the sulfur-containing byproduct of DFMS

transformation is assimilated by existing sulfur assimilating pathways.

Non-competitive inhibition was observed with sulfate, sulfite, methanesulfonate,
cystine, and methionine. These results suggest that transformation of DFMS is linked

to a sulfur-scavenging system in which DFMS is desulfonated then defluorinated.

Monofluoroacetate (MFA), difluoroacetate (DFA), and trifluoroacetate (TFA) were
selected as model compounds for study of anaerobic degradation of fluorinated
carboxylates. Denitrifying and sulfate-reducing enrichment cultures defluorinated
MFA. A bacterial isolate, designated strain M7, was capable of growth on and
defluorination of MFA under aerobic and denitrifying conditions. Strain M7 was
unable to degrade DFA or TFA. Phylogenetic analysis of the 16S rRNA sequence
shows a 96.0 to 97.5% similarity between strain M7 and the Bradyrhizobium species.
Two other bacteria with the ability to utilize MFA under aerobic conditions were also
capable of MFA degradation under denitrifying conditions, suggesting that this

capability is widespread in nature.

Dedicated to my wonderful friend, lover, and lifetime companion Lynda Rae

whom I cherish with all of my heart, mind, body, and soul.

iv

ACKNOWLEDGMENTS

I would like to express my deepest appreciation to all of the people who made the

completion of this research and dissertation possible.

A very special thanks goes to my wife Lynda for her loving support and unconditional
faith in my abilities without which I might have given up long ago. I would also like to
thank my parents, Rudy and Karolynn Key, for their support and encouragement

throughout my life.

I would like to thank my friend, mentor, and advisor Dr. Craig Criddle. His enthusiasm
for science has influenced me in many ways. I thank him for his unending
encouragement, intellectual guidance, and friendship. I am honored to have worked

with a scientist of such high caliber.

Thank you also to Dr. Robert Howell for his intellectual input and direction in several
aspects of my research. Dr. Howell's role as liaison between 3M and Michigan State
University has created a c00perative link between industry and academia. Other 3M
associates that I would like to thank are Dr. James D. Johnson, Gregory N. Maisel,
James B. Drake, and Dr. James K. Lundberg of 3M Environmental Laboratory, for their

analytical help.

A special thanks goes to Dr. Susan Masten, Dr. Larry Fomey, and Dr. Patrick Oriel for
critiquing my work and for providing guidance and direction to my research. Without

their help I would not have done many of the experiments presented here.

Thank you to the National Science Foundations, Center for Microbial Ecology (CME)
for their financial support early in my research career. I would like to commend all of
the participants of CME for creating a collaborative, cross-disciplinary research and

educational organization.

Many of my colleagues and friends deserve thanks for helping me complete my
research. If it were not for Yanlyang Pan, I am certain that I would never have finished.
His knowledge and skills to maintain and implement all of the analytical equipment in
our lab has helped me more than can be imagined. I appreciate Dr. Mike Dybas and Dr.
Greg Tatara's help with experimental design and for always keeping our lab interesting.
I would like to thank Dr. Brad Upham for his intellectual input and especially for his
encouragement and friendship. Thank you also to Mark Sneathen, Joel Klappenbach,
Mike Witt, Debbie Hogan, Xianda Zhao, Sadhana Chauhan, Lycely Sepfiveda-Torres,
Mike Apgar, Brad Kach, Janice and Bernard Weidenaar, and Myron Erickson for their

help in various aspects of my work and my life.

A very special thank you goes to Linda Steinman for graciously putting up with me and
all the other "pester flies" that are constantly requesting her help. She has been an
invaluable asset in dealing with administrative issues. I thank her for her kind and

friendly spirit that has cheered me on many occasions.

I would like to thank Dr. Mark Emptage of DuPont Corp for the MFA-degrading
strains. I would like to thank Jizhong Zhou for his work on the 16S rRNA sequencing

of strain M7.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................

LIST OF FIGURES
LIST OF SYMBOLS

CHAPTER 1

CHAPTER 2

CHAPTER 3

CHAPTER 4

ooooooooooooooooooooooooooooooo

oooooooooooooooooooooooooooooo

INTRODUCTION ....................
References ........................

FLUORINATED ORGANICS IN THE BIOSPHERE. .
Introduction ........................
Fate and Effects of Volatile Fluorinated Organics . . . .
Fate and Effects of Non-Volatile Fluorinated Organics .
A Case History - Trifluoroacetic Acid ..........
Summary and Conclusions ................
References ........................

DEFLUORINATION OF ORGANOFLUORINE
SULFUR COMPOUNDS BY PSEUDOMONAS SP.
STRAIN D2 ........................
Abstract ..........................
Introduction ........................
Methods and Materials ..................
Results ..........................
Discussion ........................
References ........................

PHYSIOLOGY OF DIFLUOROMETHANE
SULFONATE TRANSFORMATION BY
PSEUDOMONAS SP. STRAIN D2 ............
Abstract ..........................
Introduction ........................
Methods and Materials ..................
Results ..........................
Discussion ........................

References ........................

vii

ix

8

15
19
25
3O
33

45
45
46
47
49

62
62
63
64
68

CHAPTER 5 BIOTRANSFORMATION OF SULFUR-CONTAINING
ORGANOFLUORINE COMPOUNDS BY DIVERSE
ORGANISMS ....................... 81
Abstract .......................... 81
Introduction ........................ 82
Methods and Materials .................. 82
Results .......................... 86
Discussion ........................ 95
References ........................ 97
CHAPTER 6 BIODEGRADATION OF MONOFLUOROACETATE
UNDER DENITRIFYING CONDITIONS ....... 98
Abstract .......................... 98
Introduction ........................ 99
Methods and Materials .................. 100
Results .......................... 105
Discussion ........................ 1 13
References ........................ l 16
CHAPTER 7 CONCLUSIONS AND FUTURE WORK
RECOMMENDATIONS ................. 1 19
Conclusions ........................ 1 19
Future Research ...................... 121
APPENDIX A ................................... 122
GC/MS using electron impact with ion trap detector . . . 122
GC/MS using electron impact with quadrapole detector . 134
APPENDIX B ................................... 154
GC/MS using chemical ionization with quadrapole
detector .......................... 154
APPENDIX C ................................... 158
GC/Atomic emission detection .............. 158

viii

LIST OF TABLES

Chapter 1

No Tables

Chapter 2

Table 2.1. Examples of fluorinated aliphatic compounds and their applications.
Table 2.2. Examples of fluorinated aromatic compounds and their applications.
Table 2.3. Fluorinated chemical application for crop protection in US, 1995.
Calculated from the Agricultural Chemical Usage - 1995 Field Crops Summary,
published by the USDA. (Note: this data only includes applications to corn, cotton,

soybeans, and wheat for major producing states).

Table 2.4. Fluorocarbon production worldwide.

Chapter 3
Table 3.1. Emission wavelengths and plasma gases used for atomic emission detection.

Table 3.2. Possible volatile fluorinated products of H-PFOSA transformation by strain
D2.

Chapter 4

No Tables

Chapter 5

No Tables

ix

Chapter 6

Table 6.1. Kinetic parameters for Bradyrhizobium sp. strain M7

Table 6.2. Glycolate determination in cell free extract of strain M7. Values were
obtained by ion exclusion chromatography. Glycolate levels were confirmed by

colorimetric assay.

Table 6.3. Substrate degradation and growth by Bradyrhizobium sp. strain M7

Chapter 7

No Tables

Appendix A

Table A]. GC/MS listing for peak #1 of strain D2 incubated with glucose and H-
PFOSA.

Table A2. GC/MS listing for peak #2 of strain D2 incubated with glucose and H-
PFOSA.

Table A3. GC/MS listing for peak #3 of strain D2 incubated with glucose and H-
PFOSA.

Table A.4. GC/MS listing for peak #4 of strain D2 incubated with glucose and H-
PFOSA.

Table A5. GC/MS listing for peak #5 of strain D2 incubated with glucose and H-
PFOSA.

Table A6. GC/MS listing for peak #6 of strain D2 incubated with glucose and H-
PFOSA.

Table A7 GC/MS listing for peak #7 of strain D2 incubated with glucose and H-
PFOSA.

Table A8. GC/MS listing for peak #8 of strain D2 incubated with glucose and H-
PFOSA.

Table A9. GC/MS listing for peak #9 of strain D2 incubated with glucose and H-
PFOSA.

Table A.10. GC/MS listing for peak #10 of strain D2 incubated with glucose and H-
PFOSA.

Table A.11.
PFOSA.

Table A.12.
PFOSA.

Table A.13.
PFOSA.

Table A.14.
PFOSA.

Table A.15.
PFOSA.

Table A.16.
PFOSA.

Table A.17.
PFOSA.

Table A.18.
PFOSA.

Table A.19.
PFOSA.

GC/MS listing for peak A of strain D2 incubated with glucose and H-
GC/MS listing for peak B of strain D2 incubated with glucose and H-
GC/MS listing for peak C of strain D2 incubated with glucose and H-
GC/MS listing for peak D of strain D2 incubated with glucose and H-
GC/MS listing for peak E of strain D2 incubated with glucose and H-
GC/MS listing for peak F of strain D2 incubated with glucose and H-
GC/MS listing for peak G of strain D2 incubated with glucose and H-
GC/MS listing for peak H of strain D2 incubated with glucose and H-

GC/MS listing for peak I of strain D2 incubated with glucose and H-

Appendix B

No Tables

Appendix C

No Tables

xi

LIST OF FIGURES

Chapter 1

No Figures

Chapter 2
Figure 2.1. Examples of aromatic fluorinated compounds.

Figure 2.2. Biogeochemical cycling of organofluorine compounds.

Chapter 3

Figure 3.1. Defluorination of DFMS by Pseudomonas sp. strain D2. Error bars
represent the standard deviation of triplicate samples.

Figure 3.2. Growth of Pseudomonas sp. strain D2 on DFMS. Error bars represent the
standard deviation of triplicate samples.

Figure 3.3. Fluoride release from TES. Numbers represent ratio of moles of fluoride to
moles of TES. Triplicate samples were measured, but error bars are not visible due to
scale.

Figure 3.4. Fluoride release from H-PFOSA. Numbers represent ratio of moles of
fluoride to moles of H-PFOSA. Triplicate samples were measured, but error bars are
not visible due to scale.

Figure 3.5. Growth of Pseudomonas sp. strain D2 on TBS. Error bars represent the
standard deviation of triplicate samples.

Figure 3.6. Growth of Pseudomonas sp. strain D2 on H-PFOSA. Error bars represent
the standard deviation of triplicate samples.

Figure 3.7. Atomic Emission Detection of volatile fluorinated byproducts of H-PFOSA
biodegradation.

xii

Chapter 4

Figure 4.1. Growth of Pseudomonas sp. strain D2 with glucose and acetate under
aerobic conditions. No growth was observed under anaerobic conditions. Error bars
represent the standard deviation of triplicate samples.

Figure 4.2. Growth of Pseudomonas sp. strain D2 with DFMS as sole source of sulfur.
DFMS cannot be used as carbon source. Error bars represent the standard deviation of
triplicate samples.

Figure 4.3. Effects of removal of glucose or ammonium, from growth medium, on the
transformation of DFMS in stationary phase cells. Error bars represent the standard
deviation of triplicate samples.

Figure 4.4. Non-competitive inhibition of DFMS transformation rates by sodium
sulfate. Error bars represent the standard deviation of triplicate samples.

Figure 4.5. Non-competitive inhibition of DFMS transformation rates by sodium
sulfite. Error bars represent the standard deviation of triplicate samples.

Figure 4.6. Non-competitive inhibition of DFMS transformation rates by sodium
methane sulfonate. Error bars represent the standard deviation of triplicate samples.

Figure 4.7. Non-competitive inhibition of DFMS transformation rates by cystine. Error
bars represent the standard deviation of triplicate samples.

Chapter 5

Figure 5.1. Defluorination of DFMS (32 1.1M) by various bacteria and yeast under
aerobic and sulfur-limiting conditions. Error bars represent the standard deviation of
triplicate samples.

Figure 5.2. Defluorination of 2,2,2-trifluoroethane sulfonic acid (32 uM) by various
bacteria and yeast under aerobic and sulfur-limiting conditions. Error bars represent the
stande deviation of triplicate samples.

Figure 5.3. Defluorination of lH,lH,2H,2H-perfluorooctane sulfonic acid (32 uM) by
various bacteria and yeast under aerobic and sulfur-limiting conditions. Error bars
represent the standard deviation of triplicate samples.

Figure 5.4. GC/ECD chromatogram of the natural area incubation with glucose and H-
PFOSA.

Figure 5.5. GC/ECD chromatogram of strain D2 incubated with glucose and H-
PFOSA.

Figure 5.6. GC/MS chromatogram of the natural area incubation with glucose and H-
PFOSA.

xiii

Figure 5.7. GC/MS spectrum number 604 of the natural area incubation with glucose
and H-PFOSA.

Figure 5.8. GC/MS chromatogram of strain D2 incubated with glucose and H-PFOSA.
Figure 5.9. GC/MS spectrum number 604 of strain D2 incubated with glucose and H-
PFOSA.

Chapter 6

Figure 6.1. Defluorination of MFA by denitrifying and sulfate-reducing enrichments.
Error bars represent the standard deviation of triplicate samples.

Figure 6.2. Growth of Bradyrhizobium sp. strain M7 on MFA under denitrifying
conditions: (a) consumption of MFA and production of fluoride, (b) consumption of
nitrate and growth of cells. Error bars represent the standard deviation of triplicate
samples.

Figure 6.3. Phylogenetic tree showing the location of Bradyrhizobium sp strain M7 in
relation to other Bradyrhizobium species.

Figure 6.4. Comparison of specific substrate utilization rates for aerobically and
anaerobically grown Bradyrhizobium sp. strain M7. Error bars represent the standard
deviation of triplicate samples.

Chapter 7

No Figures

Appendix A
Figure A.1. GC/MS chromatogram of strain D2 incubated with H-PFOSA and glucose.

Figure A.2. GC/MS spectrum for peak #1 of strain D2 incubated with glucose and H-
PFOSA.

Figure A.3. GC/MS spectrum for peak #2 of strain D2 incubated with glucose and H-
PFOSA.

Figure A.4. GC/MS spectrum for peak #3 of strain D2 incubated with glucose and H-
PFOSA.

Figure A.5. GC/MS spectrum for peak #4 of strain D2 incubated with glucose and H-
PFOSA.

xiv

Figure A.6.
PFOSA.

Figure A.7.
PFOSA.

Figure A.8.
PFOSA.

Figure A.9.
PFOSA.

Figure A.10.
PFOSA.

Figure A.11.
H-PFOSA.

Figure A.12.

glucose.

Figure A.13.
PFOSA.

Figure A.14.
PFOSA.

Figure A.15.
PFOSA.

Figure A.16.
PFOSA.

Figure A.17.
PFOSA.

Figure A.18.
PFOSA.

Figure A.19.
PFOSA.

Figure A20.
PFOSA.

Figure A.21.
PFOSA.

GC/MS spectrum for peak #5 of strain D2 incubated with glucose and H-

GC/MS Spectrum for peak #6 of strain D2 incubated with glucose and H-

GC/MS spectrum for peak #7 of strain D2 incubated with glucose and H-

GC/MS spectrum for peak #8 of strain D2 incubated with glucose and H-

GC/MS spectrum for peak #9 of strain D2 incubated with glucose and H-
GC/MS spectrum for peak #10 of strain D2 incubated with glucose and
GC/MS chromatogram of strain D2 incubated with H-PFOSA and
GC/MS spectrum for peak A of strain D2 incubated with glucose and H-
GC/MS spectrum for peak B of strain D2 incubated with glucose and H-
GC/MS spectrum for peak C of strain D2 incubated with glucose and H-
GC/MS spectrum for peak D of strain D2 incubated with glucose and H-
GC/MS spectrum for peak E of strain D2 incubated with glucose and H-
GC/MS spectrum for peak F of strain D2 incubated with glucose and H-
GC/MS spectrum for peak G of strain D2 incubated with glucose and H-
GC/MS spectrum for peak H of strain D2 incubated with glucose and H-

GC/MS spectrum for peak I of strain D2 incubated with glucose and H-

XV

Appendix B
Figure B.l. GC/MS chromatogram of strain D2 incubated with glucose and H-PFOSA.

Figure 8.2. GC/MS spectrum for peak #1 of strain D2 incubated with glucose and H-
PFOSA.

Figure B.3. GC/MS spectrum for peak #2 of strain D2 incubated with glucose and H-
PFOSA.

Figure B.4. GC/MS spectrum for peak #3 of strain D2 incubated with glucose and H-
PFOSA.

Figure B.5. GC/MS spectrum for peak #4 of strain D2 incubated with glucose and H-
PFOSA.

Figure 8.6. GC/MS spectrum for peak #S‘Of strain D2 incubated with glucose and H-
PFOSA. '

Figure 8.7. GC/MS spectrum for peak #6 of strain D2 incubated with glucose and H-
PFOSA.

Figure B.8. GC/MS spectrum for peak #7 of strain D2 incubated with glucose and H-
PFOSA.

Figure B.9. GC/MS spectrum for peak #8 of strain D2 incubated with glucose and H-
PFOSA.
Appendix C

Figure C.1. AED chromatogram for fluorine (at 690 nm) strain D2 incubated with
glucose and H—PFOSA.

Figure C.2. AED chromatogram for sulfur (at 181 nm) strain D2 incubated with
glucose and H-PFOSA.

Figure C.3. AED chromatogram for hydrogen (at 486 nm) strain D2 incubated with
glucose and H-PFOSA.

Figure C.4. AED chromatogram for carbon (at 496 nm) strain D2 incubated with
glucose and H-PFOSA.

Figure C.5. AED chromatogram for oxygen (at 777 nm) strain D2 incubated with
glucose and H-PFOSA.

xvi

I

k
protein/hr)
Ks

Ki

LIST OF SYMBOLS

specific substrate utilization coefficient (umoles /mg protein/hr)
rate-limiting substrate concentration (uM)
inhibitor concentration (uM)

maximum specific substrate utilization coefficient (umoles /mg

half-velocity coefficient (uM)
inhibition constant (uM)

total protein concentration (mg/L)
initial protein concentration (mg/L)
specific growth rate (d'l)
maximum specific growth rate (d‘l)
endogenous decay rate (d‘l)

time in hours (hr)

xvii

CHAPTER 1

INTRODUCTION

Environmental Significance of Organofluorine Compounds

The commercial and domestic use of organofluorine compounds has dramatically
increased in recent years. These compound are used as propellants, surfactants,
agrochemicals, lamprey larvicide, insecticides, adhesives, refrigerants, fire retardants,
and medicines [2, 3, 5, 7, 8, 9, 10, 17, 20, 21, 23, 27, 29]. Many of these compounds
are used because they are chemically stable and are perceived as more inert biologically
and therefore less likely to have an impact on human health or the environment.
However, inert molecules tend to persist and accumulate, and they are more difficult to
remediate. In addition, several fluorinated organics are subject to at least limited
biotransformation under appropriate environmental conditions. Moreover, as discussed
in Chapter 2, organofluorine molecules actually do exhibit significant biological effects,
as inhibitors of enzymes, cell-cell corrrmunication, membrane transport, and processes

for energy generation [3, 4, 6, 11, 22, 24, 28].

Choice of Model Compounds

Two categories of organofluorine compounds with particularly useful properties are the
fluorinated sulfonates and the fluorinated carboxylates. Perfluorinated sulfonates and
perfluorinated carboxylates are used as industrial surfactants and as catalysts in

synthetic chemistry. Perfluorooctane sulfonate (PFOSA; C8F17SO3Na) has excellent

2

chemical and thermal stability and is important commercially as a surfactant and as a
precursor of other fluorinated surfactants and pesticides [l]. Shorter chained
perfluorinated compounds, such as trifluoromethane sulfonate (TFMS; CF3SO3Na), are
used as oligomerization or polymerization catalyst. TFMS is one of the strongest acids
known, has great thermal stability, does not release fluoride in the presence of strong
nucleophiles, and resists both oxidation and reduction [25]. Fluorinated carboxylates of
industrial significance include perfluorooctanoic acid (PFOA), perfluorodecanoic acid
(PFDA), and trifluoroacetic acid (TFA). In addition, monofluoroacetate (MFA) is used

as a potent pesticide throughout the world.

PFOSA, TFMS and their partially fluorinated analogues difluoromethane sulfonate
(DFMS; CHFst3Na), lH,lH,2H,2H-perfluorooctane sulfonic acid (H-PFOSA;
C5F13C2H4SO3H), and 2,2,2-trifluoroethane sulfonic acid (TES', CF3CH2503H) were
selected as model compounds to represent the fluorinated sulfonates. MFA,
difluoroacetate (DFA), and TFA were chosen as model compounds for the fluorinated

carboxylates.

Hypotheses

The primary objective of this research was to study the biodegradability of non-volatile
fluorinated compounds. Two major classes of fluorinated molecules were investigated:

fluorinated sulfonates and fluorinated carboxylates. The following hypotheses were

evaluated:
1. Perfluorinated sulfonates and carboxylates are refractory.
2. Hydrogen-substituted fluorinated sulfonates can be utilized as sulfur

sources under aerobic and sulfur-limiting conditions.

3

3. Hydrogen-substituted fluorinated carboxylates can be utilized as carbon
sources under aerobic and denitrifying conditions.

4. Biodegradation of hydrogen-substituted fluorinated sulfonates is linked
to sulfur assimilation.

5. The capacity for transformation of hydrogen-substituted fluorinated

sulfonates and carboxylates is widely dispersed in nature.

Organization of Thesis

Chapter 2 provides a comprehensive review of research on the fate and effects of
organofluorine compounds. The review summarizes research on the fate of various
fluorinated compounds and the effects that these compounds and their metabolic
byproducts have on animals, plants, and microorganisms. In addition, the review
identifies areas that need additional attention. Chapter 3 evaluates the biodegradability
of representative fluorinated sulfonates using Pseudomonas sp. strain D2, an isolate
capable of completely defluorinating DFMS under sulfur-limiting and aerobic
conditions. Chapter 4 investigates the physiology of transformation of sulfur-
containing organofluorine compounds in greater detail using Pseudomonas sp. strain
D2. Results from Chapter 3 established that a structural or molecular limitation to
transformation occurs when the alpha-carbon of fluorinated sulfonates is completely
fluorinated (TFMS and PFOSA), while complete transformation (DFMS) or at least
partial transformation occurs when the alpha-carbon has a hydrogen atom (TES and H-
PFOSA). Chapter 4 provides additional insight into factors that affect transformation of
these compounds. In whole cell experiments oxygen was required for growth and
transformation of DFMS. While DFMS is not utilized as a source of carbon and
energy, it is used as a source of sulfur under sulfur-limiting conditions. Defluorination

of DFMS and other fluorinated sulfonates is linked to a sulfur-scavenging system that is

4

active when both a carbon source (glucose) and a nitrogen source (ammonium) are
present in excess. Inhibition studies with other sulfur sources establish a non-
competitive pattern of inhibition, suggesting that the sulfur released by DFMS

transformation is assimilated through existing sulfur assimilation pathways.

To generalize the above findings, the defluorination ability of other bacteria and yeast
were evaluated under aerobic and sulfur-limiting conditions (Chapter 5). Several
phylogenetically related Pseudomonads, Bacillus subtilis, and Escherichia coli
degraded at least one of the tested sulfur-containing organofluorine compounds.
Saccharomyces cerevisiae was not able to transform any, suggesting that the
mechanism may be specific to prokaryotes. In addition, H-PFOSA degraded in three

different soil types, but not in aquatic samples.

MFA was selected as a model compound for this work. Pseudomonads and other
bacteria, as well as some fungi, grow with MFA as a carbon source aerobically, but
transformation under anaerobic conditions was not previously evaluated [12, 13, 14, 15,
16, 18, 19, 26, 30]. The first step in aerobic degradation of MFA is hydrolytic attack of
the carbon-fluorine bond yielding glycolic acid [12]. Chapter 6 provides evidence that
many of the same organisms are also capable of growth on and defluorination of MFA
under denitrifying conditions. In addition, aerobic and anaerobic degradation of MFA
is demonstrated for a new MFA-degrading isolate, Bradyrhizobium sp. strain M7. A
hydrolytic mechanism is proposed for both aerobic and anaerobic conditions based

upon production of glycolic acid intermediate.

REFERENCES

Abe, T., and S. Nagase. 1982. Electrochemical fluorination (Simons process) as a
route to perfluorinated organic compounds of industrial interest, pp. 19-44. In R.
E. Banks (ed.), Preparation, properties, and industrial applications of
organofluorine compounds. John Wiley & Sons, New York.

Banitt, E. H., W. E. Coyne, K. T. McGurran, and J. E. Robertson. 1974.
Monofluoromethanesulfonanilides. A new series of bronchodilators. J. Med.
Chem. 17:116-120.

Cartwright, D. 1994. Recent developments in fluorine-containing agrochemicals,
pp. 237-257. In R. E. Banks, B. E. Smart, and J. C. Tatlow (ed.), Organofluorine
chemistry: Principles and commercial applications. Plenum Press, New York.

Clarke, D. D. 1991. Fluoroacetate and fluorocitrate: Mechanism of action.
Neurochemical Research 16: 1055-1058.

Commission, Great Lakes Fishery 1985. TFM vs. the sea lamprey: A
generation later. Great Lakes Fish. Corn. Spec. Pub. 85-6.

Deocampo, N. D., B. L. Upham, and J. E. Trosko. 1996. The role of gap
junction communication in the toxicity of quadricyclane, perfluorodecanoic acid,
perfluorooctanoic acid and jet fuels JP-8 and JP-4. Fundam. Appl. Toxicol.
(suppl) 30: 1065.

Elliot, A. J. 1994. Chlorofluorocarbons, pp. 145-157. In R. E. Banks, B. E.
Smart, and J. C. Tatlow (ed.), Organofluorine chemistry: Principles and
commercial applications. Plenum Press, New York.

EPA. 1989. Pesticide Fact Sheet on Sulfluramid. EPA. 205 (540/FS-89-060).

Fielding, H. C. 1979. Organofluorine surfactants and textile chemicals, pp. 214-
232. In R. E. Banks (ed.), Organofluorine chemicals and their industrial
applications. Ellis Horwood Ltd., Chichester.

10.

ll.

12.

l3.

14.

15.

16.

17.

18.

19.

20.

6

Filler, R. 1993. Fluoromedicinal chenristry - An overview of recent
developments, pp. 1-23. In R. Filler, Y. Kobayashi, and L. M. Yagupolskii (ed.),
Organofluorine compounds in medicinal chemistry and biomedical applications.
Elsevier, Amsterdam.

Gadelhak, G. G., Dissertation, Michigan State University (1992).

Goldman, P. 1965. The enzymatic cleavage of the carbon-fluorine bond in
fluoroacetate. J. Biol. Chem. 240:3434-3438.

Goldman, P. 1971. Enzymology of carbon-halogen bonds, pp. 147-165. In (ed.),
Degradation of synthetic organic molecules in the biosphere. National Academy
of Sciences, Washington DC.

Goldman, P., and G. W. A. Milne. 1966. Carbon-fluorine bond cleavage; 11.
Studies on the mechanism of the defluorination of fluoroacetate. J. Biol. Chem.
241:5557-5559.

Goldman, P., G. W. A. Milne, and D. B. Keister. 1968. Carbon-halogen bond
cleavage; III. Studies on bacterial halidohydrolases. J. Biol. Chem. 243:428-434.

Kelly, M. 1965. Isolation of bacteria able to metabolize fluoroacetate or
fluoroacetamide. Nature (London) 208:809-810.

Kissa, E. 1994. Fluorinated surfactants: synthesis, properties, and applications.
Marcel Dekker, Inc, New York.

Meyer, J. J. M., N. Grobbelaar, and P. L. Steyn. 1990. Fluoroacetate-
metabolizing Pseudomonad isolated from Dichapetalum cymosum. Appl. Environ.
Microbiol. 56:2152-2155.

Meyer, J. J. M., and D. O'Hagan. 1992. Conversion of 3-fluoropyruvate to
fluoroacetate by cell-free extracts of Dichapetalum cymosum. Phytochemistry
31:2699-2701.

Moore, G. G. I. 1974. Sulfonamides with antiinflarnmatory activity, pp. 159-176.
In R. A. Scherrer, and M. W. Whitehouse (ed.), Antiinflammatory agents:
Chemistry and pharmacology. Academic Press, New York.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

7

Moore, G. G. I. 1979. Fluoroalkanesulfonyl Chlorides. J. Org. Chem. 14:1708-
1711.

Peters, R. 1972. Some metabolic aspects of fluoroacetate especially related to
fluorocitrate, pp. 55-76. In (ed.), Carbon-fluorine compounds: Chemistry,
biochemistry, and biological activities (A Ciba Foundation Symposium).
Associated Scientific Publishers, Amsterdam.

Rao, N. S., and B. E. Baker. 1994. Textile finishes and fluorosurfactants, pp.
321-336. In R. E. Banks, B. E. Smart, and J. C. Tatlow (ed.), Organofluorine
chemistry: Principles and commercial applications. Plenum Press, New York.

Schnellmann, R. G., and O. M. Randall. 1990. Perfluorooctane sulfonamide: a
structurally novel uncoupler of oxidative phosphorylation. Biochemica Et
Biophysica Acta 1016:344-348.

Stang, P. J., and M. R. White. 1983. Triflic acid and its derivatives.
Aldrichimica Acta 16:15-22.

Tonomura, K., F. Futai, O. Tanabe, and T. Yamaoka. 1965. Defluorination of
monofluoroacetate by bacteria; Part 1. Isolation of bacteria and their activity of
defluorination. Agr. Biol. Chem. 29:124-128.

Trepka, R. D., et al. 1974. Synthesis and herbicidal activity of fluorinated N-
phenylalkanesulfonamides. J. Agr. Food Chem. 22: 1111-1119.

Upham, B. L., 1996, personal communication.

Vander Meer, R. K., C. S. Lofgren, and D. F. Williams. 1985. Fluoroaliphatic
sulfones: a new class of delayed-action insecticides for control of Solenopsis
invicta. J. Econ. But. 78: 1 190-1 197.

Walker, .1. R. L., and B. C. Lien. 1981. Metabolism of fluoroacetate by a soil
Pseudomonas sp. and Fusarium solani. Soil Biol. Biochem. 13:231-235.

CHAPTER 2

F LUORINATED ORGANICS IN THE BIOSPHERE

INTRODUCTION

Research investigating the environmental fate of halogenated compounds has largely
focused on brominated and chlorinated organics. Fluorinated organics have received
less attention because fewer are regulated, their measurement in environmental samples
is generally more difficult, and they are perceived as more inert biologically and
therefore less likely to have an impact on human health or the environment. Of course,
the perception of "inertness" and its environmental significance are debatable: inert
molecules tend to persist and accumulate, and they are more difficult to remediate. In
addition, several fluorinated organics are subject to at least limited biotransformation
under appropriate environmental conditions. Moreover, organofluorine molecules
actually do exhibit significant biological effects, as inhibitors of enzymes, cell-cell
communication, membrane transport, and processes for energy generation [16, 19, 23,

37, 77, 85, 105].

The chemistry of organofluorine molecules is unique because of the properties of
fluorine. The fluorine atom has a van der Waals radius of 1.47 A, a size more
comparable to that of oxygen (1.52 A) than to that of the other halogens (chlorine, 1.8
A; bromine, 1.95 A; and iodine, 2.15 A). Fluorine was once thought to be similar in
size to hydrogen (1.2 A), but it is now considered isosterically similar to a hydroxyl
group [90]. Compared to other halogens, fluorine is extremely electronegative having

an electronegativity of 4.0 compared to an electronegativity of 3.0 for chlorine, 2.8 for

9

bromine, and 2.5 for carbon [76]. This high electronegativity confers a strong polarity
to the carbon-fluorine bond [40]. The carbon-fluorine bond also has one of the largest
bond energies in nature (105.4 kcal/mol); other carbon-halogen bond energies are
similar to those of common metabolic intermediates [76]. The "unnatural" strength of
carbon-fluorine bonds confers unusual stability. In fact, many fluorinated
agrochenricals capable of enzyme inhibition are fluorine-stabilized analogues of the
natural enzyme substrate. A dramatic illustration of the strength and stability of the C-F
bond is monofluoroacetate, which can withstand boiling with 100% sulfuric acid
without any defluorination [82]. For many man-made fluorinated organics, such as the
perfluorinated organics, stability is probably related to the fact that the molecular

structure is unlike anything currently known in nature.

Fluorine is the most abundant halogen in the earth’s crust and ranks 13th in abundance
among all elements [75]. This may explain instances of natural organofluorine
production. The best known of these natural organofluorine compounds is
monofluoroacetate (MFA). MFA is produced by plants in the genus Dichapetalum, as
well as Palicourea marcgravii, Acacia georginae, Gastrolobium grandiflorum, and
Oxylobium species [48]. The West African plant, Dichapetalum toxicarium, also
produces m-fluorooleic acid, w—fluoropalmitic acid, and possibly w-fluorocaprate and
m—fluoromyristate [93]. Certain fungi also produce fluorinated organics; Streptomyces
clavus and Streptomyces cattleya produce the fluorine-containing antibiotic
nucleocidin and 4-fluorothreonine respectively [48, 93, 94]. Streptomyces cattleya is
also capable of producing monofluoroacetate [94]. Finally, production of CFC-11,
CFC-12, CFC-113, HCFC-21, HCFC-22, tetrafluoroethylene, and
chlorotrifluoroethylene has been reported in volcanic gases and drill wells [44, 51]. All

of the known biologically-produced fluorinated organics contain only one carbon-

10

fluorine bond. This contrasts with many man-made fluorinated organics, which often

contain many fluorine substituents and may even be fully fluorinated.

Because of their many useful properties, the number of man-made fluorinated organics
has dramatically increased over the past few years. According to a report from
Business Communications Company, Inc., sales of fluoropolymers (including
surfactants, textile finishes, fluoroelastomers, and polymer resins) were expected to
increase from $1.35 billion in 1994 to $1.76 billion by 1999 [12]. Tables 2.1 and 2.2
list several examples of aliphatic and aromatic fluorinated compounds. Representative
structures of some aromatic fluorinated compounds are illustrated in Figure 2.1. These
or related compounds have been used in aerosol propellants, surfactants, refrigerants,
plastics, anesthetics, herbicides, insecticides, rodenticides, lampricides, plant growth
regulators, medicines, adhesives, fire retardants, and even blood substitutes [4, 7, 15,
16, 26, 31, 32, 52, 54, 63, 67, 68, 69, 78, 92, 98, 99, 106, 115]. Synthesis is
accomplished using classical chemical or electrochemical processes, although

production of novel fluorochemicals using microbial pathways is also possible [79].

11

Cl

Diflubenzuron

CF3 CHzCHNHCH2CH3°HCl
or

ReduxTM
(dexfenfluramine hydrochloride)

NHCOCH3
H 3C NO2

NHS02CF3
CH3
Mefluidide

H

CF3
N02

3-Trifluoromethyl-4-nitrophenol

“1.13
,C=C co CHr‘Q’
F C

Ffi-Q HCHzCHzNHCH3-HC1

Prozac®
(fluoxetine hydrochloride)

NO2
1: c ,CHZCHZCH3
—< '>— It
3 crtzcuzcn3
no2

Trifluralin

moo-SF

dichloro(trifluoromethyl)
difluorodiphenylmethane

Series of chlorinated homologues
(1-5 chlorines)

Tefluthrin

Figure 2.1. Examples of aromatic fluorinated compounds.

12

The environmental fate of fluorinated organics depends upon the structure of the
molecule. Although some generalizations are possible, research is needed before
reliable predictions of fate will be possible for many compounds. Broadly speaking,
fluorinated organics can be classified as either volatile or nonvolatile. Volatile
fluorinated organics consist primarily of partially or completely fluorinated alkanes,
ethers, or amines. Fully halogenated fluorinated organics have very long lifetimes in
the atmosphere and can migrate to the stratosphere where they are destroyed by ozone
and photolysis [1, 66, 80]. By contrast, volatile fluorinated organics containing one or
more hydrogen atoms are susceptible to oxidation by hydroxyl radicals in the
troposphere, yielding fluoride, chloride, and partially oxidized organic species, the most
significant of which is trifluoroacetic acid (TFA) [100, 109]. TFA is also produced

industrially, as are many other commercially important nonvolatile fluorinated organics.

13

Table 2.1. Examples of fluorinated aliphatic compounds and their applications.

 

 

Compound Molecular Formula CAS # Application
Volatile
CFC-l l CFCl3 75-69-4 Refrigerant
CFC-12 Cl32(313 75-71-8 Refrigerant
HFC-134a CF3CH2F 81 1-97-2 Refrigerant
HCFC-22 CHF2C1 75-45-6 Refrigerant
Methoxyflurane CHC13CF20CH3 76-38-0 Anaesthetic
Halothane® CF3CHClBr 151-67-7 Anaesthetic
Perfluorotributyl-amine (C4F9)3N 311-89-7 Foam blowing
agent
Nonvolatile
Carboxylic Acid
Monofluoroacetic acid CHZFC02H 62-74-8 Pesticide
Trifluoroacetic acid CF3C02H 76-05-1 Reagent
Perfluorooctanoic acid C71:15C02H 335-67-1 Surfactant
Sulfonic Acid
Trifluoromethane CF3SO3H 1493-13-6 Catalyist/Reagent
sulfonic acid
Perfluorooctane C8F17SO3H 2795-39-3 Surfactant
sulfonic acid
1H, 1 H,2H,2H- C6F13CH 2CH2$O3H 27619-97-2 Surfactant
perfluorooctane
sulfonic acid
Sulfonamide
N -acetic-N-ethy1 (C 8F 17SO2N(CH2COOH) 2991 -50-6 Surfactant
perfluorooctane (CH2CH3))
sulfonamide
Sulfluramid (C8F17802NH(CH2CH3)) 4151-50-2 Insecticide
Miscellaneous
Polytetrafluoroethylene (~(CF2CF2)n-) 9002-84-0 Teflon® polymer
Perfluoropolyether (-(CF(CF3)CF20)n-) 60164-51-4 Lubricant
Zony1® Alcohol (C8F17CH2CH20H) 678-39-7 Surfactant

14

Table 2.2. Examples of fluorinated aromatic compounds and their applications.

 

 

Compound CAS # Application

Acifluorfen 50594-66-6 Herbicide; CF3-type
Diflubenzuron 35367-38-5 Insecticide

Fluometuron 2164-17-2 Herbicide; CF3-type
Fluorobenzoates N/A Pharmaceutical and agricultural
Fluorouracil 51-21-8 Chemotherapeutic agents
Fluotrimazole 3 125 1 -03-3 Fungicide

Fluoxydine 671-35-2 Chemotherapeutic agents
Flurprimidol 56425-91-3 Plant growth regulator; OCF3-type
Flutriafol 76674-21-0 Fungicide

Mefluidide 53780-34-0 Herbicide; CF3SO3-type
Nucleocidin 2475 1 -69-7 Antibiotic

Tefluthrin 79538-32-2 Insecticide

Trifluralin 1582-09-8 Herbicide; CF3-type
Trifluorobenzoates N/A Pharmaceutical and agricultural

Over the past 15 years, the number of fluorine containing agricultural chemicals have

grown from 4% to approximately 9% of all agrochemicals and have increased in

number faster than non-fluorinated agrochemicals [16]. These compounds are primarily

used as herbicides (48%), insecticides (23%), and fungicides (18%) [16]. A summary

of the fluorinated agrochemical usage for 1995 in the United States is provided for in

Table 2.3.

Given the widespread production and use of fluorocarbons, it is perhaps not surprising

that organofluorine has been detected in the blood of individuals from the general

public as well as industrial workers [8, 38]. For workers handling fluoroorganics,

organofluorine levels of 1.0 to 71 ppm have been reported in the blood serum [8].

Individuals who have not been exposed to industrial fluorochemicals have had organic

fluorine concentrations from 0.0-0.13 ppm. However, it is unclear whether trace

amounts of organic fluorine compounds found in human blood samples are from natural

or industrial sources.

15

In the following sections volatile and nonvolatile fluorinated compounds are discussed
and a current assessment of the fate and the effect of these molecules in the biosphere is

provided.

Table 2.3. Fluorinated chemical application for crop protection in US, 1995.
Calculated from the Agricultural Chemical Usage - 1995 Field Crops Summary,
published by the USDA. (Note: this data only includes applications to corn, cotton,
soybeans, and wheat for major producing states)

 

 

 

Fluorinated Agrochemical Total Applied Type of Pesticide
(1000 kg)
Acifluorfen 675 Herbicide
Bifenthrin 43 Insecticide
Cyfluthrin 59 Insecticide ‘
Diflubenzuron 56 Insecticide
Ethalfluralin l 19 Herbicide
Fluazifop-P-butyl 200 Herbicide
Flumetsulam 48 Herbicide
Fluometuron 1273 Herbicide
Fomesafen 286 Herbicide
Lactofen 1 20 Herbicide
Norflurazon 470 Herbicide
Oxyfluorfen 20 Herbicide
Primisulfuron 1 9 Herbicide
Tefluthrin 1 3O Insecticide
Trifluralin 6570 Herbicide
Total 10,088

FATE AND EFFECTS OF VOLATILE FLUORINATED ORGANICS

Chlorofluorocarbons

Volatile fluorinated organics include the chlorofluorocarbons (CFCs), the

hydrochlorofluorocarbons (HCFCs), the hydrofluorocarbons (HFCs), halothane,

fluorinated ethers, and fluorinated amines. Chemical formulas, atmospheric lifetime

16

estimates, and production values for some of these compounds are provided in Table
2.4. CFCs have long been used as refrigerants and aerosols in industrial processes and
domestic products. In 1974, CFCs were implicated as agents of depletion of
stratospheric ozone by Molina and Rowland [66] and more recently as contributors to
global warming [33]. As a result, worldwide production of the CFCs is being phased
out under the terms of the Montreal Protocol and its amendments. Nevertheless, CFCs
continue to be released into the environment due to past production and continued use.
The major sink for CFCs is expected to be stratospheric oxidation. In aerobic aquatic
environments, CFCs are recalcitrant, but they are transformed in anaerobic soils and
sediments, as well as anaerobic aquatic environments [22, 50, 55, 56, 57, 58, 88, 91].
The expected anaerobic degradation products are hydrochlorofluorocarbons (HCFCs)

and hydrofluorocarbons (HFCs).

Hydrochlorofluorocarbons and hydrofluorocarbons

The phase out of CFC production and use has inspired a major research effort to assess
the environmental fate of the CFC alternatives. The HCFC and HFC alternatives are
one- and two-carbon aliphatics, similar in structure and physical properties to the CFCs,
but containing one or more hydrogen atoms. Tropospheric oxidation is expected to be
the most significant sink for the HCFCs and the HFCs. The presence of hydrogen
makes HCFCs and HFCs more susceptible to tropospheric oxidation than the CFCs and
therefore less likely to enter the stratosphere. In the troposphere, HCFCs and HFCs are
oxidized by hydroxyl radicals, yielding HF, C02, and HCl (in the case of HCFCs) and
in some cases trifluoroacetic acid (TFA) [1, 36]. Some volatile fluorinated organics
(HCFC-134a, HFC-123, HFC 143a, and halothane) are oxidized by hydroxyl radicals to
TFA [62, 109, 110]. It is likely that TFA is produced biochemically when these

compounds are oxidized by monooxygenase activities, such as cytochrome P450 in the

17

liver and methane monooxygenase [17, 59, 70]. Rainfall is believed to be the primary

mechanism for removal of TFA from the atmosphere [87, 100, 101, 110].

A minor sink for HCFCs and HFCs is biochemical reduction or oxidation in aquatic
systems. Lesage et al. [56] reported reductive transformation of HCFC-123a to
chlorotrifluoroethane (HCFC-133 and HCFC-l33b) under methanogenic conditions.
Oremland et al. [73] have also reported reductive dechlorination of HCFC-123
(CF3CHC12) to chlorotrifluoroethane under anaerobic conditions and degradation of
HCFC-21 (CHFC12) under both aerobic and anaerobic conditions. The products of
HCFC-21 oxidation in these experiments were not investigated [73]. Oxidative
transformations mediated by monooxygenases are known, but these reactions proceeded
slowly compared to the reactions with analogous chlorinated compounds. Oxidative
pathways are hypothesized for the transformation of HFC-134a (1,1,1,2-
tetrafluoroethane) by rat liver microsomes [70]. DeFlaun et a1. [21] reported oxidative
transformation of HCFC-21 (CHFC12), HCFC-l4lb (CFC12CH3), HCFC-131
(CFC12CH2C1), and HFC-143 (CHF2CH2F) by Methylosinus trichosporium OB3b.
HCFC-123 (CF3CHC12), HCFC-142b (CF2C1CH3), HFC-134a (CF3CH2F), HFC-134
(CHFzCHFz), and CFC-ll (CFC13) were not degraded. Chang and Criddle [17]
observed slow and limited oxidation of HCFC-123, HFC-134a, and HCFC-142b by a
defined methanotrophic consortium. They also reported oxidation of HCFC-22
(CHF2C1), with indirect evidence of product toxicity for HCFC-22 transformation and

production of TFA from HFC-134a.

18

Table 2.4. Fluorocarbon production worldwide

 

 

Compound Molecular Estimated Total Cumulative Cumulative
Formula Atmospheric World Production for
Lifetime21 (metric tons)b years
(years)
HCFC-22 CHFzCl 6.7 3,602,365 1 14,536 1970 to 1994
HFC-134a CF3CFH2 14 85,717 1 1,194 1990 to 1994
HCFC-l41b CFClzCH3 7.1 139,382 1 1,479 1990 to 1994
HCFC-142b CF2C1CH3 17.8 193,324 1 1,177 1981 to 1994
CFC-11 CFC13 60 8,583,266 1 15,398 1931 to 1994
CFC-12 CF2C12 105 11,195,032 1 19,542 1931 to 1994
CFC-113 CF2C1CFC12 90 2,244,779 1 8,797 1980 to 1994
CFC-114 CF2C1CF2C1 185 185,111 1 923 1980 to 1994
CFC-115 CF3CF2C1 380 167,459 1 684 1980 to 1994

a Wallington EST 28:320A-326A, and [26].
b AFEAS Report: Production, sales, and atmospheric release of fluorocarbons through
1994 [3].

Fluorinated Anesthetics

Methoxyflurane (2,2—dichloro-1,l-difluoroethyl methyl ether) is used very little in
human medicine, but it is used in some veterinary applications [30]. The environmental
fate of methoxyflurane is not known, but likely involves tropospheric oxidation by
hydroxyl radicals. In rats, methoxyflurane is metabolized by two different pathways
[61]. The first is an o-demethylation reaction with release of fluoride to form
dichloroacetic acid. The second is hydroxylation at the B-carbon with release of

chloride and the formation of methoxydifluoroacetic acid.

Halothane (l-bromo-l-chloro-2,2,2-trifluoroethane), a bromofluoroalkane is used as a

human anesthetic and as a fire extinguishing agent. It is oxidized in the troposphere to

l9

TFA. In fact, TFA currently detected in the environment is thought to have largely

originated from tropospheric oxidation of halothane [62].

FATE AND EFFECTS OF NONVOLATILE F LUORINATED ORGANICS

Trifluoromethyl substituted aromatics

The majority of the fluorinated organics used in agricultural applications are
trifluoromethyl-substituted aromatics (54.5%) [16]. Of these, trifluralin (1,1,1-trifluoro-
2,6-dinitro-N,N-dipropyl-p-toluidine), a pre-emergent herbicide, is the most commonly
used in the United States. Williams et al. [111] observed reduction of the nitro groups
of trifluralin by rumen microflora, but no loss of the trifluoromethyl group and no
cleavage of the ring was observed. Trifluralin had little effect on rumen microbe
populations, as determined by volatile fatty acid production and endogenous gas

evolution [1 1 1].

The pre-emergent herbicides norflurazon and fluridone, both trifluoromethyl-containing
aromatics, inhibit carotenoid biosynthesis [16]. This inhibition causes overoxidation of
chlorophyll and subsequently the loss of the ability to photosynthesize. Flurprimidol, a
fluorine containing plant growth regulator, interferes with Gibberellin (plant growth
hormone) biosynthesis [16]. Fluotrimazole and the structurally similar flutriafol, are
fungicides that weaken the cell membrane by blocking the carbon-1,4 alpha

demethylation step in ergosterol biosynthesis [114].

Several trifluoromethyl-substituted aromatics have been detected in sediments and in
fish from the Niagara River and Lake Ontario [52]. Various trifluoromethyl-substituted

polychlorinated biphenyls, dichloro(trifluoromethyl)-benzophenone, and

20

dichloro(trifluoromethyl)-difluorodiphenylmethane were found. These compounds
originated at a dump site containing 55,000 tons of halogenated waste, of which 10%
was from the production of 4-chloro-(trifluoromethyl)benzene [52].
Dichloro(trifluoromethyl)-difluorodipheny1methane was present in fish at
concentrations as high as 850 ng/g and in sediments from a creek near the dump site at
concentrations as high as 35,000 ng/g. The trifluoromethyl-substituted PCBs are
believed to bioaccumulate and partition into the sediment more effectively than non-
fluorine containing PCBs based on octanol/water partition coefficients (log Kow from

6.8 to 9.0) [52].

The larnpricide, 3-trifluoromethyl-4-nitrophenol, has been used since 1958 to combat
the sea lamprey problem in the Great Lakes basin. It is released into rivers and streams
containing lamprey larvae. From 1991 through 1995 the five year average use of 3-
trifluoromethyl-4-nitrophenol was 40,823 kg/year (active ingredient) [20]. Carey and
Fox [14] observed defluorination of 3-trifluoromethyl-4-nitrophenol by photolysis, but
reported that only 15% was degraded assunring that complete defluorination would

yield 3 moles of fluoride.

Fluorinated aromatics

Diflubenzuron (1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl) urea), a urea-based
larvicide, inhibits chitin synthesis and the molting process in a broad spectrum of
insects. Four fungal isolates (F usarium sp., Penicillium sp., Rhodotoruia sp., and
Cephalosporium sp.) are capable of degrading diflubenzuron [89]. The proposed
pathway for this transformation is through 4-chlorophenylurea and 2,6-difluorobenzoic
acid. Although 4-chlorophenylurea is completely metabolized, 2,6-difluorobenzoic acid
is persistent and lethal to soil microbes [89]. Several investigators have also shown that

diflubenzuron affects non-target aquatic organisms [112].

21
Several bacterial isolates defluorinate fluorobenzoic acids [28, 29, 40, 71, 83, 84, 86].
The general pathway for degradation of fluorobenzoic acids is attack by a 1,2-
dioxygenase followed by decarboxylation to yield fluorocatechols. These catechols are

then subject to ring cleavage followed by defluorination.

Fluorinated sulfonamides

Sulfluramid (N-ethylperfluorooctane sulfonamide), a fluorinated insecticide used to
control cockroaches and ants, is deethylated to perfluorooctane sulfonamide in rats,
dogs, and rabbit renal mitochondria [5, 45, 60, 85]. Perfluorooctane sulfonamide has
not been shown to undergo further transformation, but will most likely be converted to
perfluorooctane sulfonic acid (PFOSA; C3F17SO3H) which is believed to be highly
recalcitrant. Schnellman et al. [85] demonstrated that perfluorooctane sulfonamide and
sulfluramid are potent uncouplers of oxidative phosphorylation in rabbit renal
mitochondria. They also reported that the metabolite perfluorooctane sulfonamide was
three times more potent than sulfluramid at uncoupling oxidative phosphorylation.
Other fluorinated sulfonamides have demonstrated delayed action toxicity in red
imported fire ants [106]. This delayed action toxicity allows the insecticide to be
applied in baits which are taken back to the colony by foraging members that distribute

it throughout the ant colony, allowing for better control of ants.

Fluorinated sulfonic acid

Perfluorinated sulfonic acids are used as industrial surfactants and as catalysts
depending on their chain length. Trifluoromethane sulfonic acid (triflic acid;
CF 3SO3H) is an excellent oligomerization/polymerization catalyst. Triflic acid is one

of the strongest organic acids known, has great thermal stability, does not release

22
fluoride in the presence of strong nucleophiles, and resists both oxidation and reduction
[92]. Perfluorooctane sulfonic acid (PFOSA) also has excellent chemical and thermal
stability. PFOSA is important commercially as a surfactant and as a precursor of other
fluorinated surfactants [2]. Unfortunately, production values for most of the
perfluorinated compounds are considered proprietary information by manufacturers and
are not disclosed to the public [34]. Perfluorooctane sulfonic acid and triflic acid are
resistant to biological attack. However, a surfactant sirrrilar to PFOSA, lH,lH,2H,2H-
perfluorooctane sulfonic acid (H-PFOSA), was partially degraded by a Pseudomonad
under aerobic and sulfur-limiting conditions yielding 1-2 moles fluoride per mole of H-
PFOSA (Chapter 3). The degradation of H-PFOSA produced several volatile
fluorinated compounds that have not yet been identified. 2,2,2-Trifluoroethane
sulfonate (TES) was also partially degraded with equimolar release of fluoride (Chapter
3). Another fluorinated sulfonate, difluoromethane sulfonate (DFMS; CHFZSO3Na),
was completely metabolized by this Pseudomonad yielding stoichiometric amounts of
fluoride (Chapter 3). Transformation of DFMS, TES and H-PFOSA was subsequently
observed with Bacillus subtilus and Escherichia coli (Chapter 5). However, E. coli was
not capable of utilizing H-PFOSA. Evidence of H-PFOSA degradation was also
observed in soil incubations. This and other work suggests that the transformation of

fluorinated sulfonates requires the presence of hydrogen.

Although PFOSA is resistant to metabolism, it is not biologically inactive. For
example, PFOSA was shown to inhibit gap junction intercellular communication (GJIC)
in rat liver epithelial cells cultured in vitro [105]. In addition, Gadelhak [37] showed
that perfluorooctane sulfonic acid was an uncoupler of phosphorylation in rat liver
mitochondria. Although PFOSA alone was not as potent of an uncoupler as

perfluorooctane sulfonamide, when PFOSA was ion-paired with various monoamines,

23

polyamines, and phospholipids, the effect of uncoupling was in some instances as high

as that of perfluorooctane sulfonamide [37].

Fluorinated alcohols

While some of the perfluorinated organics undergo limited biotransformation, none
undergo extensive defluorination. An example of a highly fluorinated molecule that has
shown some limited defluorination is lH,lH,2H,2H-perfluorodecanol. lH,lH,2H,2H-
perfluorodecanol was metabolized first to 2H,2H-perfluorodecanoic acid and then to
perfluorooctanoic acid (PFOA) in adult male rats [47]. Hagen et a1 [47] suggest that the
overall reaction is production of PFOA with the release of 2 moles of fluoride per mole
of 1H,1H,2H,2H-perfluorodecanol. PFOA is metabolically stable in rats [72] and has

been found in the blood serum of humans [47].

Fluorinated carboxylic acids

Monofluoroacetate (MFA) is one of the most toxic substances known, based on a lethal
dose (LD50) of 0.7-2.1 mg/kg for man [6]. Its toxicity is due to "lethal synthesis" of
fluorocitrate which inhibits the aconitase enzyme of the Kreb's cycle [77] although
recent investigations implicate fluorocitrate as a "suicide" substrate instead of a
competitive inhibitor [19]. Given that certain plants can produce MFA, it is not
surprising that several microorganisms can metabolize MFA. Pseudomonads and other
bacteria, as well as some fungi, have been shown to grow with MFA and
monofluoroacetamide (a systemic pesticide and rat poison) as a carbon source
aerobically [39, 40, 41, 42, 53, 64, 65, 97, 108]. The first step in degradation of MFA
has been shown to be a hydrolytic attack of the carbon-fluorine bond yielding glycolic

acid [39]. Many of the same organisms are also capable of growth on and

24

defluorination of MFA under denitrifying conditions (Chapter 6). Gregg et al. [43]
demonstrated defluorination of MFA by genetically modified rumen bacteria
Butyrivibrio fibrisolvens. This was accomplished by transferring the plasmid
responsible for MFA defluorination from Moraxella sp. strain B into B. fibrr'solvens.
We know of only one other report of anaerobic MFA degradation. Visscher et al. [107]
reported reductive dehalogenation of MFA to acetate, under methanogenic conditions,
but this transformation was not reproducible in subsequent investigations [74]. MFA
has been shown to inhibit methanogenesis in anaerobic digestor sludge, rumen fluid,
and freshwater mud [27]. Another fluorinated carboxylate, the herbicide flupropranate

(CHFzCFzCOzH), is persistent in soils [113].

Other fluorinated organic acids of industrial significance include perfluorooctanoic acid
(PFOA), perfluorodecanoic acid (PFDA), and trifluoroacetic acid (TFA). No evidence
of PFOA or PFDA transformation has been reported. Although, PFOA and PFDA are
not metabolized they have been found to inhibit gap junction intercellular
communication (GJIC) in rat liver epithelial cells and human kidney epithelial cells at
concentrations of 100 and 250 uM respectively [23]. The inhibition of GJIC has been
implicated in tumor promotion during carcinogenesis [49, 104, 116], teratogenesis

[103], and reproductive dysfunction [9, 102, 117].

Significant hydrolytic defluorination has so far been observed only for monofluorinated
molecules. Another biotransformation of importance is decarboxylation. Meyer and
O'Hagan have demonstrated decarboxylation of 3-fluoropyruvate to MFA by cell-free
extracts of D. cymosum [65]. In addition, Chauhan et a1 [18], have demonstrated
decarboxylation of TFA by Azoarcus tolulyticus TOL-4. An intensive international
effort is currently underway to explore the fate and effects of TFA, and some results of

this effort are summarized in the next section.

25

A CASE HISTORY - TRIFLUOROACETIC ACID

Three of the chlorofluorocarbon (CFC) alternatives, HCFC-123, HCFC-124, and HFC-
134a are expected to yield TFA as an atmospheric degradation product (Figure 2.2) [25,
100, 109, 110]. As a result, TFA is expected to become a ubiquitous low-level global
contaminant. Estimates based on 100% conversion of HCFC-123, and HCFC-124 and
20% conversion of HFC-134a to TFA predict 0.1 rig/L TFA in rainwater [13] although
it is possible that TFA levels in urban areas could reach levels as high as 2 to 20 ug/L
[87]. Average TFA concentrations of 50 pg/m3 in air, 100 ng/L in rain water, and 140
ng/L in surface waters have been reported [35]. TFA is concentrated in plant leaves and
it has the potential to accumulate in ecosystems such as saline lakes, vernal and
temporary rain pools, closed basin lakes, playa lakes, and aestival ponds and certain
prairie lakes that exhibit high evaporative potential [100, 101]. Environmental factors
such as climate, geology, topography, biota, and time are factors affecting
evapoconcentration of solutes. TFA adsorbs weakly to most soils, and, therefore, soils
are not expected to be a permanent sink for TFA. However, adsorption is favored in
low pH soils which are enriched with iron and aluminum oxides [24]. Photo-oxidation
of TFA-iron complexes by near-UV radiation in clouds and the ocean was dismissed
because of the low concentrations of iron in these environments [13]. Sinks for TFA
would include groundwater due to leaching from soils and vegetation due to
bioaccumulation [l3]. TFA can be degraded by the enzyme acetyl-CoA synthetase, but
rates are low even with high concentrations of enzyme and substrate (> 10 mM) [27].
As a result, this pathway is unlikely in the environment where TFA is at nM levels [27].
Hydrolysis of TFA is also an unlikely mechanism for degradation due to the extreme
stability of the carbon-fluorine bond and the shielding effect of three fluorine atoms on

the carbon atom.

26

Even small quantities of TFA in solution can influence the pH of unbuffered solutions
because TFA behaves like a strong inorganic acid at environmental pH [13].
Bioaccumulation in animals is thought to be unlikely due to a low octanol/water
partition coefficient (log Kow = -4.21) [13], but TFA can accumulate in plants through
root uptake. Estimates of the concentration factor give approximately 10-32 times the
soil concentration in plant leaves with virtually no degradation in the plant [13, 96].
Vascular plants seem to be affected by TFA when bioaccumulation through roots to

leaves occurs, however, little toxicity was shown for seed germination [96].

Toxicological tests have been performed on five species of algae. Three species of
microalgae (freshwater diatom Navicula, marine diatom, Skeletonema, and freshwater
blue-green Anabaena) showed no toxic effects from TFA concentrations up to 1000
mg/L. The freshwater green alga Chlorella sp. also showed no toxicity up to 1200
mg/L. A second freshwater green alga, Selenastrum capricornutum , exhibited toxicity
at concentrations as low as 0.36 mg/L [96]. Bott and Standley reported that TFA did
not significantly affect the metabolism of acetate by microbial communities at
environmentally expected concentrations [11]. However, Visscher et al. reported
inhibitory effects on methanogenic activity at TFA concentrations 2 luM [107]. TFA

appears to be non-mutagenic in bacteria [10, 81].

The LD 50 of TFA is reported to be between 200-400 mg/kg (oral exposure to rats), and
sodium TFA is only slightly toxic when administered intraperitoneally to mice with no
deaths at doses up to 5000 mg/kg [81]. Trifluoroacetic acid does not interfere with
homeostasis of rat liver epithelial cell by inhibiting gap junction intercellular

communication (GJIC) [105].

27

Although defluorination of monofluoroacetate by bacteria is well established, few
reports of biodegradation of trifluoroacetic acid are available. Visscher et al. [107]
reported reductive defluorination of TFA under methanogenic and sulfate-reducing
conditions. They report that TFA is sequentially defluorinated to difluoroacetic acid
(DFA), monofluoroacetic acid, and finally to acetic acid. Under aerobic conditions,
Visscher et al [107] observed production of fluorofonn (CHF3). However, these results
with TFA were not reproducible in subsequent experiments leading the investigators to
conclude that TFA is widely recalcitrant to biodegradation [74]. Chauhan et al. [18]
have demonstrated that Azoarcus tolulyticus TOL—4 is capable of decarboxylating TFA.
Cells were grown under denitrifying conditions with toluene as the carbon source and
then incubated aerobically without nitrate. No fluoride or volatile fluorinated products
were detected [18]. The fate of the trifluoromethyl moiety of TFA is unknown. This is
also true of most other trifluoromethyl-substinrted compounds. Photolysis of the
trifluoromethyl moiety has been reported for trifluoromethyl-benzoate and 3-
trifluoromethyl-4-nitrophenol [14, 95]. Taylor et al. [95] observed equimolar
concentrations of fluoride from trifluoromethyl-benzoate. Carey and Fox [14] observed
defluorination of 3-trifluoromethyl-4-nitrophenol by photolysis, but reported that only
15% was degraded assuming that complete defluorination would yield 3 moles of
fluoride. In both of these cases there appears to be only partial defluorination of the
trifluoromethyl moiety. Of particular interest is the fate of these trifluoromethyl grbups
and their photo-degradation products and the role of photolysis as a sink for these
compounds. In the absence of photolysis, it seems plausible that the trifluoromethyl
group remains intact during biotransformation of trifluoromethyl-substituted aromatics,

possibly contributing to the TFA load on the environment.

28

Figure 2.2. Biogeochemical cycling of organofluorine compounds.

29

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30

SUMMARY AND CONCLUSIONS

It is clear that fluorinated molecules are unique with regard to their physical, chemical,
and biological properties and they do not fit with the usual paradigm of chemicals of
environmental concern. For example, the fluorinated surfactants have distinctly
different characteristics to those of their hydrocarbon counterparts. Fluorinated
surfactants are simultaneously oleophobic and hydrophobic, but solubilizing moieties
such as carboxylic acids, sulfonic acids, phosphates, and quaternary ammonium groups
can change their solubility in water [4, 54]. In addition, the perfluorinated alkyl chain is
more "rigid" due to fluorine atoms on the molecule [46]. This rigidness almost
certainly interferes with molecule/enzyme interactions, protecting fluorocarbon
molecules from biological attack. Currently, little is known of the sorption/partitioning

prOperties of fluorinated organics.

Instances in which the carbon-fluorine bond is ruptured by direct attack are only known
for a rarely observed reductive defluorination of tri-, di-, and monofluoroacetate [107]
and for hydrolytic defluorination of monofluorinated organics [39]. Reductive
defluorination seems to require extreme and uncommon reducing conditions, and so far
has been observed only under methanogenic conditions that proved impossible to
replicate [74]. In addition, hydrolytic defluorination of carbon atoms with two or more
fluorine substituents appears to be too slow to be of environmental significance [27].
More often, transformation of highly fluorinated organics requires attack at functional
groups or bonds attached to the fluorinated moiety. Attack on adjacent functional
groups can be hydrolytic, oxidative, or reductive, and can result in decarboxylation,
desulfonation, deamination, and fluoride elimination.

Although there have been instances of partial defluorination of perfluoroalkyl chains,

complete mineralization in the biosphere has not been reported. The products of these

3]

partial transformations are of interest with regard to their lifetimes in the environment
and their effects on the biosphere directly or indirectly (see Figure 2.2). For example, it
has been shown that several nonvolatile compounds such as sulfluramid and
1H,lH,2H,2H-perfluorooctane sulfonic acid (Chapter 3 and 5) are transformed to
volatile fluorinated compounds [5]. In addition, volatile compounds such as HCFC-
123, HCFC-124, and HFC-134a are expected to yield TFA (a nonvolatile fluorinated
compound) as a tropospheric degradation product [25, 100, 109, 110]. The CFCs are
degraded in the stratosphere and are responsible for ozone depletion and global
warming. While several of the fluorinated agrochemicals are reported to "dissipate" in
soils, mineralization of these chemicals has not been demonstrated in most cases.
Similarly, the environmental fate of the trifluoromethyl group, which is utilized in many
fluorinated organic compounds, is largely unknown. The potential for TFA production
from these trifluoromethyl-substituted aromatics should be investigated. The
biogeochemical cycling of fluorinated compounds is just now being discovered and is

not yet clearly understood.

The stability that makes fluorinated compounds desirable for commercial use also
makes them potentially significant environmental contaminants due to their persistence.
Very little research into the toxicological effects of these compounds and their
byproducts on animals and plants has been done. Therefore, caution is warranted when
evaluating these compounds because although they may not be chemically or even

biochemically reactive, they may still be biologically active.

To successfully study the fate and the effects of fluorinated compounds in the
environment, sufficiently sensitive analytical methods need to be developed and
published. Currently, very few methods are available to measure or detect nonvolatile

fluorinated organics in environmental samples. In addition, many of the perfluorinated

32

organics are produced as mixtures of straight and branched homologues making
analysis even more challenging. Although traditional analytical methods for
determining hydrocarbon analogues have been used, most environmental samples have
co-eluting ions and metabolic products that confound analysis. Inorganic fluoride
measurement, by ion-selective electrode or ion chromatography, has been an effective
means of indirectly detecting transformation of these compounds, but this method
requires defluorination which may not occur with many perfluorinated compounds.
Destructive conversion of organofluorine compounds to inorganic fluoride ions can
provide information on the ratio of fluorine in the samples, but this method cannot
distinguish between different homologues. Another indirect method is monitoring of
oxygen consumption during aerobic incubation. Methane and carbon dioxide
production can be measured with anaerobic incubations. Again these methods fail to
answer key questions about the fate of fluorinated compounds. Real progress on these

fate issues may require synthesis of radiolabelled compounds.

It is likely that industrial, agricultural, domestic, and medical applications for fluorine
containing compounds will continue to increase in the coming years. As we continue to
use and manufacture molecules containing carbon-fluorine bonds, several questions
arise pertaining to the fate of such compounds. Are they recalcitrant in the environment
or do they possess substituents, such as the trifluoromethyl group, which persists as
mobile, recalcitrant functional groups? In which environments do they accumulate?
What are the products of biological or abiotic reactions? What is the fate of reaction
products? What is the toxicity and mutagenicity of such reaction products? How can
we accurately measure and detect these compounds in environmental samples? Clearly,
considerable research is needed to better understand the fate and effects of fluorinated

organics in the biosphere.

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14:817-832.

CHAPTER 3

DEFLUORINATION OF ORGANOFLUORINE SULFUR
COMPOUNDS BY PSEUDOMONAS SP. STRAIN D2

ABSTRACT

Little is known of the potential for biodegradation of sulfur containing fluorinated
surfactants. Difluoromethane sulfonate (DFMS, CHF2803Na) was chosen as a
representative model compound. A Pseudomonad (strain D2) was isolated that
completely defluorinated DFMS under aerobic and sulfur-limiting conditions in minimal
medium. Strain D2 was subsequently used to evaluate the potential for
biotransformation of the following fluorinated sulfonates: trifluoromethane sulfonate
(TFMS; CF3SO3Na), 2,2,2-trifluoroethane sulfonic acid (TES; CF3CHzSO3H),
perfluorooctane sulfonate (PFOSA; CgF] 7803 K), and 1H,]H,2H,2H-perfluorooctane
sulfonic acid (H-PFOSA; C6F13C2H4803H). Strain D2 was capable of utilizing those
compounds containing hydrogen (TES and H-PFOSA), but only partial defluorination
was observed. When TES was the sole sulfur source, one mole of fluoride was released
per mole of TES transformed. Transformation of H-PFOSA yielded one mole of
fluoride per mole of H-PFOSA when incubations were open to the atmosphere and 1.4
moles per mole when incubations were closed to the atmosphere. Eight volatile and
fluorinated byproducts of H-PFOSA were detected by GC/mass spectrometry, but
volatile transformation products were not detected for TES or DFMS. This is the first
report of biotic transformation of fluorinated sulfonates resulting in defluorination. These

findings suggest that the initial steps of transformation are linked to sulfur metabolism

45

46

with subsequent defluorination of the molecule. The results also suggest that hydrogen

substitution is required for transformation.

INTRODUCTION

The commercial use of organofluorine compounds has dramatically increased in recent
years. Many of these compound are used as propellants, surfactants, agrochemicals,
adhesives, refrigerants, fire retardants, and medicines [2, 3, 4, 5, 6, 7, ll, 14, 15, 16, 22,
25]. One category with particularly useful properties is the fluorinated sulfonates.
Perfluorinated sulfonates are used as industrial surfactants and as catalysts in synthetic
chemistry. Perfluorooctane sulfonate (PFOSA; C3F17SO3Na) has excellent chemical
and thermal stability and is important commercially as a surfactant and as a precursor of
other fluorinated surfactants and pesticides [1]. Shorter chained perfluorinated
compounds, such as trifluoromethane sulfonate (TFMS; CF3SO3H), are used as
oligomerization or polymerization catalyst. TFMS is one of the strongest acids known,
has great thermal stability, does not release fluoride in the presence of strong
nucleophiles, and resists both oxidation and reduction [19]. Difluoromethane sulfonate
(DFMS; CHFZSO3N a), 1H,1H,2H,2H-perfluorooctane sulfonic acid (H-PFOSA;
C6F13C2H4803H), and 2,2,2-trifluoroethane sulfonic acid (TES; CF3CH2803H) are
partially fluorinated analogues of the perfluorinated sulfonates. Because of the apparent
stability of these compounds and their potential for accumulation in the environment, it is
important to understand their environmental fate and the mechanisms by which they are
degraded. The biodegradability of TFMS, TBS, PFOSA, and H-PFOSA was evaluated
using a Pseudomonad that is capable of completely defluorinating DFMS under aerobic

and sulfur-limiting conditions.

47

MATERIALS AND METHODS

Media and chemicals. A defined rrrineral medium containing (in grams per liter):
glucose, 2.0; KZHPO4, 3.5; KH2P04, 2.0; NH4C1, 1.0; MgC12°6H20, 0.5; 0.15M
CaC12°2H20 stock, 1.0 ml/liter; trace elements stock I and II, 1.0 mllliter. Trace elements
stock 1 contained (in grams per liter): FeCl3, 1.36; CoC12-6H20, 0.2; MnC12-4H20,
0.122; ZnClz, 0.07; N azMoO4°2H20, 0.036; NiC1206H20, 0.12; B3(OH)3, 0.062;
CuC12-2H20, 0.017. Concentrated H2SO4 was added at 2.5 ml/L to the trace elements
solution 1. Trace elements solution 11 contained (in grams per liter): NazSeO3°5H20,
0.006; N aWO4-2H20, 0.033; NazMoO4o2H20, 0.024. To this medium was added the
appropriate organofluorine sulfonate as the sulfur source. Sodium difluoromethane
sulfonate (DFMS) was provided by 3M, St Paul, MN. Potassium perfluorooctane
sulfonate (PFOSA) and lH,lH,2H,2H-perfluorooctane sulfonic acid (H-PFOSA) were
obtained from ICN Pharmaceuticals Inc., Costa Mesa, CA. 2,2,2-Trifluoroethane
sulfonyl chloride was obtained from Sigma Chemical, St. Louis, MO. Hydrolysis of
2,2,2-trifluoroethane sulfonyl chloride by addition to water and autoclaving yielded
trifluoromethane sulfonic acid (TES). All other carbon sources and chemicals were

obtained from Sigma Chemical.

Growth conditions for Pseudomonas sp. strain D2. Plate colonies of strain D2 were
inoculated into 5 ml of nutrient broth (Difco, Detroit, MI) and incubated at 30° C for 24
hours. A 1% inoculum of this was used in above medium (pH of 6.9-7.0) with the
specified organofluorine compound as the sole sulfur source. Cells were grown

aerobically at 30°C and were shaken on a rotorary shaker at 160 rpm.

Fluoride and DFMS analysis. Fluoride and DFMS were measured on a Dionex ion

chromatography model 2000i/sp fitted with an IonPac AS4A ion exchange column and a

48

Dionex IonPac AG4A guard column. This system utilizes an anion micromembrane
suppressor with a Dionex Conductivity Detector-H (CDM). The eluant was a carbonate
buffer (1.8 mM N azCO3 and 1.7 mM N aHCO3) with a flow rate of 2 ml/min. Fluoride
concentration was used as an indirect method of detecting biotransformation of the
fluorinated sulfonates. Fluoride was also measured using an ion selective electrode
(Orion 96-09 BN). A total ionic strength adjuster (TISA) was used to stabilize fluoride
measurements in samples. TISA was prepared by adding 57 m1 of glacial acetic acid and
58 g reagent grade sodium chloride into 500 ml of deionized water. This mixture was
cooled on ice, adjusted to a pH of 5.0-5.5 with 5M sodium hydroxide, and diluted with
water to a final volume of one liter. One part TISA to one part sample was used to

measure fluoride.

Mass spectrometry and atomic emissions analysis. GCIMS data were obtained using
a Hewlett-Packard 5995 series H GC/MS. A DB624 capillary column (30 m x 0.25 mm
x 1.4 pm) was used for separation of volatile byproducts (J & W Scientific, Inc.,
Folsom, CA). Operating conditions were as follows: flow of 30 cm/sec linear velocity,
initial temperature 40° C for 4 minutes followed by a 10° C/minute ramp to 200° C,
injector temperature of 250° C, transfer line temperature of 225° C. Samples were taken
by injecting a solid phase microextraction (SPME) fiber assembly with a 100 uM
polydimethylsiloxane coating (Supelco Inc., Bellefonte, PA) in through the septum of the
sample. This assembly was allowed to equilibrate for 30 rrrinutes within the sample

headspace before injecting onto the GC/MS.

A Hewlett-Packard 5890 series H GC with operating conditions identical to those above
was used with a Hewlett-Packard 5921A atomic emission detector (AED) for elemental
analysis of volatile products of H-PFOSA. Table 3.1 lists the emission wavelengths and

plasma gases used for the various elements.

49

Table 3.1. Emission wavelengths and plasma gases used for atomic emission detection.

 

 

Element Monitored Emission Wavelength (nm) Plasma Reagent Gases
Sulfur 181 oxygen and hydrogen
Carbon 496 oxygen
Hydrogen 486 oxygen

Fluorine 690 hydrogen

Oxygen 777 hydrogen with auxillary gas

(10% methane/90% nitrogen)

RESULTS

Enrichment and identification of bacteria. A Pseudomonad, designated as strain D2,
was isolated from a mixed culture of bacteria that fortuitously contaminated a laboratory
batch of carbon rich medium containing DFMS as the sole source of sulfur. Strain D2
was isolated by streaking on nutrient agar plates. This isolate is a motile, Gram-negative
rod capable of using select fluorinated sulfonates as a source of sulfur for growth under
aerobic and sulfur-limiting conditions. It is catalase positive and oxidase positive.
Optimal growth was observed at 30° C. Fatty acid profiles for strain D2 were performed
by Microbial ID, Inc. (MIDI), Newark, Delaware. This analysis gave a similarity index
of 0.788 for Pseudomonas chloroaphis and a similarity index of 0.692 for Pseudomonas
fluorescens. Biolog, Inc. (Hayward, CA), identified strain D2 as Pseudomonas

fluorescens.

Biotransformation of difluoromethane sulfonate. Cells of strain D2 were grown for
24 hours in nutrient broth (Difco, Detroit, MI), and a 1% inoculum of this culture was

added to medium containing DFMS. Fluoride release, disappearance of DFMS, and

50

optical density were monitored. Figure 3.1 shows complete defluorination of DFMS
with stoichiometric yield of fluoride. Figure 3.2 demonstrates the corresponding growth
associated with the utilization of DFMS as the sole sulfur source. Control samples (not

shown) did not defluorinate DFMS and showed no change in optical density.

 

 

 

 

50 _ DFMS —) _
(as fluoride) (— fluoride

 

-....~.—.-—».«..—.._..—.. -....-—

40

 

Concentration (uM)

 

 

41 . \I\*I 4n ‘
20 30 40

Time (hours)

 

 

 

 

 

Figure 3.1. Defluorination of DFMS by Pseudomonas sp. strain D2. Error bars
represent the standard deviation of triplicate samples.

51

 

 

 

 

 

 

 

 

' fa 0-8
a 50 ‘11-“...1. 0.6 g
E" ‘ DFMS —) ‘ optical 'I Q,
B 40 - (as fluoride) density E
a 0.4 a
o 30 'I or
3 , - o
8 20 - 3'
, 0.2 '8-
O

10 q/ \\ I
0 is 1 r . r 1 r 1 r .. . . 00
0 10 20 30 40 50 60

Time (hours)

Figure 3.2. Growth of Pseudomonas sp. strain D2 on DFMS. Error bars represent the
standard deviation of triplicate samples.

Biotransformation of other fluorinated sulfonates. Pseudomonas sp. strain D2 was
used to evaluate the biotransformation of other sulfur containing fluorinated compounds
such as trifluoromethane sulfonate (TFMS), 2,2,2-trifluoroethane sulfonic acid (TES),
perfluorooctane sulfonate (PFOSA), and 1H,lH,2H,2H-perfluorooctane sulfonic acid
(H-PFOSA). Biotransformation experiments were similar to those used for DFMS
studies except that TES and H-PFOSA experiments were conducted in bottles sealed
with teflon lined septa (1:6 liquid to gas volume). Strain D2 was only capable of utilizing

the hydrogen-substituted sulfonates - DFMS, TES, and H-PFOSA .

Unlike the transformation of DFMS, TES and H-PFOSA were only partially
defluorinated. When TES was the sole sulfur source for growth, one mole of fluoride
was released per mole of TES degraded. Transformation of H-PFOSA yielded

approximately one mole of fluoride per mole of H-PFOSA transformed when

52

incubations were open to the atmosphere and 1.4 moles per mole when incubations were
closed to the atmosphere. Figures 3.3 and 3.4 show fluoride released from various
concentrations of TES and H-PFOSA, respectively. As with biotransformation of
DFMS, transformation of TES and H-PFOSA correlated with growth of strain D2
indicating that sulfur was released and assimilated. Figures 3.5 and 3.6 show growth of

strain D2 with TES and H-PFOSA as the sole sources of sulfur.

 

 

 

 

 

 

 

400 T I T r r r . r v
I. 3:1
300 ...-.._-_....-M. 1.1.-..111--_-_1._---- "-1. -. .--__--.-..M,.,1 m--.-........-1-. .---._.---,_’ I r I I
Fluoride x'” I, 23]
Released 200 ’," ’,,-"'
(”M) I I” I ’ ' ' a
’,-" ,-””' ’ 11
100 ”*’ ,x“ ,,,,,, "" A
” ’ ’ ””””” ‘
I ’ - - ’ ’ _ ‘ ’ - 4
A- - - -
0 n I n I 1 l n l n
20 4O 6O 80 100 120

Initial 2,2,2-trifluoroethane sulfonate (uM)

Figure 3.3. Fluoride release from TES. Numbers represent ratio of moles of fluoride to
moles of TES. Triplicate samples were measured, but error bars are not visible due to
scale.

53

 

 

 

 

 

 

 

 

100 V r I I l I U
2:1 ’1
1’
1”
’1
75 ,’
I,” 1.4:1_
b ’1‘ ””" ‘
Fluoride / x”
I’ ”
Released so ,4 -- -.1
(HM) ’1’ ’,v’ """" -
I ’ ’o
I' ’1 ’1’ a'
” I" ””” I
I’ a” ...-p"
25 -’ ,- “-4—
0 1 l n I u 1 1
10 20 30 40 50

Inital lH,lH,2H,2H-perfluorooctane sulfonic acid (uM)

Figure 3.4. Fluoride release from H-PFOSA. Numbers represent ratio of moles of
fluoride to moles of H-PFOSA. Triplicate samples were measured, but error bars are not
visible due to scale.

 

 

 

 
 
  

 

 

 

 

 

 

20 ' I ' I ' I ' I ' I ' 0.30
fluoride ‘ ...
15 («Ch 2* o
A - 0 20 8
i - ' s.
g 10 0.15 g
2 optical density ‘ a
m - 0.10 a
5 g
j - 0.05 o

O n J 1 l n I n l n I 1 0m

0 20 40 6O 80 100 120

Time (hours)

Figure 3.5. Growth of Pseudomonas sp. strain D2 on TBS. Error bars represent the
standard deviation of triplicate samples.

54

 

 

 

 

 

 

 

 

 

 

 

 

I ‘ _ 0.4
:3,
- 0.3 5
A D
E . 8
g i
. 0.2 ....
3 1%
m d
.- 55';
. / (———- optical density - 0,1 3
2 8'
0 1 1 ‘ 1 ‘ 1 ‘ l 4 l T 0.0
O 20 40 60 80 100 120

Time (hours)

Figure 3.6. Growth of Pseudomonas sp. strain D2 on H-PFOSA. Error bars represent
the standard deviation of triplicate samples.

Eight volatile and fluorinated byproducts of H-PFOSA were detected with GC/MS and
AED. However, no transformation products were detected for TES. As shown in
Figure 3.7, all of the volatile products of H-PFOSA contained carbon, oxygen, hydrogen,
and fluorine. Sulfur was not detected in any volatile products of H-PFOSA. Complete
GC/MS data and spectrums (both electron impact and chemical ionization) can be found

in Appendices A and B. In addition, complete AED plots are also found in Appendix C.

55

 

 

 

 

 

 

 

 

12000-
100004
8000-1
4
I
6000-1
40001
I carbon
2000 1 .’ hydrogen I. ' '1. -
1"“
j “I I.
0‘ _fluorine A flit“ _ _ |
75.11%. "10" -1,21..1,4- - -1,6.-11.8.--2,0

Time (minutes)

Figure 3.7. Atomic Emission Detection of volatile fluorinated byproducts of H-PFOSA
biodegradation.

DISCUSSION

Several researchers have demonstrated the utilization of aliphatic sulfonates as sole sulfur
source [9, 10, 12, 18, 20, 21, 23, 24], but there are no reports establishing that fluorinated
sulfonates can serve as the sole source of sulfur for growth. In general, fluorinated
organics are perceived as refractory in natural environments with the potential for
accumulation. Therefore, it is important to understand the details of how these
compounds are metabolized and/or degraded. It is demonstrated in this report that
specific fluorinated sulfonates are at least partially degraded. Presumably, the growth

associated with these transformations is due to the utilization of the sulfur in these

56

molecules (no growth was observed in their absence). As shown in Figures 3.5 and 3.6,
growth continues even after the release of fluoride has st0pped. A likely explanation is
that the initial transformation and defluorination are rapid and not rate-limiting, while the
assimilation of the sulfur byproduct into biomass is slower and rate-limiting. These
findings suggest that the initial steps of transformation are linked to sulfur scavenging

with subsequent defluorination of the molecule.

Kelly et al. [10] have shown that sulfite and formaldehyde are the products of degradation
of methanesulfonic acid (MSA) by a methylotrophic bacterium. The methylotroph was
not able to utilize sulfonates with more than three carbons when it was pregrown with
MSA and grew poorly on compounds containing more than one carbon. An NADl-I-
dependent monooxygenase was found to be responsible for the reaction. Based on these
results and those of other investigators [21, 24], a possible mechanism for degradation of
the fluorinated sulfonates is desulfonation of the molecule, with the production of sulfite
or bisulfite and an aldehyde. The sulfite would then be subsequently used as a source of
sulfur for growth (no free sulfite was detected in incubation of strain D2). An aldehyde
formed from the degradation of H-PFOSA may or may not be stable and could
decompose to other products. The discrepancy in fluon‘ned stoichiometry between closed
and open incubations of H-PFOSA with strain D2 could be explained by subsequent
transformation of one or more of the volatile fluorinated products. Postulation of a
mechanism that removes the sulfur moiety from the molecule is supported by atomic
emissions spectroscopy data that failed to detect sulfur in any of the volatile fluorinated
byproducts of H-PFOSA biodegradation. Table 3.2 illustrates possible structures for the

volatile products from H-PFOSA transformation.

57

Table 3.2. Possible volatile fluorinated products of H-PFOSA transformation by strain

D2.

Possible H-PFOSA products (-0 fluoride)
C6F13CH2CH2-O-CH3

C6F13CH2-O-CH2CH3

C5F13CH2CCH3

C6F13CH2CH20H

Possible H-PFOSA roducts -l fluoride

 

C5F1 1CF=CH—CH=O
C5F1 1CF=C=O

C 5F1 1CF=C=C=O

C5F11CF\-C117H
0

Possible H-PFOSA products (-2 fluoride)

C5F1 1CH=(|3-CH=O
OH

C5F1 1EH-CH2-CH 3
0

Molecular Weight
378

378

376

364

Molecular Weight

342

328

340

343

Molecular Weight

340

327

The fate of volatile fluorinated compounds in the environment has been studied

internationally during the past twenty years. The chlorofluorocarbons (CFCs) have been

implicated in the destruction of the ozone layer and global warming [13, 17].

58
Replacements for the CFCs, hydrofluorocarbons (HFCs) and hydrochlorofluorocarbons
(HCFCs), are expected to degrade before reaching the stratosphere and the ozone layer,
but some of these can still contribute to global warming [26]. Although the identity of
the volatile fluorinated products of H-PFOSA transformation is unknown, their
production is important because it suggests an alternative route for the production of
volatile fluorinated compounds. Until the identity of these compounds is determined, it is

unclear what effect, if any, they might have on the environment or on global warming.

Another finding of this research was the observation that only fluorinated compounds
with one or more hydrogen substituents are transformed by strain D2. It is likely that the
enzyme responsible for the transformation of these compounds must interact at or near
the carbon-sulfur bond. In the case of molecules with only fluorine at the alpha-carbon,
the strong electronegativity of fluorine (4.0) may preclude attachment of the enzyme to
the molecule and thus limit biotransformation. The fluorocarbon chain is also more
"rigid" due to fluorine atoms on the molecule [8]. This rigidness almost certainly
interferes with molecule/enzyme interactions, protecting fluorocarbon molecules from

biological attack.

The results of this study clearly demonstrate that hydrogen-substituted fluorinated
sulfonates are susceptible to biodegradation and defluorination. In addition, growth of
bacteria may be supported by the sulfur byproduct of transformation under aerobic and
sulfur-limiting conditions. By understanding the specific nature of the enzyme(s) or co-
factor(s) involved in the transformation of these molecules, we may be able to gain
insight into the mechanism of degradation of fluorinated sulfonates as well as non-
fluorinated sulfonates. This insight may make it possible to construct fluorinated

surfactants that are more readily biodegradable to harmless end products.

REFERENCES

Abe, T., and S. Nagase. 1982. Electrochemical fluorination (Simons process) as
a route to perfluorinated organic compounds of industrial interest, pp. 19-44. In
R. E. Banks (ed.), Preparation, properties, and industrial applications of
organofluorine compounds. John Wiley & Sons, New York.

Banitt, E. H., W. E. Coyne, K. T. McGurran, and J. E. Robertson. 1974.
Monofluoromethanesulfonanilides. A new series of bronchodilators. J. Med.
Chem. 17:116-120.

Cartwright, D. 1994. Recent deve10pments in fluorine-containing
agrochemicals, pp. 237-257. In R. E. Banks, B. E. Smart, and J. C. Tatlow (ed.),
Organofluorine chemistry: Principles and commercial applications. Plenum
Press, New York. .

Elliot, A. J. 1994. Chlorofluorocarbons. DP. 145-157. In R. E. Banks, B. E.
Smart, and J. C. Tatlow (ed.), Organofluorine chemistry: Principles and
commercial applications. Plenum Press, New York.

EPA. 1989. Pesticide Fact Sheet on Sulfluramid. EPA. 205 (540/FS-89-060).

Fielding, H. C. 1979. Organofluorine surfactants and textile chemicals, pp. 214-
232. In R. E. Banks (ed.), Organofluorine chemicals and their industrial
applications. Ellis Horwood Ltd., Chichester.

Filler, R. 1993. Fluoromedicinal chemistry - An overview of recent
developments, pp. 1-23. In R. Filler, Y. Kobayashi, and L. M. Yagupolskii (ed.),
Organofluorine compounds in medicinal chemistry and biomedical applications.
Elsevier, Amsterdam.

Guo, W., T. A. Brown, and B. M. Fung. 1991. Micelles and aggregates of
fluorinated surfactants. J. Phys. Chem. 95: 1829-1836.

Higgins, T. P., M. Davey, J. Trickett, D. P. Kelly, and J. C. Murrell. 1996. .
Metabolism of methanesulfonic acid involves a multicomponent monooxygenase
enzyme. Microbiol. 142:251-260.

59

10.

ll.

12.

13.

14.

15.

16.

17.

18.

19.

20.

60

Kelly, D. P., S. C. Baker, J. Trickett, M. Davey, and J. C. Murrell. 1994.
Methanesulphonate utilization by a novel methylotrophic bacterium involves an
unusual monooxygenase. Microbiol. 140: 1419-1426.

Kissa, E. 1994. Fluorinated surfactants: synthesis, properties, and applications.
Marcel Dekker, Inc, New York.

Laue, H., J. A. Field, and A. M. Cook. 1996. Bacterial desulfonation of the
ethanesulfonate metabolite of the chloroacetanilide herbicide metazachlor.
Environ. Sci. Technol. 30: 1 129-1 132.

Molina, M. J ., and F. S. Rowland. 1974. Stratospheric sink for
chlorofluoromethanes: chlorine atom-catalysed destruction of ozone. Nature
249:810—812.

Moore, G. G. I. 1974. Sulfonamides with antiinflarnmatory activity, pp. 159-
176. In R. A. Scherrer, and M. W. Whitehouse (ed.), Antiinflammatory agents:
Chemistry and pharmacology. Academic Press, New York.

Moore, G. G. I. 1979. Fluoroalkanesulfonyl Chlorides. J. Org. Chem. 14:1708-
1711.

Rao, N. S., and B. E. Baker. 1994. Textile finishes and fluorosurfactants, pp.
321-336. In R. E. Banks, B. E. Smart, and J. C. Tatlow (ed.), Organofluorine
chemistry: Principles and commercial applications. Plenum Press, New York.

Rowland, F. S., and M. J. Molina. 1975. Chlorofluoromethanes in the
environment. Rev. Geophys. Space Phys. 13:1-35.

Seitz, A. P., E. R. Leadbetter, and W. Godchaux III. 1993. Utilization of
sulfonates as sole sulfur source by soil bacteria including Comamonas
acidovorans. Arch. Microbiol. 159:440-444.

Stang, P. J ., and M. R. White. 1983. Triflic acid and its derivatives.
Aldrichimica Acta 16:15-22.

Thompson, A. S., N. J. P. Owens, and J. C. Murrell. 1995. Isolation and
characterization of methanesulfonic acid-degrading bacteria from the marine
environment. Appl. Environ. Microbiol. 61 :2388-2393.

21.

22.

23.

24.

25.

26.

61

Thysee, G. J. E., and T. H. Wanders. 1974. Initial steps in the degradation of
n-alkane-l -sulphonates by Pseudomonas. Antonie Leeuwenhoek J. Microbiol.
40:25-37.

Trepka, R. D., et al. 1974. Synthesis and herbicidal activity of fluorinated N-
phenylalkanesulfonamides. J. Agr. Food Chem. 22:11] 1-1 1 19.

Uria-Nickelsen, M. R., E. R. Leadbetter, and W. Godchaux III. 1993.
Sulphonate utilization by enteric bacteria. J. Gen. Microbiol. 139:203-208.

Uria-Nickelsen, M. R., E. R. Leadbetter, and W. Godchaux Ill. 1994.
Comparative aspects of utilization of sulfonate and other sulfur sources by
Escherichia coli K12. Arch. Microbiol. 161:434—438.

Vander Meer, R. K., C. S. Lofgren, and D. F. Williams. 1985. Fluoroaliphatic
sulfones: a new class of delayed-action insecticides for control of Solenopsis
invicta. J. Econ. Ent. 78:1190-1197.

Wallington, T. J ., et al. 1994. The environmental impact of CFC replacements-
HFC's and HCFCs. Environ. Sci. Technol. 28:320A-326A.

CHAPTER 4

PHYSIOLOGY OF DIFLUOROMETHANE SULFONATE
TRANSFORMATION BY PSEUDOMONAS SP. STRAIN D2

ABSTRACT

Difluoromethane sulfonate (DFMS; CHFZSO3Na), a fluorinated sulfonate, was chosen
as a model compound to investigate the physiology of transformation of fluorinated
sulfonates by Pseudomonas sp. strain D2. Previous research has established that a
structural or molecular limitation to transformation is observed when the fluorinated
sulfonates are completely fluorinated and do not possess hydrogen substituents. The
present study investigates further the limitations on biotransformation of these
compounds. In whole cell experiments, oxygen was required for growth and
transformation of DFMS. It is also shown that DFMS cannot be utilized as a source of
carbon and energy, but can be used as a sole source of sulfur under sulfur-limiting
conditions with an added carbon source. It appears that actively metabolizing cells are
required for the transformation of DFMS. This conclusion is based on the complete
absence of transformation activity when glucose or ammonium were removed from the
medium. It is hypothesized that transformation of DFMS and other fluorinated
sulfonates is linked to a sulfur-scavenging system. Inhibition studies suggest that the
sulfur-containing byproduct of DFMS transformation is assimilated through existing
sulfur assimilation pathways. Non-competitive inhibition kinetics were observed with Ki

values of 3-4 “M for sulfate, sulfite, methane sulfonate, cystine, and methionine.

62

63

INTRODUCTION

Although fluorinated compounds are used more frequently today, the environmental fate
of many of these compounds is largely unknown. Reductive defluorination has been
observed for tri-, di-, and monofluoroacetate .[22]. However, Oremland et al. [15] were
not able to replicate this work during further studies and concluded that trifluoroacetic
acid was "generally refractory to microbial degradation." Hydrolytic defluorination of
monofluoroacetate by bacteria has been reported [3, 4, 5, 6, 10, 13, l4, 19, 23]. More
often, the transformation of fluorinated organics appears to require attack on ring

structures and functional moieties such as sulfonic, carboxylic, and amine groups.

Defluorination of and growth on sulfur-containing organofluorine compounds under
aerobic and sulfur-limiting conditions by Pseudomonas sp. strain D2 (chapter 3) and
other bacteria (chapter 5) has been demonstrated. Among the compounds degraded were
difluoromethane sulfonate (DFMS; CHFZSO3N a), 1H,1H,2H,2H-perfluorooctane
sulfonic acid (II-PFOSA; C6F13C2H4803H), and 2,2,2-trifluoroethane sulfonic acid
(TES; CF3CH2803H). Trifluoromethane sulfonate (TFMS; CF3SO3Na) and
perfluorooctane sulfonate (PFOSA; C3F17SO3K) were not degraded. This work
suggested that the transformation of these compounds is restricted to molecules
containing hydrogen. In this report other limitations to the transformation of sulfur-
containing organofluorine compounds are examined using DFMS as a model compound.
It is hypothesized that the transformation of DFMS is linked to sulfur metabolism or
sulfur assimilation. Sulfur inhibition studies are used to evaluate whether utilization of
DFMS as a sulfur source shares some intermediates or characteristics of sulfur

assimilation pathways.

64

MATERIALS AND METHODS

Media and chemicals. A defined mineral medium containing (in grams per liter):
glucose, 2.0; Kzl-IPO4, 3.5; KH2P04, 2.0; NH4C1, 1.0; MgC12°6H20, 0.5; 0.15M
CaC12-2H20 stock, 1.0 ml/liter; trace elements stock I and II, 1.0 mllliter. Trace elements
stock I contained (in grams per liter): FeCl3, 1.36; CoC12°6H20, 0.2; MnC1204H20,
0.122; ZnC12, 0.07; NazMoO4-2H20, 0.036; NiClzo6H20, 0.12; B3(OH)3, 0.062;
CuC12°2H20, 0.017 . Concentrated H2804 was added at 2.5 ml/L to the trace elements
solution 1. Trace elements solution 11 contained (in grams per liter): NaZSeO3°5H20,
0.006; NaWO4-2H20, 0.033; Na2M004-2H20, 0.024. Sodium difluoromethane
sulfonate (DFMS) was provided by 3M, St Paul, MN. All other carbon sources and

chemicals were obtained from Sigma Chemical.

Fluoride and DFMS analysis. Fluoride and DFMS were measured on a Dionex ion
chromatography model 2000i/sp fitted with an IonPac AS4A ion exchange column and a
Dionex IonPac AG4A guard column. This system utilizes an anion micromembrane
suppressor with a Dionex Conductivity Detector-II (CDM). The eluant was a carbonate
buffer (1.8 mM Na2C03 and 1.7 mM NaHCO3) with a flow rate of 2 ml/min. Fluoride
concentration was used as an indirect method of detecting biotransformation of the
fluorinated sulfonates. Fluoride was also measured using an ion selective electrode
(Orion 96-09 BN). A total ionic strength adjuster (TISA) was used to stabilize fluoride
measurements in samples. TISA was prepared by adding 57 ml of glacial acetic acid and
58 g reagent grade sodium chloride into 500 ml of deionized water. This mixture was
cooled on ice, adjusted to a pH of 5.0-5.5 with 5M sodium hydroxide, and diluted with
water to a final volume of one liter. One part TISA to one part sample was used to

measure fluoride.

65

Growth conditions for Pseudomonas sp. strain D2. Plate colonies of strain D2 were
inoculated into 5 ml of nutrient broth (Difco, Detroit, MI) and incubated at 30° C for 24
hours. A 1% inoculum prepared in this manner was introduced into the defined mineral
medium (pH of 6.9-7.0) with DFMS as the sole sulfur source. Cells were typically
grown aerobically at 30°C and were shaken on a rotary shaker at 160 rpm. To test
whether strain D2 could grow under denitrifying conditions, growth medium was
amended with 10 mM sodium nitrate, degassed with a 98% N2 and 2% H2 gas mixture,
and capped with teflon-faced butyl rubber septa. Culture manipulations were performed
in a Coy anaerobic glove box (Coy Laboratories, Ann Arbor, MI). Glucose or acetate
provided the carbon and energy source for these incubations. To test whether DFMS
could simultaneously serve as carbon source and sulfur source, medium (without
glucose) was amended with 5 or 10 mM DFMS. To test whether DFMS could serve as
carbon source, medium (without glucose) was amended with 50 uM sulfate and 5 or 10
mM DFMS. A 1% inoculum of nutrient broth grown cells was used for these

experiments.

Growth conditions and preparation of cells for inhibition experiments. Because
fluoride concentration correlated with growth of strain D2, both fluoride and optical
density measurements were used to determine stationary phase. Stationary phase cells
were harvested by centrifugation (15 minutes at 12,100 X g in a Beckman SS-34 rotor at
4°C), washed in medium without a sulfur source, and resuspended to one tenth the
original culture volume in the same medium to a cell density of approximately 1000-
1500 mg protein/L. One milliliter of the resulting 10X concentrated cell suspension was
added to 4 ml of medium. Specific substrate utilization curves were determined by
varying the concentration of DFMS as well as the concentration of the tested sulfur
sources. Fluoride was measured as an indirect measurement of transformation over a 20

minute time period and total cell protein was determined using the modified Lowry

66

method, with bovine serum albumin as the standard [12]. Specific rates of substrate
utilization were computed as the mass degraded over a given time period divided by the

total mass of protein used in the assay.

Modeling of Kinetic parameters. Difluoromethane sulfonate transformation rate

coefficients were determined using a model for specific substrate utilization rate:

U = (k*S) / (Ks + S) = -(dS/dt)/X

where U is the specific substrate utilization coefficient (umoles DFMS/mg protein/hr), k
is maximum Specific substrate utilization coefficient (pmoles DFMS/mg protein/hr), S is
the rate-limiting substrate concentration (pM), Ks is the half-velocity coefficient (uM), t is
time in hours (hr), and X is the total protein concentration (mg protein/L). Values for k
and Ks were estimated using a nonlinear curve fit obtained from Systat version 5.2.1,

based on initial DFMS degradation rates at a fixed cell protein concentration.

Two models were used to determine the nature of the inhibition by other sulfur sources.

The first model was the competitive inhibition model:

U = (k * S) / (Ks*(l + I/Ki) + S) = -(dS/dt)/X

where I is the inhibitor concentration (uM) and Ki is the inhibition constant (uM). The

second model used was the non-competitive inhibition model:

U = (k*S) / ((I/Ki + l)*(Ks + S)) = -(dS/dt)/X

The inhibition rate coefficients were determined using a nonlinear fit of the initial DFMS
degradation rates at a fixed cell protein concentration. Rates of transformation were
determined at concentrations of DFMS varying from 1.3 uM to 65 pM and inhibitor

concentrations from 2 uM to 200 uM.

67

Sonicated crude cell extract preparation. Cultures of strain D2 were grown in IL
batches aerobically in 2L Erlenmeyer flasks for approximately 36 hours from a 1%
inoculum. Pseudomonas strain D2 was screened for defluorination activity by
measuring transformation rates prior to preparation of cell extracts. Actively
transforming cultures were transferred to 250 m1 centrifuge tubes and centrifuged at
12,100 X g for 15 minutes at 4°C. Cells were then washed twice and resuspended in 50
ml of buffered growth medium without DFMS. Thirty milliliters of this concentrated
cell suspension was sonicated on ice, for 10 minutes at 1 second bursts at stage 5 (50%
time on and 50% time off). The effective sonication time was 5 minutes. The sonicate
was centrifuged at 12,100 X g for one hour at 4°C and divided into a supernatant and cell
pellet. Whole cells, whole sonicate, supernatant, and cell pellet (resuspended in 30 ml
medium) were used to assay DFMS transformation. DFMS was added to these samples

at 32 pM and 20 11M B-NADH was added to a subset of these samples.

French press crude cell extract preparation. Cells grown and harvested as described
above were resuspended in 5 ml 20 mM Tris-buffer (pH 7.0) with 1 mM EDTA, placed
on ice, and supplemented with the following protease inhibitors: 1 til/ml of leupeptin
solution and Sal/ml aprotinin. The cells were then passed three times through a chilled
French pressure cell at 1000-1200 psi. The cell extract was diluted to 20 ml. Three
reaction mixtures were used to evaluate cell free activity. The first consisted of growth
medium, as described above, amended with glucose and 1 mM DFMS. The second was
a mixture containing 50 pM ascorbic acid, 50 11M FeC12 , and 10 mM imidazole buffer
(pH 6.75) amended with 1 mM DFMS. The third was 20 mM Tris-buffer (pH 7 .0)
amended with 1 mM DFMS. Three different cofactors, ATP, B-NADH, and or-
ketoglutarate were chosen to evaluate biotransformation requirements. A portion of the
cell extract was centrifuged at 27,200 X g for 20 minutes and 0.5 ml of the supernatant

was added to 1.5 ml of reaction mixture with one of the three cofactors. Controls

68
consisted of samples with no cofactor, samples with no cell extract, and cell extract that
was not centrifuged. The 2 ml samples were placed into 12 ml screw-capped tubes and

shaken for 20 minutes to one hour at 21 °C.

RESULTS

Although Pseudomonas sp. strain D2 can grow under denitrifying conditions with
sulfate as the sole sulfur source, it was not capable of growth under denitrifying

conditions when the sole sulfur source was difluoromethane sulfonate (Figure 4.1).

 

 

 

 

 

 

 

  

 

  

 

 

 

 

 

0.8 1 I ' I I I v I
L glucose & aerobic
V
N
5. 0.6 /F
o
r:
so .
© acetate & aerobic
e 0.4 -- -
a l
O _ .1
% fll
'E‘, 0.2
0 glucose & anaerobic acetate & anaeI'ObiC
I 4
0.0 Eb ‘ ‘ TB ‘ d
O l 2 3 4 5
Time (days)

Figure 4.1. Growth of Pseudomonas sp. strain D2 with glucose and acetate under
aerobic conditions. No growth was observed under anaerobic conditions. Error bars
represent the standard deviation of triplicate samples.

69

As shown in Figure 4.2, Pseudomonas sp. strain D2 was not capable of using DFMS as
a source of both carbon and sulfur nor was it capable of utilizing DFMS as a source of

carbon when sulfur was provided in the form of sulfate.

 

 

 

 

    

 

 

 

 

0.8 ' I ' l . r .
g 0'6 ‘ DFMS
o (5 or 10 mM) as only
a l sulfur source + glucose
.E‘ 0 4
E ' -carbon - sulfur + DFMS
8 or
'3 -carbon + SOuM sulfate + DFMS
‘33. 02 . _---. -. .---..“ l m.-- “.-----“
o l
,
0.0
O l 2 3 4
Time (days)

Figure 4.2. Growth of Pseudomonas sp. strain D2 with DFMS as sole source of sulfur.
DFMS cannot be used as carbon source. Error bars represent the standard deviation of
triplicate samples.

As shown in Figure 4.3, transformation did not occur when cells that had been grown to
stationary phase with DFMS as sole sulfur source were incubated without a source of
carbon and energy (no glucose). In addition, transformation of DFMS did not occur

when ammonium, the nitrogen source, was removed from the medium.

7O

 

21 ' I ' I r I

 

15
. Control (+g1ucose,+ammonium)—7/ ,

 

 

 

 

 

 

 

 

 

 

7; 12 WWW-—
:8 ° / 1
u, Ammonrum (-) GIUCIOSC H
. 45g ._ . _-
O l l l

0.0 0.5 1.0 1.5 2.0

Time (hours)

Figure 4.3. Effects of removal of glucose or ammonium, from growth medium, on the
transformation of DFMS in stationary phase cells. Error bars represent the standard
deviation of triplicate samples.

To determine whether transformation of DFMS was liked to sulfur metabolism or sulfur
assimilation, sulfur inhibition studies were initiated to further elucidate the physiology of
DFMS utilization. Sulfate, sulfite, methane sulfonate (a structural analogue of DFMS),
cystine, and methionine were used as inhibitors of transformation. N on-competitive
inhibition kinetics were observed. Figure 4.4 illustrates non-competitive inhibition of
DFMS transformation for varying concentrations of sulfate. Figures 4.5, 4.6, and 4.7
show the effects of sulfite, methane sulfonate, and cystine respectively on DFMS

transformation. Methionine had a similar effect on the transformation of DFMS.

71

 

 

 

 

 

 

 

 

 

0.025 . . . . . , .
__1' 0 “M
g 0.020 ? -
'9 Ki = 3.24 i 0.92
E.
g 0.015 _
a
E El 2 uM
o 0.010 -
A
5 0.005 10 “M -
0.000 ' * L
40 60

 

Concentration (uM)

Figure 4.4. Non-competitive inhibition of DFMS transformation rates by sodium

sulfate. Error bars represent the stande deviation of triplicate samples.

80

72

 

 

 

 

 

 

 

 

 

0.025 . . . . . ,
‘1’? O “M 4
3 0.020 -
B
8 .
g Ki = 3.59 i 0.20
g 0.015 ‘
272
E
Q 0.010 ‘
In 5 [1M
.9 D
E I 10 uM
0.005 . . -
V (A
D 15 uM
0.000 ' ‘ ' ‘
40 60 80

Concentration (uM)

Figure 4.5. Non-competitive inhibition of DFMS transformation rates by sodium sulfite.
Error bars represent the standard deviation of triplicate samples.

73

 

 

 

 

 

 

 

 

0.025 ' I ' ' ' 1
OuM
3 0.020 ~ 0 ' ' ‘
.3
8
E.
g 0.015 ~ ,, Ki=2.87tO.56 ‘
275 [j ZuM
E _ D U 4uM _
Q 0.010 I
n I.
.9 ’ .
3 0.005 if "
D _A_ A 20 “M 4
A
0.000 ’ ‘ ‘ ‘ ‘ ‘
0 2O 40 60 80
Concentration (uM)

Figure 4.6. N on-competitive inhibition of DFMS transformation rates by sodium
methane sulfonate. Error bars represent the standard deviation of triplicate samples.

74

 

 

 

 

 

 

 

 

 

0.025 ' I I I f I I
E T 0 “M
3 0.020 ? a
s
8
9' Ki=3.64:0.61
g 0.015 -
:75
E
a 0.010 CI 5 uM ~
.2
a 0.005 10 “M -
E; 15 uM

0.000 ‘ L ' ‘

4O 6O 8O
Concentration (uM)

Figure 4.7. Non-competitive inhibition of DFMS transformation rates by cystine. Error
bars represent the standard deviation of triplicate samples.

DISCUSSION

We have previously shown that Pseudomonas sp. strain D2 is capable of growth with
difluoromethane sulfonate (DFMS), 2,2,2-trifluoroethane sulfonic acid (TES), and
lH,lH,2H,2H-perfluorooctane sulfonic acid (ll-PFOSA) as sole sulfur source (Chapter
3). DFMS was completely defluorinated while TES and H-PFOSA were only partially
defluorinated. Molecules that were completely fluorinated and did not contain hydrogen
(trifluoromethane sulfonate; TFMS and perfluorooctane sulfonic acid; PFOSA) were not
utilized as a sulfur source and were not defluorinated. These results suggest that

hydrogen-substitution is required for transformation.

75

Most reports on the utilization of sulfonates by bacteria have been restricted to aerobic
conditions [7, 9, 11, 16, 17, 18, 20, 2]]. However, Chien et a1 [1] have shown that a
fermenting Clostridium isolate can grow with taurine and isethionate as sulfur source,
and a fermenting Klebsiella isolate can grow with cysteate as a sulfur source.
Pseudomonas sp. strain D2 was not able to grow on or transform DFMS under
anaerobic conditions even though it was capable of growth under denitrifying conditions
with sulfate as the sulfur source. Evidently, whole cells of strain D2 require molecular
oxygen to grow with fluorinated sulfonates as the sole sulfur source. Additional evidence

is needed to establish whether molecular oxygen is required for the transformation.

These results show that DFMS could not be used as a carbon and energy source. In
most of these experiments, glucose served as the source of carbon and energy. When
glucose was removed from the medium, cells did not grow or transform DFMS. When
pregrown cells were used, removal of glucose prevented transformation (Figure 4.3).
This suggests that energy from glucose metabolism or some metabolic product of
glucose utilization may be required for transformation. In all experiments, ammonium
was the source of nitrogen. Removal of ammonium from the medium also prevented
transformation (Figure 4.3). It is unclear what role ammonium has on the transformation
activity of pregrown cells. Perhaps it is related to enzymatic activation, energy

production, or some transport mechanism.

Presumably, the transformation of DFMS, as well as the other fluorinated sulfonates, is
related to sulfur scavenging. A plausible hypothesis is that the first step in transformation
of DFMS is desulfonation yielding sulfite (or another sulfur byproduct) and a fluorinated
intermediate that spontaneously decomposes releasing fluoride. The sulfur byproduct is
then assimilated into biomass through normal sulfur assimilation pathways. To evaluate

this hypothesis, the effects of other sulfur sources on the transformation of DFMS were

76

investigated. Sulfate, sulfite, methane sulfonate, cystine, and methionine were used as
possible inhibitors of DFMS transformation. As is shown in Figures 4.4 through 4.7, all
of these sulfur sources were inhibitory to transformation (methionine not shown). The
data obtained from these experiments did not fit the competitive inhibition model.
However, the non—competitive model fit well, with an average correlation coefficient of
0.98 t 0.013. Of interest is the similarity in Ki values (3-4 uM) for these different sulfur
sources. This suggests some commonality in the mechanism of inhibition. One
possibility is the inhibition of sulfur transport mechanism(s). This inhibition could affect
binding proteins in the periplasm or cell membrane complexes that are used for sulfur
uptake. Perhaps the high electronegativity of the carbon-fluorine bond interferes with
proper binding to transport proteins or other sulfur sources compete for binding proteins
in the periplasm but not within the cell. The overall effect of competition outside of the
cell combined with abscence of competition on the inside could result in non-competitive
pattern of inhibition. It is important to note that the Michaelis-Menten model used to
model non-competitive inhibition is based on the assumption of one enzyme interacting
with one substrate and a inhibitor molecule. In the case of whole cells experiments, a one
step reaction is unlikely to explain the observed non-competitive inhibition. In any case,
these results strongly suggest that the transformation of DFMS is related to sulfur-
scavenging activities of the cell and that the sulfur byproduct(s) enter via existing sulfur

assimilation pathways.

In an attempt to clarify the mechanism of transformation of DFMS, crude cell extract
experiments were conducted. These experiments investigated the hypothesis that an
NADH-dependent oxygenase was responsible for the initial attack on the molecule. This
is similar to what has been shown by others [7, 8, 9, 18]. In addition, the possibility of
an energy requirement was evaluated and ATP was used as a cofactor for these

experiments. Finally, a-ketoglutarate was used to determine if a dioxygenase similar to

77

that encoded by the tfdA gene of Alcaligenes eutrophus (responsible for the first step in
2,4-dichlorophenoxyacetic acid degradation) was present [2]. No transformation activity
was observed in cell extracts, even with added cofactors. Perhaps additional cofactors are
required or critical membrane or cellular components were destroyed during lysis of
cells. Because inhibition was observed with various forms of sulfur found in intact cells
(such as cysteine) the negative results might be partially explained by sulfur release

during cell lysis.

This work establishes several limitations on the biotransformation of sulfur-containing
organofluorine compounds and establishes the importance of sulfur assimilation
processes in the transformation of DFMS. Structural or molecular limitations evidently
prevent transformation when the molecule is completely fluorinated and the presence of

hydrogen facilitates transformation under aerobic and sulfur-limiting conditions.

REFERENCES

Chien, C. C., E. R. Leadbetter, and W. Godchaux III. 1995. Sulfonate-sulfur
can be assimilated for ferrnentative growth. FEMS Microbiol. Lett. 129:189-194.

Fukumori, F., and R. P. Hausinger. 1993. Purification and characterization of
2,4-dichlorophenoxyacetic acid/a—ketoglutarate dioxygenase. J. Biol. Chem.
268:24311-24317.

Goldman, P. 1965. The enzymatic cleavage of the carbon-fluorine bond in
fluoroacetate. J. Biol. Chem. 240:3434—3438.

Goldman, P. 1971. Enzymology of carbon-halogen bonds, pp. 147-165. In
(ed.), Degradation of synthetic organic molecules in the biosphere. National
Academy of Sciences, Washington DC.

Goldman, P., and G. W. A. Milne. 1966. Carbon-fluorine bond cleavage; H.
Studies on the mechanism of the defluorination of fluoroacetate. J. Biol. Chem.
241:5557-5559.

Goldman, P., G. W. A. Milne, and D. B. Keister. 1968. Carbon-halogen bond
cleavage; III. Studies on bacterial halidohydrolases. J. Biol. Chem. 243:428-434.

Higgins, T. P., M. Davey, J. Trickett, D. P. Kelly, and J. C. Murrell. 1996.
Metabolism of methanesulfonic acid involves a multicomponent monooxygenase
enzyme. Microbiol. 142:251-260.

Junker, F., T. Leisinger, and C. A.M. 1994. 3-Sulphocatechol 2,3-dioxygenase
and other dioxygenases (EC 1.13.112 and EC 1.14.12.-) in the degradative
pathways of 2-aminobenzesulphonic, benzesulphonic and 4-toluenesu1phonic
acids in Alcaligenes sp. strain O-l. Microbiol. 140:1713-1722.

Kelly, D. P., S. C. Baker, J. Trickett, M. Davey, and J. C. Murrell. 1994.
Methanesulphonate utilization by a novel methylotrophic bacterium involves an
unusual monooxygenase. Microbiol. 140:1419-1426.

78

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

79

Kelly, M. 1965. Isolation of bacteria able to metabolize fluoroacetate or
fluoroacetamide. Nature (London) 208: 809-810.

Laue, H., J. A. Field, and A. M. Cook. 1996. Bacterial desulfonation of the
ethanesulfonate metabolite of the chloroacetanilide herbicide metazachlor.
Environ. Sci. Technol. 30:1129-1132.

Markwell, M. A., S. M. Hass, N. E. Tolbert, and L. L. Bieber. 1981. Protein
determination in membrane lipoprotein samples: manual and automated
procedures. Methods Enzymology 72:296-301.

Meyer, J. J. M., N. Grobbelaar, and P. L. Steyn. 1990. Fluoroacetate-
metabolizing Pseudomonad isolated from Dichapetalum cymosum. Appl.
Environ. Microbiol. 56:2152-2155.

Meyer, J. J. M., and D. O'Hagan. 1992. Conversion of 3-f1uoropyruvate to
fluoroacetate by cell-free extracts of Dichapetalum cymosum. Phytochemistry
3 1:2699-2701.

Oremland, R. S., L. J. Matheson, J. R. Guidetti, J. K. Schaefer, and P. T.
Visscher. 1995. Summary of research results on bacterial degradation of
trifluoroacetate (TFA), November, 1994-May,1995. USGS. OF 95-0422.

Seitz, A. P., E. R. Leadbetter, and W. Godchaux III. 1993. Utilization of
sulfonates as sole sulfur source by soil bacteria including Comamonas
acidovorans. Arch. Microbiol. 159:440-444.

Thompson, A. S., N. J. P. Owens, and J. C. Murrell. 1995. Isolation and
characterization of methanesulfonic acid-degrading bacteria from the marine
environment. Appl. Environ. Microbiol. 61 :2388-2393.

Thysee, G. J. E., and T. H. Wanders. 1974. Initial steps in the degradation of
n-alkane-l-sulphonates by Pseudomonas. Antonie Leeuwenhoek J. Microbiol.
40:25-37.

Tonomura, K., F. Futai, O. Tanabe, and T. Yamaoka. 1965. Defluorination
of monofluoroacetate by bacteria; Part 1. Isolation of bacteria and their activity of
defluorination. Agr. Biol. Chem. 29:124—128.

20.

21.

22.

23.

80

Uria-Nickelsen, M. R., E. R. Leadbetter, and W. Godchaux III. 1993.
Sulphonate utilization by enteric bacteria. J. Gen. Microbiol. 139:203-208.

Uria-Nickelsen, M. R., E. R. Leadbetter, and W. Godchaux III. 1994.
Comparative aspects of utilization of sulfonate and other sulfur sources by
Escherichia coli K12. Arch. Microbiol. 161:434-438.

Visscher, P. T., C. W. Culbertson, and R. S. Oremland. 1994. Degradation
of trifiuoroacetate in oxic and anoxic sediments. Nature 369:729-731.

Walker, J. R. L., and B. C. Lien. 1981. Metabolism of fluoroacetate by a soil
Pseudomonas sp. and Fusan'um solani. Soil Biol. Biochem. 13:231-235.

CHAPTER 5

BIOTRANSFORMATION OF SULFUR-CONTAINING
ORGANOFLUORINE COMPOUNDS BY DIVERSE ORGANISMS

ABSTRACT

Previous work (Chapters 3 and 4) established that difluoromethane sulfonate (DFMS,
CHF2803Na), 2,2,2-trifluoroethane sulfonic acid (TES; CF3CH2$O3H), and
1H,1H,2H,2H-perfluorooctane sulfonic acid (H-PFOSA; C6F13C2H4$O3H) are
degraded by a Pseudomonad under aerobic and sulfur-limiting conditions in minimal
medium. The present study focuses on generalizing this observation to other bacteria and
yeast. The phylogenetically related Pseudomonasfluorescens, Pseudomonas
chloroaphis, and Pseudomonas stutzeri KC were tested. Bacillus subtilis, Escherichia
coli, and Saccharomyces cerevisiae were also evaluated. The bacteria were capable of
transforming sulfur-containing organofluorine compounds. However, E. coli was not
able to degrade H-PFOSA. Yeast was unable to utilize any of the organofluorine
compounds as a source of sulfur. Transformation was also evaluated in aquatic samples
and soil. No transformation was observed in the aquatic samples, but transformation
was detected in the soil samples. It is concluded that the ability to degrade fluorinated
sulfonates is not unique to strain D2 and in fact is widely distributed in nature. However,
the results also suggest that transformation in the environment requires aerobic and

sulfur-limiting conditions.

81

82

INTRODUCTION

Sulfur-containing organofluorine compounds are commonly used as catalysts, reagents,
surfactants, and pesticides [1, 2, 3, 4, 5, 6, 7, 9, 10]. Several of these are perfluorinated
compound such as perfluorooctane sulfonate (PFOSA; C3F17SO3Na), perfluorooctane
sulfonamide (C3F17SOzNH2), and trifluoromethane sulfonic acid (TFMS; CF3SO3H).
TFMS is one of the strongest organic acids known, has great thermal stability, does not
release fluoride in the presence of strong nucleophiles and resists both oxidation and
reduction [6]. These compounds generally have excellent chemical and thermal stability.
Partially fluorinated analogues of the perfluorinated sulfonates include difluoromethane
sulfonate (DFMS; CHF2803N a), 1H,lH,2H,2H-perfluorooctane sulfonic acid (H-
PFOSA; C5F13C2H4803H), and 2,2,2-trifluoroethane sulfonic acid (TES;
CF3CH2503').

A previous report (Chapter 3) describes experiments to assess biodegradation of TFMS,
DFMS, TES, H-PFOSA, and PFOSA by Pseudomonas strain D2. Under aerobic and
sulfur-limiting conditions, strain D2 utilized compounds with hydrogen substituents
(DFMS, TES, and H-PFOSA) as sulfur sources. D2 completely defluorinated DFMS
and partially defluorinated TES and H-PFOSA. The present study generalizes these
earlier findings to various other bacteria, yeast, and complex environmental samples

(activated sludge, soil, and water).

MATERIALS AND METHODS

Media and chemicals. A defined mineral medium containing (in grams per liter):
glucose, 2.0; K2HPO4, 3.5; KH2P04, 2.0; NH4C1, 1.0; MgC12°6H20, 0.5; 0.15M

CaC12°2H20 stock, 1.0 mllliter; trace elements stock I and II, 1.0 mllliter. Trace elements

83
stock I contained (in grams per liter): FeCl3, 1.36; CoC12-6H20, 0.2; MnC12°4H20,
0.122; ZnClz, 0.07; N azMoO4°2H20, 0.036; NiC12°6H20, 0.12; B3(OH)3, 0.062;
CuC12°2H20, 0.017. Concentrated H2SO4 was added at 2.5 ml/L to the trace elements
solution 1. Trace elements solution 11 contained (in grams per liter): N aZSeO3°5H20,
0.006; NaWO4-2H20, 0.033; Na2M004-2H20, 0.024. To this medium was added the
appropriate organofluorine sulfonate as the sulfur source. Ferrnenting cultures of E. coli
were prepared similar to that described above except that 5.5 g/L of Kzl-IPO4 and 3.0 g/L
KH2P04 were used. Nitrate-respiring cultures were prepared as described above with
the addition of 3.88 g/L NaNO3. Both the fermenting medium and nitrate-respiring
media were degassed under vacuum. Glucose ( 1 g/L) was used as a carbon source for
fermenting cultures and glycerol (0.5 g/L) was used as carbon source in aerobic and
nitrate-respiring cultures of E. coli. For experiments with yeast, 10 m1/L of a vitamin
stock and 5 ml/L of a amino acid stock were added. The vitamin stock contained (in
grams per liter): folate, 0.021; pyridoxine (B6), 0.21; nicotinic acid, 2.1; riboflavin; 0.18;
pantothenic acid, 0.53; p-arrrinobenzoic acid 0.025; and B12, 0.025. The amino acid
stock solution contained 4.2 g/L glycine and 4.2 g/L of the 21 essential L-amino acids
except methionine, cystine, and cysteine. Sodium difluoromethane sulfonate (DFMS)
was provided by 3M, St Paul, MN. Potassium perfluorooctane sulfonate (PFOSA) and
1H,lH,2H,2H-perfluorooctane sulfonic acid (H-PFOSA) were obtained from ICN
Pharmaceuticals Inc., Costa Mesa, CA. 2,2,2-Trifluoroethane sulfonyl chloride was
obtained from Sigma Chemical (St. Louis, MO). Hydrolysis of 2,2,2-trifluoroethane
sulfonyl chloride by addition to water and autoclaving yielded trifluoromethane sulfonate

(TES). All other chemicals were obtained from Sigma Chemical.

Experiments with diverse cell types. Strain D2 was identified as Pseudonwnas
fluorescens previously (Chapter 3). Pseudomonasfluorescens (ATCC deposit no.

17400) and Pseudomonas chloroaphis (ATCC deposit no. 9446) were obtained from

84

American Type Culture Collection (ATCC), Rockville, MD. Bacillus subtilis (ATCC
deposit no. 6051) and Escherichia coli (ATCC deposit no. 10798) were obtained from
the culture collection of the Microbiology Department at Michigan State University.
Pseudomonas stutzeri KC (ATCC deposit no. 55595) is routinely cultured in our
laboratory. Plate colonies of bacteria were inoculated into 5 ml of nutrient broth (Difco,
Detroit, MI) and incubated at 30° C for 24 hours. A 1% inoculum of bacteria was used
in the above medium (pH of 6.9-7.0) with the specified organofluorine compound as the
sole sulfur source. Cells were grown aerobically at 30 °C and were shaken on a rotary
shaker at 160 rpm. Saccharomyces cerevisiae (American Ale, type no. 1056) was
obtained from Wyeast Laboratories, Inc., Mt Hood, OR. Saccharomyces cerevisiae was
grown in malt extract for 36 hours and then inoculated at 1% of medium. Yeast were
grown aerobically 21 °C and were shaken on a rotary shaker at 160 rpm. E. coli was
used to determine if degradation of DFMS would occur under anaerobic conditions. A
1% inoculum of nutrient broth grown cell of E. coli were added to fermenting medium
and nitrate-respiring medium. Control cultures were prepared in medium that was

amended with sulfate as the source of sulfur.

Experiments with activated sludge, river water, and soil. To evaluate the fate of
fluorinated sulfonates in the environmental samples, samples of activated sludge were
obtained from the East Lansing Wastewater Treatment Facility, East Lansing, MI and
river water was obtained from the Red Cedar River, on the campus of Michigan State
University, East Lansing, MI. In addition three soils samples were also obtained from
various locations on campus including a sample from a woodland swamp (natural area),
a sample from a botanical garden (agricultural area), and a sandy soil from the banks of
the Red Cedar River. The river and activated sludge samples were prepared by placing
nine milliliters of the sample into sterile flasks (50 ml). One subset was amended with 1

ml sterile water (no added carbon source), another subset was amended with 1 ml of 20

85

g/L stock of glucose, and another was amended with 1 ml of 10 times concentrated
growth medium with glucose (20 g/L). Triplicate samples were prepared with 32 pM
DFMS as a source of sulfur and incubated at 21 °C. These samples were shaken on a
rotary shaker at 160 rpm. Soil samples were prepared by adding 2 g of soil to sterile
balch tubes (28 ml) and amending with 100 pl of 0.1 pg/pl of H-PFOSA stock solution
(+ H-PFOSA) or 100 pl sterile water (- H-PFOSA). In addition subsets of these
samples were amended with 200 pl of 10 mM glucose stock (+carbon) or 200 pl sterile
water (-carbon). For all tested conditions and soil types, killed controls were prepared by

autoclaving at 121°C for 30 minutes.

Analysis of biotransformation. Fluoride was measured using an ion selective electrode
(Orion 96-09 BN). A total ionic strength adjuster (TISA) was used to stabilize fluoride
measurements in samples. TISA was prepared by adding 57 ml of glacial acetic acid and
58 g reagent grade sodium chloride into 500 ml of deionized water. This mixture was
placed on ice for cooling, and the pH was adjusted to between 5055 with 5M sodium
hydroxide. This mixture was then diluted with 500 ml of water to a final volume of one

liter. One part TISA to one part sample was used to measure fluoride.

Mass spectrometry, GCIECD, and GC/AED analysis. GC/MS data were obtained
using a Perkin-Elmer GC with a Finnigan Ion Trap Mass Spectrometer. A DB624
capillary column (30 m x 0.25 mm x 1.4 pm) was used for separation of volatile
byproducts (J & W Scientific, Inc., Folsom, CA). Operating conditions were as follows:
flow of 30 cm/sec linear velocity, initial temperature 40° C for 4 minutes followed by a
10° C/minute ramp to 200° C, injector temperature of 250° C, transfer line temperature of
225° C. Samples were taken by injecting a solid phase rrricroextraction (SPME) fiber
assembly with a 100 pM polydimethylsiloxane coating (Supelco Inc., Bellefonte, PA) in

through the septum of the sample. This assembly was allowed to equilibrate for 30

86
minutes within the sample headspace before injecting onto the GC/MS. Identical
conditions were used for GC/ECD analysis with a Hewlett-Packard 5890 series 11
GC/ECD and a DB624 capillary column. A Hewlett-Packard 5890 series 11 GC with
operating conditions identical to those above was used with a Hewlett-Packard 5921 A
atomic emission detector (AED) for elemental analysis of volatile products of H-
PFOSA. Emission wavelengths and plasma gases used for the various elements were

previously detailed (Chapter 3).

Sulfate and fluoride measurement. Sulfate was measured on a Dionex ion
chromatograph model 2000i/sp fitted with an IonPac AS4A ion exchange column and a
Dionex IonPac AG4A guard column. This system utilizes an anion micromembrane
suppressor with a Dionex Conductivity Detector-II (CDM). The eluant was a carbonate
buffer (1.8 mM N azCO3 and 1.7 mM N aHCO3) with a flow rate of 2 ml/min. Fluoride

was measured using an ion selective electrode (Orion 96-09 BN).

RESULTS

Biotransformation of fluorinated sulfonates by diverse cell types. In previous
studies, Pseudomonas sp. strain D2 completely defluorinated DFMS and partially
defluorinated TES and H-PFOSA. In the present study, the phylogenetically related
Gram-negative and oxidase-positive bacteria, P. fluorescens , P. chloroaphis, and P.
stutzeri KC were all capable of complete defluorination of DFMS (Figure 5.1, P.
chloroaphis and P. stutzeri KC are not shown) with stoichiometric yield of fluoride.
These organisms showed partial defluorination of TES and H-PFOSA (Figure 5.2 and
Figure 5.3, P. chloroaphis and P. stutzeri KC are not shown). None of the tested
Pseudomonad strains were capable of growth on or transformation of the completely

fluorinated sulfonates TFMS and PFOSA.

87

Bacillus subtilis was chosen as a representative of the Gram-positive bacteria. This
organism was capable of defluorination of DFMS as shown in Figure 5.1. However, it
was not capable of degrading as much DFMS as the Pseudomonas strains in this
medium. Defluorination was also observed with TES (Figure 5.2) and H-PFOSA
(Figure 5.3), but again a smaller amount was degraded, as compared to the
Pseudomonas strains. B. subtilis was not capable of growth on or transformation of

TFMS or PFOSA.

Escherichia coli K-12 was selected as a representative Gram-negative, oxidase-negative,
facultative anaerobe. As shown in Figure 5 .1, when grown aerobically, E. coli K-12 was
capable of defluorinating DFMS, but like B. subtilis, it did not degrade as much DFMS
as the Pseudomonas strains. E. coli K-12 also showed a similar partial defluorination of
TES like that of the Pseudomonas strains and B. subtilis. However, E. coli K-12 was
not capable of growth on or transformation of H-PFOSA. In addition, K-l2 was not
capable of growth on or transformation of TFMS or PFOSA, when these compounds
were added as the sole source of sulfur. Furthermore, E. coli was not capable of growth
or transformation of DFMS under anaerobic conditions. Both fermenting and nitrate-
respiring conditions were evaluated with DFMS as the sole source of sulfur. When
sulfate was added to these anaerobic cultures, growth was observed yet no transformation

of DFMS occurred.

Yeast were not capable of degrading any of the tested fluorinated sulfonates even when

supplemented with amino acids and vitamins.

W
m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2: ZWA

 

 

 

 

 

Control P. DZ P. fluorescens B. subtilus E. coli S. cerevisiae
Organism
Figure 5.1. Defluorination of DFMS (32 pM) by various bacteria and yeast under

aerobic and sulfur-limiting conditions. Error bars represent the standard deviation of
triplicate samples.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

‘: EEEE

 

 

 

Control P. DZ P. fluorescens B. subtilus E. coli S. cerevisiae

Organism

Figure 5.2. Defluorination of 2,2,2-trifluoroethane sulfonic acid (32 pM) by various
bacteria and yeast under aerobic and sulfur-limiting conditions. Error bars represent the
standard deviation of triplicate samples.

 

 

 

 

 

 

 

 

 

 

.m. E E, Emm‘

 

 

 

 

 

 

 

Control P. DZ P. fluorescens B. subtilus E. coli S. cerevisiae

Organism

Figure 5.3. Defluorination of 1H,lH,2H,2H-perfluorooctane sulfonic acid (32 pM) by
various bacteria and yeast under aerobic and sulfur-limiting conditions. Error bars
represent the standard deviation of triplicate samples.

91

Biotransformations in aqueous samples. Two different aquatic samples were used to
determine the fate of fluorinated sulfonates in natural microbial communities: activated
sludge from a wastewater treatment facility and river water. To insure that these samples
were not carbon limited 2 g/L glucose was added to all but the no carbon control. It was
shown previously (Chapter 4) that ammonium is also required for the transformation of
DFMS, so complete growth medium was added to the carbon plus ammonium
replicates. No transformation was observed in any of the activated sludge and river
samples. Sulfate concentration in these samples was 1.1 mM for the activated sludge and

0.90 mM for the river water.

Biotransformations in soils. A volatile transformation product was detected by gas
chromatography with an electron capture detector (Figure 5.4) and GC/MS ion trap
(Figures 5.6 and 5.7) in all soil samples with and without added carbon. This peak was
not observed in the autoclaved controls nor in the samples without H-PFOSA. This peak
was identical to a peak found as a transformation product of H-PFOSA by Pseudomonas
sp. strain D2 (Figures 5.5, 5.8 and 5.9). Atomic emission analysis of these peaks

indicates that they are fluorinated and do not contain sulfur.

1.2e61
1.0e6j

l

ti0e5€
t10e5€
4.0e5‘:
2.0e5‘:

0-

92

Peak“

 

 

 

0

L... M

6 a 10 12 1h 16
Time (min.)

Figure 5.4. GC/ECD chromatogram of the natural area incubation with glucose and H-

PFOSA.

Peak :1 ]

 

 

 

 

 

 

 

 

 

 

 

W ”W

 

15
Time (min.)

Figure 5.5. GC/ECD chromatogram of strain D2 incubated with glucose and H-

PFOSA.

93

Scan Range: 1 - 984 Int = 968 1002 = 10144
mg -
Peak #1
TOI- -
l b

 

 

 

 

,.I.,.I.,.l.,.]...,
200 400 800 800 1000
3:21 8:41 10:01 13:21

Figure 5.6. GC/MS chromatogram of the natural area incubation with glucose and H-
PFOSA.

 

 

 

 

 

 

fluerageol‘l 110805 l‘lims258510589 1002:1533
m; .

i l-
5’9’4 77 .
11KB.

‘ 51 we 13: '

. 93' 119 .. 219 255

J.,“. I. .I 141 1". 1 1-. I 2??

'|'IT ‘1']‘l‘l‘l'l'l']‘l'l'l'l'l‘l'l'l‘lrl'l'l'l'l

so we 150 200 250

Figure 5.7. GC/MS spectrum number 604 of the natural area incubation with glucose
and H-PFOSA.

94

 

 

 

 

 

 

 

8cm Range! 1 - 1200 Int = 548 1002 = 218844
100; 4 10
1
1
1'01- Peak #1
8
. 67 9
1 2
3 5 .
1._._,~1.L1 M
I A'fij—‘WJ . I , I ,— I I 1
3M 51': 900
5:01 10:01 15:01

Figure 5.8. GC/MS chromatogram of strain D2 incubated with glucose and H-PFOSA.

 

 

 

 

 

average oftfltom Hims3821101m=23526
1M;
1
sari
mg!
1 1 77
119 . 277
1.1913. 1 1" 2117.2332151
fl'l' rtzri]'I'I'I'I'VITI'I'I']'I'I'T'T'I'I'I'I’I‘]
50 m 150 200 250 300

Figure 5.9. GC/MS spectrum number 604 of strain D2 incubated with glucose and H-
PFOSA.

95

DISCUSSION

As is shown in Figures 5.1 through 5.3, Pseudomonads related to strain D2 showed
transformation patterns that were similar to those of strain D2 with respect to DFMS,
TES, and H-PFOSA. B. subtilis was not as effective at transforming all of the
fluorinated sulfonate present. A minimal medium was used for all of these experiments,
so that the inability of B. subtilis to degrade all of the sulfonate present may be associated
with depletion of some essential nutrient or carbon source. This limited transformation
may also be the result of production of a toxic byproduct. A similar result was observed
with E. coli when DFMS was the sole sulfur source but not when TES was the sole
sulfur source. Interestingly, E. coli was not capable of growth on or transformation of
H-PFOSA. E. coli may lack the necessary transport mechanism to transport H-PFOSA
across the cell membrane or a different mechanism(s) of transformation may be present
in E. coli. Uria-Nickelsen et al. [8] demonstrated growth of S. cerevisiae on various
sulfonates such as taurine, isothionate, and cysteate. However, S. cerevisiae was not
capable of growth on any of the fluorinated sulfonates tested. The electronegativity of the
carbon-fluorine bond may inhibit the uptake of organofluorine sulfur compound or it

may inhibit the transformation of these compounds.

No transformation was observed in the river water or activated sludge samples. This is
most likely due to high background sulfate concentrations (0.9-1.1 mM sulfate) that
inhibits transformation of DFMS. However, in soil incubations with H-PFOSA a
volatile peak was observed. This product had a mass spectrum identical to one of the
peaks generated by incubations of Pseudomonas sp. strain D2 with H-PFOSA. The
mass/charge ratio for many of the fragments from this product are typical of fluorinated
compounds (i.e. 69, 100, 119, 131, 169, and 219). Figures 5.4 and 5.6 illustrate that this

product had the same run time and response to GC/ECD. In addition to these results,

96

atomic emission analysis of this peak indicates that it is fluorinated and does not contain
sulfur. These observations support the conclusion that H-PFOSA is transformed in soils
with the production of a volatile fluorinated compound that is identical to one of the
products of H-PFOSA degradation by Pseudomonas sp. strain D2. Further

characterization of this product may provide clues to the mechanism(s) of transformation.

The results of this work demonstrate that fluorinated sulfonates can be degraded by a
wide range of bacteria as well as within complex communities of microorganisms. In
addition, this data suggest that if the environmental restrictions of sulfur limitation and
aerobic conditions are satisfied then degradation of these compounds is possible. Future
work is needed to identify the mechanism(s) of transformation and to explain why
transformation is apparently limited to bacteria, with differences in the extent of

transformation and types of sulfonates susceptible to transformation.

10.

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Langlois, B. R. 1990. Difluoromethanesulfonic acid. Part II. A two-step route to
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Stang, P. J., and M. R. White. 1983. Triflic acid and its derivatives. Aldrichimica
Acta 16:15-22.

Trepka, R. D., et al. 1974. Synthesis and herbicidal activity of fluorinated N -
phenylalkanesulfonamides. J. Agr. Food Chem. 22:11] 1-1 1 19.

Uria-Nickelsen, M. R., E. R. Leadbetter, and W. Godchaux III. 1993.
Sulphonate-sulfur assimilation by yeasts resembles that of bacteria. FEMS
Microbiol. Lett. 114:73-78.

Vander Meer, R. K., C. S. Lofgren, and D. F. Williams. 1985.F1uoroaliphatic
sulfones: a new class of delayed-action insecticides for control of Solenopsis
invicta. J. Econ. Ent. 78:1190—1197.

Vander Meer, R. K., C. S. Lofgren, and D. F. Williams. 1986. Control of
Solenopsis invicta with delayed-action fluorinated toxicants. Pestic. Sci. 17:449-
455.

97

CHAPTER 6

BIODEGRADATION OF MONOFLUOROACETATE UNDER
DENITRIFYING CONDITIONS

ABSTRACT

This research investigated the potential for biodegradation of monofluoroacetate
(MFA), a fluorinated pesticide, under anaerobic conditions. Enrichment cultures were
evaluated for MFA degradation under denitrifying and sulfate-reducing conditions.
Defluorination was detected under both conditions. Although no isolates were obtained
from the sulfate-reducing enrichments, a bacterium, designated strain M7, was isolated
from the denitrifying enrichment. The isolate was capable of growth on MFA and
defluorination under denitrifying conditions. Phylogenetic analysis of the 16S rRNA
sequence for the isolate indicated a 96.0 to 97.5% match for the Bradyrhizobium genus.
When incubated with MFA, crude cell extracts of aerobically or anaerobically grown
strain M7 released glycolate and fluoride indicating hydrolytic defluorination. Strain
M7 was not capable of defluorinating molecules with more than one fluorine
substituent, but was capable of dechlorinating monochloroacetate, dichloroacetate, and
trichloroacetate. Other bacteria with the ability to utilize MFA under aerobic conditions
were evaluated under denitrifying conditions. Two of five isolates were capable of
growth on MFA under denitrifying conditions. These findings demonstrate that
defluorination of MFA is not limited to aerobic conditions and is likely widespread in

the biosphere.

98

99

INTRODUCTION

Monofluoroacetate (MFA) is one of the most toxic substances known, based on a lethal
dose (LD50) of 0.7-2.1 mg/kg for man [1]. Its toxicity is due to "lethal synthesis" of
fluorocitrate which inhibits the aconitase enzyme of the Kreb's cycle [22] although
recent investigations implicate fluorocitrate as a "suicide" substrate instead of a
competitive inhibitor [3]. MFA is produced naturally by plants in the genus
Dichapetalum, as well as Palicourea marcgravii, Acacia georginae, Gastrolobium
grandiflorum, and Oxylobium species [11, 12, 23]. The West African plant,
Dichapetalum toxicarium, produces m-fluorooleic acid, w-fluoropalmitic acid, and
possibly co-fluorocaprate and m-fluoromyristate [24]. Certain fungi are known to
produce fluorinated organics: Streptomyces clavus and Streptomyces cattleya produce
the fluorine containing antibiotics, nucleocidin and 4-fluorothreonine, respectively [12,

24, 25]. Streptomyces cattleya is also capable of producing MFA [25].

Given that certain plants can produce MFA, it is not surprising that several
microorganisms can metabolize MFA. While many investigators have reported aerobic
defluorination of MFA by bacteria (mainly Pseudomonads ) and fungi [6, 7, 8, 9, 15,
18, 19, 26, 29], few reports of MFA biodegradation under anaerobic environments are
available. Gregg et al. [10] demonstrated defluorination of MFA by genetically
modified rumen bacteria Butyrivibrio fibrisolvens. The engineered strain contained a
plasmid encoding MFA defluorination genes derived from Moraxella sp. strain B. The
goal of this research was to protect cattle from MFA poisoning by accidental ingestion
of MFA-containing plants. Evidently, there is only one other report of anaerobic MFA
degradation. Visscher et al. [28] reported reductive dehalogenation of MFA to acetate
under methanogenic conditions, but this transformation was not reproducible in

subsequent investigations [21].

100

The present report documents isolation and characterization of Bradyrhizobium sp.
strain M7, a facultative aerobe that is capable of growth on and defluorination of MFA
under denitrifying conditions. The defluorination reaction is shown to proceed by a
hydrolytic pathway like that utilized by aerobic organisms. The observation of MFA

degradation is then generalized to other bacterial species.

MATERIALS AND METHODS

Chemicals. Sodium monofluoroacetate, 95 %, was obtained from Aldrich Chemical
(Milwaukee, WI). Sodium difluoromethane sulfonate, sodium difluoromalonate, and
sodium tetrafluorosuccinate were provided for by 3M, St Paul, MN. All other carbon
sources and chemicals were obtained from Sigma Chemical (St. Louis, MO). Nutrient

agar and nutrient broth were purchased from Difco (Detroit, MI)

Denitrifying and sulfate-reducing enrichments. Primary effluent from the East
Lansing Wastewater Treatment Facility, East Lansing, Michigan was used as a source
of organisms for enrichment culture. Denitrifying enrichments were prepared by
amending this effluent with 2.35 mM sodium nitrate as electron acceptor and 5 mM
sodium monofluoroacetate as a carbon source. Sulfate-reducing enrichments were
initiated with 2.80 mM sodium sulfate as an electron acceptor. Enrichments were
sealed with teflon lined stoppers, and incubated at 20° C for approximately four weeks.
Subsequent enrichments and subculturing were performed in defined mineral medium
containing (in grams per liter 18 Mohm water): K2HPO4, 0.7; KHZPO4, 0.4; NaNO3,
0.4; NH4SO4, 0.2; MgSO4-7H20, 0.1; 0.15M CaC12-2H20 stock, 0.2 mllliter; trace
elements stock, 0.2 mllliter; yeast extract, 0.05. Trace elements stock contained (in

grams per 500 ml of 18 Mohm water): FeSO4, 0.68; NazMoO4-2H20, 0.12;

101
CuSO4°5H20, 0.125; ZnSO4-7H20, 0.29; Co(NO3)2°6H20, 0.145; NiSO4°6H20, 0.11;
NaSeO3, 0.018; B3(OH)3, 0.031; NH4VO3, 0.06; and MnSO4°H20, 0.505.
Concentrated H2804 was added at lml/L to the trace metals solution. After initial

subculturing, yeast extract was removed from the medium.

Bacterial strains and growth conditions. Strain M7 was grown to stationary phase
aerobically in medium containing 10 mM MFA, unless otherwise noted. Cells were
typically grown at 30 °C and shaken at 150 rpm on a rotary shaker. For MFA
degradation experiments, cells were harvested by centrifugation (15 minutes at 12,100
X g in a Beckman SS-34 rotor at 4°C), washed in buffered medium then resuspended in
fresh medium. For anaerobic incubations, bottles with cells and medium were degassed
with nitrogen, amended with MFA, and capped with teflon stoppers. Protein was
assayed using the modified Lowry method, with bovine serum albumin as the standard
[17]. Five isolates capable of aerobic growth on MFA were provided by M. Emptage
(DuPont Central Research and Development, Wilmington, DE). All of these isolates
were Gram-negative rods. Strain WSZ6b-12-Z and strain CFR1-16-BO have not been
identified, strain WS3-12-Z has been identified as a Pseudomonad by Vitek and Biolog,
strain N Zl4-5a has been identified asAncylobacter aquaticus by Biolog, and strain
DWI-9-G has been identified as Agrobacterium tumefaciens by MIDI (M. Emptage,
personal communication). These isolates were grown in the same medium used for

strain M7.

Chemical analysis. MFA, nitrate, nitrite, and fluoride were measured on a Dionex ion
chromatography model 2000i/sp fitted with an IonPac AS4A ion exchange column and
a Dionex IonPac AG4A guard column. This system utilizes an anion micromembrane
suppressor with a Dionex Conductivity Detector-II (CDM). The eluant was a carbonate

buffer (1.8 mM NazCO3 and 1.7 mM NaHCO3) with a flow rate of 2 ml/min. Fluoride

102

was also measured using an ion selective electrode (Orion 96-09 BN). In addition, ion
exclusion chromatography was used to determine fluoride and MFA using a Dionex
IonPac ICE-AS] column. The eluant for ion exclusion chromatography was 1.0 mM
octanesulfonic acid / 2% 2-propanol at a flow of 0.8 ml/min. Nitrous oxide was
detected using a thermal conductivity detector (TCD). A Hewlett-Packard 5890 GC
fitted with a molecular sieve column (13x, 80/100 mesh, Alltech Associates, Inc.,
Deerfield, IL) was operated isotherrnally at 150 C. Headspace samples (100 pl) were

injected with a 1 ml gas-tight syringe.

Metabolite analysis. Strain M7 was grown in 500 ml batches in IL Erlenmeyer flasks
for 5 days from a 1% inoculum. Actively transforming cultures were centrifuged at
12,100 X g for 15 rrrinutes in a Beckman SS-34 rotor at 4°C. Cells were then washed
twice and resuspended in 8 ml of buffered growth medium without MFA. Six
milliliters of this concentrated cell suspension was placed on ice and was disrupted by a
550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) for 10 minutes at 1 second
bursts at stage 5 (50% time on and 50% time off). The effective sonication time was 5
minutes. The sonicate was centrifuged for 14,000 X g for 30 minutes at 4°C and
divided into a supernatant and cell pellet. The supernatant (final protein concentration
of 35 pg/ml) from this crude cell extract was then incubated with 1 mM MFA in growth
medium and shaken at 150 rpm at 30 °C. One fraction was degassed with nitrogen, and
the other fraction remained aerobic. As a control for glycolate accumulation, whole
cells were also incubated with 1 mM MFA aerobically and anaerobically. Samples
were withdrawn periodically from both fractions and assayed for glycolate. Glycolate
was measured by ion exclusion chromatography as described above and by the

colorimetric method of Calkins [2].

103
Kinetic characterization. Growth kinetic parameters were determined using van
Uden's modified version of Monod's microbial growth model which includes

endogenous decay of microorganisms :

p = “max * (S / (Ks + S )) -b = (dX/dt)/X

where p is specific growth rate (d'l), llmax is the maximum specific growth rate (d'1 ),
S is the rate-limiting substrate concentration (mg/L), Ks is the half-velocity coefficient
(mg/L), b is the endogenous decay rate (d'1 ), t is time in days (d), and X is the
concentration of microorganisms (mg/L) [27]. The model used for substrate

consumption was:

U = (k * S) / (Ks + S) = -(dS/dt)/X

where U is the specific substrate utilization coefficient (d‘l) and k is maximum specific
substrate utilization coefficient (d'l ). Values for k and Ks were estimated using a
nonlinear curve fit obtained from Systat version 5.2.1 based on initial MFA degradation
rates at a fixed cell protein concentration. Yield (Y; mg protein/mg MFA) was
determined by dividing the change in total cell protein by the change in MFA
concentration. Maximum specific growth rate was calculated by taking the slope of the

equation:

111(X/Xo) = 11 max *1

where X0 is the initial cell concentration (mg/L).

104

The endogenous decay rate was calculated by taking the slope of the equation:

ln(X) = b*t.

Molecular characterization. To establish the phylogenetic relationship for M7, 16S
rRNA gene-based molecular analyses were performed. The total genomic DNA from
strain M7 was isolated using the SDS-based lysis method [30]. The 16S rRNA gene
was amplified as described previously [30]. The PCR amplified products were purified
and directly used as the templates for automated fluorescent sequencing [31]. The DNA

sequence was determined using 10 primers from both directions [30].

Sequences were assembled using assembling programs in the Genetic Computer Group
(GCG) software package [4], and a preliminary analysis was done by searching the
current databases (GenBank release 91.0 and EMBL release 44.0) using the program
FASTA. Sequences were then aligned manually to the 16S rDNA sequences of the
species, which showed high similarity scores in the outputs of FASTA, in the
previously aligned l6S rDNA sequence database, RDP (Ribosomal Database Project)
[16] using the GDE multiple sequence editor program from RDP. Initial phylogenetic
screening was constructed using the DNA distance program, Neighbor-Joining, in the
PHYLIP package [5] based on all 16S rDNA sequences from alpha subdivision. Based
on the initial phylogenetic results, appropriate subsets of 16S rDNA sequences were
selected and subjected to final phylogenetic analyses through maximum parsimony
method with a bootstrap procedure of 500 replicates, DNA distance matrix method,
Neighbor-Joining, and maximum-likelihood method. The SEQBOOT program was

used to obtain confidence levels.

105

RESULTS

Evaluation of initial enrichments and isolation of denitrifying bacteria. Both the
denitrifying and sulfate-reducing enrichments demonstrated defluorination of MFA
(Figure 6.1). The sulfate-reducing enrichments turned black as evidence of sulfate
reduction with removal of sulfate as measured by ion chromatography. Nitrate and
nitrite were completely depleted in the denitrifying enrichments. No isolates were
obtained from the sulfate-reducing enrichments. However, an isolate designated strain
M7 was isolated aerobically from the denitrifying enrichment by streaking on 1.5 %
(wt/vol) nutrient agar plates. Colonies were small (approximately 1 mm) and appeared
only after a lengthy incubation period. Colonies were opaque specks and did not

spread.

Figure 6.2 illustrates defluorination of MFA, with the corresponding reduction of nitrate
and accumulation of fluoride ion. A minor accumulation of nitrite during log growth
phase was observed followed by subsequent utilization. Nitrous oxide was detected in
the headspace of anaerobically grown cultures. Growth kinetic data for this strain M7

under both aerobic and anaerobic conditions are provided in Table 6.1.

106

 

 

 

 

 

 

 

 

 

 

 

 

6 1
.. . :3“ E
E I sulfate control E /
t; B sulfate +MFA E /
:3 1 II]! nitrate control ; E /
g D nitrate + MFA E /
11.. / __ /

2 E E /

r = r
E a e
r a E
O W // f — n é

 

 

 

 

 

 

O
:3
34:
Am
D.
u
x<
w
v
0‘
C

Figure 6.1. Defluorination of MFA by denitrifying and sulfate-reducing enrichments.
Error bars represent the standard deviation of triplicate sammes.

Concentration (mM)

Concentration (mM)

0.6

    

0.2

107

 

  

 

fluoride

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time (days)

10 12
I r 8.2
(_— log(CFU/ml) - 8.1
1- 8.0
~ 7.9
L I l l 7.8
4 6 8 10 12

Figure 6.2. Growth of Bradyrhizobium sp. strain M7 on MFA under denitrifying
conditions: (a) consumption of MFA and production of fluoride, (b) consumption of
nitrate and growth of cells. Error bars represent the standard deviation of triplicate

samples.

10g(CFUlm1)

108

Table 6.1. Kinetic parameters for Bradyrhizobium sp. strain M7

 

 

Kinetic parameter Denitrifying conditions Aerobic conditions
k(d'1) 3.0 :t 0.30 4.9 1 0.40

Ks (mg MFA/L) 4.9 3: 0.20 26 :1: 7.0

b1 (d'l) 0.14 i 0.02 0.14 x 0.02

b2 (d'l) 0.013 :t 0.002 N/A

Y (mg protein/mg MFA) 0.13 t 0.01 0.15 i 0.01

“max (d'l) 0.29 i 0.02 0.52 1 0.03

 

Physiological characterization . Strain M7 is a motile, Gram-negative rod (2.5-4 by
0.8 pM) that utilizes MFA as its sole source of carbon under aerobic and denitrifying
conditions. Liquid cultures of strain M7 grow slowly, requiring 2-3 days to enter log
phase with a 1% inoculum in nutrient broth. It is both catalase positive and oxidase
positive. Optimal growth is observed at 30° C. Strain M7 was classified as a denitrifier
based on a growth yield that was proportional to the amount of nitrate reduced, use of
both nitrate and nitrite as electron acceptors for MFA degradation, and detection of
N20 during anaerobic growth. Fatty acid profiles were performed by Microbial ID,
Inc. (MIDI), Newark, Delaware. Although no match to the MIDI library was obtained
there were some unique fatty acids present: 48% of the fatty acids were present as cis-
11-octadecenoic acid, 18.5% as octadecenoic acid, and 14.3% as cis-9-hexadecenoic

acid.

Phylogenetic analysis. Phylogenetic analyses established by bootstrap parsimony
method showed that strain M7 is affiliated with the Rhizobium-Agrobacterium group of
the alpha subdivision of the Proteobacteria, and very closely related to Bradyrhizobium

species (Figure 6.3). Similar trees were also obtained by DNA distance matrix methods

109

and maximum likelihood methods. Strain M7 has 96.0 to 97.5% similarities to
Bradyrhizobium species. Since the similarities of 16S rRNA genes from most
Bradyrhizobium species range from 95.5 to 99.9%, it is proposed that strain M7 is a

new unidentified species of the genus Bradyrhizobium.
_E Rhodopseudomonas palustris strain ATH 2.1.6
75
F1. palustrist

Bradyrhizobium sp. strain LMG 9980
8. sp. strain LMG 9966

 
   
  

—*34

B. sp. strain LMG 9514
8. sp. strain LMG 10689

 

 

*
_ 100 '8. sp. strain M7|

 

B. japonicum strain LMG 6138

B. japonicum strain USDA 136

    
  
 

 

‘ 1100 1 Nitrobacter sp. strain LL

N. winogradskyi strain W

. hamburgensis strain X14

_EAgmbacterium tumefaciens strain ATCC 4720
100
A. rubi LMG 156

— Escherichia coli

 

 

Figure 6.3 Phylogenetic tree showing the location of Bradyrhizobium sp. strain M7 in
relation to other Bradyrhizobium species.

110

MFA degradation. When aerobically grown cells were converted to oxygen-free
conditions, specific substrate utilization rates were reduced by a sixth (k, 0.83 :t 0.17 d'
1; Ks, 11 :i: 2 mg/L) as compared to cells that were incubated aerobically (see Figure
6.4). When anaerobically grown cells were converted to aerobic conditions however,
there was no detectable inhibition of rates as compared to cells that were incubated
anaerobically (data not shown). Cells that were grown anaerobically and then incubated
anaerobically with MFA had a lower specific substrate utilization rate (R) and a lower
half-velocity coefficient as compared to aerobically grown and aerobically incubated

cells.

111

 

I 1 I ' r ' I ' I

 

5 -0 aerobic growth/aerobic incubation

. 'r 7
4.0 I.

 

  

 

T
3.0
anaerobic growth/anaerobic incubation T
4

 

2.0

 

aerobic growth/anaerobic incubation

1.0 i _I

q

Specific substrate utilization me
(mg MFAimg proteiniday)

 

 

 

 

0. 0 1 l . l 1 I l l n l
O 100 200 300 400 500

MFA (mg/L)

Figure 6.4. Comparison of specific substrate utilization rates for aerobically and
anaerobically grown Bradyrhizobium sp. strain M7. Error bars represent the standard
deviation of triplicate samples.

Identification of glycolate as a metabolite of MFA degradation. Several
investigators have demonstrated that the first step in degradation of MFA is hydrolytic
attack of the carbon-fluorine bond resulting in glycolic acid as an intermediate [6, 7,
26]. Aerobic and anaerobic cell extracts and whole cells of strain M7 were incubated
with MFA to assay glycolate production. No accumulation of glycolate was observed
for aerobic or anaerobic whole cells. However, supernatant from crude cell extracts
from aerobically grown cells, incubated both anaerobically and aerobically, showed

stoichiometric accumulation of glycolate and fluoride (see Table 6.2)

112

Table 6.2. Glycolate determination in cell free extract of strain M7 .
Values were obtained by ion exclusion chromatography. Glycolate
levels were confirmed by colorimetric assay.

 

 

Glycolate (uM) MFA (uM)
produced converted

Anaerobic
whole cells 0 1215
Anaerobic
supernatant 276 282
Aerobic
whole cells 0 1200
Aerobic
supernatant 305 326

 

Degradation of other halogenated compounds. As shown in Table 6.3, strain M7
was unable to defluorinate compounds with more than one fluorine substituent.
However, chlorinated analogues of MFA monochloroacetate, dichloroacetate, and
trichloroacetate degraded both aerobically and anaerobically using cells that had been
grown on MFA. Bradyrhizobium sp. strain M7 was not capable of growth on the

chlorinated analogues under aerobic or anaerobic conditions.

MFA degradation by other bacteria under denitrifying conditions. Five strains
capable of MFA degradation aerobically were evaluated for MFA degradation under
denitrifying conditions. Two of the five tested strains, strains DWl-9-G and WS3-12-
Z, were capable of growth on MFA under denitrifying conditions. Strain WS3-l2-Z
showed some accumulation of nitrite with MFA degradation. None of the other strains
were capable of growth under denitrifying conditions with MFA as the sole carbon

source.

113

Table 6.3. Substrate degradation and growth by Bradyrhizobium sp. strain M7

 

 

 

Denitrifying conditions Aerobic conditions
substrate substrate

Substrate degradation growth degradation growth
monofluoroacetate + + + +
difluoroacetate - - - -
trifluoroacetate - - - -
difluoromalonate - - - -
tetrafluorosuccinate - - - -
trifluoromethane

sulfonate - - - -
difluoromethane

sulfonate - - - ..
monochloroacetate + - + -
dichloroacetate + - + -
trichloroacetate + - + -
glycolate + + + +
glyoxylate + + + +
oxalate + + + +

DISCUSSION

Although many bacteria are known to use MFA as a growth substrate under aerobic
conditions [6, 7, 8, 9, 15, 18, 19, 26, 29], this is the first report of a naturally occurring
bacterium using MFA as a growth substrate under anaerobic conditions. It is also the

first report of a Bradyrhizobium species possessing this trait. The results with other

114

MFA-degrading bacteria demonstrate that the ability to degrade MFA under anaerobic
conditions is not unique to strain M7. For example, strains DWI—9-G and WS3-l2-Z
were also able to grow on MFA under denitrifying conditions. Although no isolate was
obtained from the sulfate—reducing enrichments, defluorination of MFA was observed
under this condition. Therefore, it may be possible to obtain a sulfate-reducing isolate
that is capable of degrading MFA. Clearly, the ability to degrade MFA under anaerobic
conditions is not restricted to the Bradyrhizobium and in fact several other bacteria can
degrade MFA under anaerobic conditions. These findings suggest that this trait is

widely distributed in nature.

Defluorination of MFA, by strain M7 under denitrifying conditions appears to proceed
by the same hydrolytic mechanism and pathway as aerobic MFA degradation although,
it remains to be seen whether a single enzyme system is responsible for the
transformation under both conditions. MFA was defluorinated hydrolytically with
production of glycolate as an intermediate under both aerobic and anaerobic conditions.
The results of this study suggest that the same or a similar halidohydrolase produced by
aerobes capable of MFA degradation is also produced by strain M7 and other
denitrifying bacteria. The pattern of hydrolytic defluorination, with the production of
glycolate, and degradation of the chlorinated analogues , is similar to results obtained
by others [6, 9, 29]. The existence of a transmittable plasmid could explain the broad
distribution of MFA-degrading capabilities among different genera of bacteria.
Kawasaki et a1.[l3] demonstrated that the halidohydrolase responsible for
defluorination of MFA is plasmid encoded in Moraxella sp. strain B. Kawasaki et a1.
and others have cloned this plasmid into Escherichia coli [14] and Buryrivibrio
fibrisolvens. [10]. In addition, Meyer and van Rooyen [20] have transferred the plasmid

responsible for MFA defluorination from Pseudomonas cepacia into Bacillus subtilis

115

and have shown that the genetically engineered B. subtilis was also capable of

defluorinating MFA.

As shown in Table 6.1, the maximum specific growth rate (u max ), the maximum
specific substrate utilization rate (k), and the half-velocity coefficient constant (Ks) are
different for aerobically grown cells as compared to the anaerobically grown cells.
Another difference between aerobic and anaerobic degradation of MFA can be seen in
Figure 6.4. When aerobically grown cells are incubated under anaerobic conditions,
transformation of MFA is inhibited. However, the contrary is not true when anaerobic
cells are incubated aerobically. The inhibition of aerobically grown cells incubated
under anaerobic conditions is dramatic, with a one sixth reduction of maximum specific
utilization rates. These findings are not readily explained. It is possible that some
transport system that functions under aerobic conditions is affected under anaerobic
conditions or perhaps different enzyme systems are involved under aerobic and

anaerobic conditions.

The results of this study demonstrate defluorination of MFA and growth on MFA as
sole carbon and energy source under aerobic and anaerobic conditions by
Bradyrhizobium sp. strain M7, a genus previously unknown to possess dehalogenating
activity. In addition this work establishes defluorination of MFA by other MFA-
degrading organisms and by a sulfate-reducing enrichment. The mechanism for
degradation of MFA by strain M7 is hydrolytic attack yielding glycolate and fluoride
under both aerobic and anaerobic conditions. As observed in aerobic systems the
hydrolytic activity is not observed with more highly fluorinated analogues, but is

observed with chlorinated analogues.

10.

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Oremland, R. S., L. J. Matheson, J. R. Guidetti, J. K. Schaefer, and P. T.
Visscher. 1995. Summary of research results on bacterial degradation of
trifluoroacetate (TFA), November, 1994-May,1995. USGS. OF 95-0422.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

118

Peters, R. 1972. Some metabolic aspects of fluoroacetate especially related to
fluorocitrate, pp. 55-76. In (ed.), Carbon-fluorine compounds: Chemistry,
biochemistry, and biological activities (A Ciba Foundation Symposium).
Associated Scientific Publishers, Amsterdam.

Peters, R., and R. J. Hall. 1960. Fluorine compounds in nature; the distribution
of carbon-fluorine compounds in some species of Dichapetalum. Nature
187:573-575.

Suida, J. F., and J. F. DeBernardis. 1973. Naturally occurring halogenated
organic compounds. Lloydia 36:107-143.

Tamura, T., M. Wada, N. Esaki, and K. Soda. 1995. Synthesis of
fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by
Streptomyces cattleya. J. Bacteriol. 177:2265-9.

Tonomura, K., F. Futai, 0. Tanabe, and T. Yamaoka. 1965. Defluorination
of monofluoroacetate by bacteria; Part 1. Isolation of bacteria and their activity
of defluorination. Agr. Biol. Chem. 29:124-128.

van Uden, N. 1967. Transport-limited growth in the chemostat and its
competitive inhibition; a theoretical treatment. Archiv fur Mikrobiologie
58:145-154.

Visscher, P. T., C. W. Culbertson, and R. S. Oremland. 1994. Degradation of
trifluoroacetate in oxic and anoxic sediments. Nature 369:729-731.

Walker, J. R. L., and B. C. Lien. 1981. Metabolism of fluoroacetate by a soil
Pseudomonas sp. and Fusarium solani. Soil Biol. Biochem. 13:231-235.

Zhou, J.-Z., M. R. Fries, J. C. Chee-Sanford, and J. M. Tiedje. 1995.
Phylogenetic analyses of a new group of denitrifiers capable of anaerobic
growth on toluene: Description of Azoarcus tolulyticus sp. nov. Int. J. Syst.
Bacterol. 45:500-506.

Zhou, J.-Z., and J. M. Tiedje. 1995. Gene transfer from a bacterium injected
into an aquifer to an indigenous bacteria. Mol. Ecol. 4:613-638.

CHAPTER 7

CONCLUSIONS AND FUTURE WORK RECOMMENDATIONS

CONCLUSIONS

1. The ability to transform fluorinated sulfonates is restricted at a molecular or
structural level. Completely fluorinated sulfonates were not degraded by the organisms
evaluated in this work. However, fluorinated sulfonates containing hydrogen (DFMS,

TES, and H-PFOSA) were defluorinated and used as sources of sulfur for growth.

2. The ability to transform fluorinated sulfonates was restricted by physiological
conditions. No transformation of fluorinated sulfonates was observed under anaerobic
conditions. Transformation was inhibited by other sulfur sources and was completely
inhibited when other sulfur sources were present at levels sufficient to support growth.
DFMS was not utilized as a carbon source. A carbon and nitrogen source were required

for transformation by whole cells.

3. Transformation of fluorinated sulfonates under aerobic and sulfur-limiting
conditions was linked to a sulfur-scavenging system. Based upon inhibition studies
with other sulfur sources it is suggested that desulfonation of the molecule is followed

by sulfur assimilation through existing sulfur assimilation pathways.

119

120

4. Although DFMS was completely defluorinated, with stoichiometric yield of fluoride,
TES and H-PFOSA were only partial defluorinated. Transformation of H-PFOSA by
Pseudomonas sp strain D2 generated several volatile and fluorinated products. None of

these products contained sulfur.

5. In addition to strain D2, several other Pseudomonas species could utilize DFMS,
TES and H-PFOSA as sulfur sources for growth. The gram positive species Bacillus
subtilis was also capable of transformation of DFMS, TES, and H-PFOSA. Escherichia
coli was capable of degrading DFMS and TES, but did not grow on or transform H-
PFOSA. Saccharomyces cerevisiae was not able to utilize fluorinated sulfonates as a

sulfur source.

6. Samples of river water and activated sludge showed no transformation of DFMS.
However, soil samples incubated with H-PFOSA generated a volatile product that is
most likely fluorinated. It appears that the ability to degrade partially fluorinated
sulfonates is widely distributed among bacteria, but this ability may not be expressed if

environmental conditions are not suitable.

7. The bacterium Bradyrhizobium sp. strain M7 was shown to have MFA.degrading
abilities. Strain M7 used MFA as a carbon source under both aerobic and anaerobic

conditions.

8. A few bacteria that use MFA as a carbon source under aerobic conditions can also
use it as the carbon source under denitrifying conditions. In addition, defluorination
was observed under sulfate-reducing conditions. These findings suggest that the ability
to defluorinate MFA is most likely widespread throughout nature including some

anaerobic environments.

121

9. The pathway for MFA degradation under denitrifying conditions was hydrolytic
attack, with release of fluoride and production of glycolate. This pathway is similar

under both aerobic and anaerobic conditions.

FUTURE RESEARCH

1. The volatile byproducts of H-PFOSA transformation should be identified.

2. Improved analytical methods for direct detection and quantification of fluorinated
sulfonates in environmental samples are needed. This should include improved

methods for sample preparation, separation of mixtures, and detection.

3. The enzyme(s) or cofactors responsible for the transformation of the fluorinated
sulfonates should be characterized. This could provide insight into the reasons for
inhibition by other sulfur sources and will likely provide clues into the nature of the
reaction mechanism ( i.e. oxidative, hydrolytic, nucleophilic, etc.). It will also help to
explain why some organisms are capable of the transformation (bacteria) while others

are not (yeast).

4. Further evaluation of the fate of these compounds in environmental samples is
needed. In addition, the fate of fluorinated sulfonates in anaerobic environments should

be evaluated.

5. Further work is needed to determine the fate and identity of the byproduct(s) of TES

transformation.

APPENDIX A

APPENDIX A

GC/MS DATA USING ELECTRON IMPACT WITH ION TRAP DETECTOR

GC/MS (Ion Trap) analysis:

GC:
MS:

Column:

Operation:

Injector:

Transfer Line:

Sample:

Perkin Elmer Autosystem GC

Perkin Elmer Ion Trap Detector

DB624 capillary column (30 m x 0.25 mm x1.4 pm) from
J & W Scientific (Folsom, CA)

40°C for 4 minutes, 10°C/minute ramp to 200°C

250°C

225°C

Headspace for 15-30 minutes with a solid phase
microextraction (SPME) fiber assembly with a 100 uM
polydimethylsiloxane coating (Supelco, Inc.,

Bellefonte, PA)

122

123

Scanllangei 1-1200 lnt=548 lm=218844
100; 4 10

1'01- Peak #1

 

 

 

 

 

 

 

300
5201

 

Figure A.l. GC/MS chromatogram of strain D2 incubated with H-PFOSA and glucose.

124

 

 

 

 

 

 

 

 

Mange ofiflto I7 Hinus282110025 1002=23528
100% 00
mi
0881
+51 100 131
‘ 77
118 . 277
119?. I 1'. 47.233275 I
I|Tl' 'I'I'[’I'l'l'l'l'l'l'l'l'I'erT'l—FI'I'T‘I'I'I']
50 100 150 200 250 300

Figure A.2. GC/MS spectrum for peak #1 of strain D2 incubated with glucose and H-
PFOSA.

 

 

Hass Intensity 2 Base
50 4.584 13 13
51 8.426 24.24
65 4,213 12.12
68 34,758 100.00
75 3.158 8.08
77 4,315 14.14
80 2,808 8.08
83 3.510 10.10
84 2,108 0.00

100 8,479 27.27
131 8,777 25.25
163 2,808 8.08
207 2,808 8.08
255 3,153 3.03
277 4.564 13.13

 

 

 

Table A.1. GC/MS listing for peak #1 of strain D2 incubated with glucose and H-
PFOSA.

125

 

 

 

 

 

 

 

 

liver-age or: 71810722 Minus: 70710711 1002:4234
m; as _
1
. 95
sup. .
m.
55 77 131
118' .
1| .11 ..I 10231 295.
1'1 'I'I‘IJIITIFTT'TTI'I'I li'l'rl'l'r'l'l'lrftl't'v
so m 150 200 250 see

Figure A.3. GC/MS spectrum for peak #2 of strain D2 incubated with glucose and H-
PFOSA.

 

Hess Intensity 2 Base Mass Intensity 2 Base
50 454 4.71 100 1,565 16.23
51 1,565 16.23 118 808 8.42
54 404 4.18 127 1,816 16.75
55 1,212 12.57 131 2.020 20.84
56 707 7.33 181 353 3.66
57 5.76 285 303 3.14
64 858 8.80 305 303 3.14
65 3,283 34.03
67 505 5.24
00 8,648 100.00
70 353 3.66
74 404 4.18
75 1,010 10.47
77 1,111 11.52
83 1,212 12.57
85 4.182 43.46

 

 

 

 

 

Table A2. GC/MS listing for peak #2 of strain D2 incubated with glucose and H-
PFOSA.

126

 

 

 

 

 

 

 

fluerage or: 738 to 742 Minus: 74710 751 100: = 1543
mg 75 _
,
9?. r
m.
‘ 127
1" 157
1- 276
11.1.: I. 1-191 257 m9
l'l’ IJ'ITI'TTT r'r'r'
so 100 159 200 250 an

 

Figure A.4. GC/MS spectrum for peak #3 of strain D2 incubated with glucose and H-
PFOSA. -

 

 

Hess Intensity 2 Base ass Intensity 2 Base
50 180 5.53 112 120 3.68
51 587 17.87 118 256 7.83
57 256 7.83 124 165 5.07
65 150 4.61 127 602 18.43
67 120 3.68 131 873 26.73
68 3,268 100.00 137 135 4.15
71 120 3.68 157 361 11.06
75 2,801 85.71 168 210 6.45
76 150 4.61 181 451 13.82
77 813 24.88 218 105 3.23
83 286 8.76 257 120 3.68
85 225 6.81 276 185 5.88

100 487 15.21 281 225 6.81
107 331 10.14
108 165 5.07
111 75 2.30

 

 

 

 

 

Table A3. GC/MS listing for peak #3 of strain D2 incubated with glucose and H-
PFOSA.

127

 

 

 

Huerage or: 753 to 753 11171115: 747 to 751 1333 = 43252
133; 75
5111’.
mo.

1 127 157

137 278 327
“9 133 213 257 293
so 103 15o 233 253

Figure A.5. GC/MS spectrum for peak #4 of strain D2 incubated with glucose and H-
PFOSA.

 

3'
E!
5

_li_as_s Intensity ZBase

 

 

 

 

50 2,800 4.32 157 10.181 15..
51 7,372 11.35 257 2,67 3.78
57 6,318 8.73 275 2,67 3.78
65 6,318 8.73 276 6.319 8.73
29,11! 44.86 327 8.075 12.43
70 2,106 3.24
75 64,852 100.00
76 3,510 5.41
77 13,341 20.54
1“ 6,670 10.27
107 6,318 8.73
108 3,158 4.86
113 2,106 3.24
124 3,862 5.85
127 11,235 17.30
131 2,608 4.32

 

Table A.4. GC/MS listing for peak #4 of strain D2 incubated with glucose and H-
PFOSA.

 

128

 

 

 

 

Mrageol‘27661077011ims275010754 1332:3333
133; 31
”4
3113.

‘ 75

133
“9 157 213 243 275233VMW
so no 153 zoo 253 333

Figure A.6. GC/MS spectrum for peak #5 of strain D2 incubated with glucose and H-
PFOSA.

 

 

Mass Intensity 2 Base Mass Intensity 2 Base
50 144 2.55 101 144 2.55
51 372 6.57 107 186 3.28
56 124 2.18 118 310 5.47
58 248 4.38 131 578 10.22
00 186 3.28 181 331 5.84
61 5,672 100.00 207 144 2.55
62 165 2.82
00 372 6.57
65 227 4.01
00 3,208 56.57
70 124 2.18
75 000 15.33
77 268 4.74
78 805 6.83
83 310 5.47

100 486 8.76

 

 

 

 

 

 

Table A5. GC/MS listing for peak #5 of strain D2 incubated with glucose and H-
PFOSA.

129

Mrageoft77613780111ms278410788 1333:2317
133; 33

 

 

 

 

 

135 121
1 ‘35 213

II
[IlrlrltIrrvlr'I'r'UIW'TrIIIIUITTrIIIIIIIIIIII'lll

150 2” 250 3“

 

 

 

 

Figure A.7. GC/MS spectrum for peak #6 of strain D2 incubated with glucose and H-
PFOSA.

 

 

 

Hass Intensity 2 Base Mass Intensity 2 Base
50 4 7.78 81 5.84
51 532 13.64 81 3,142 80.52
53 354 8.08 82 1,78 46.10
55 633 16.23 83 3,802 100.00
62 152 3.80 84 30 7.78
63 177 4.55 100 22 5.84
65 28 8.44 105 456 11.68
66 481 12.34 106 202 5.18
67 887 22.73 107 202 5.18
68 177 4.55 121 532 13.64
68 1,485 38.31 131 532 13.64
70 126 3.25 136 228 5.84
77 1,875 48.05
78 760 18 48
78 2,230 57 14
8 557 14 28

 

 

 

 

 

Table A6. GC/MS listing for peak #6 of strain D2 incubated with glucose and H-
PFOSA.

130

 

 

average of: 782 to 786 Minus: 800 to 804 1002 = 3588
100;

sari

BKCJ

59 77 91 245
1' 281
1 19131 195
218 327
50 133 150 200 250 30

Figure A.8. GC/MS spectrum for peak #7 of strain D2 incubated with glucose and H-
PFOSA.

 

 

 

Mass Intensitu 2 Base 0355 Intensity 2 Base
5 518 6.67 100 1,178 15.15
51 1,743 22.42 108 1,036 13.33
5 658 8.48 118 612 7.88
58 1,508 18.38 131 1,318 16.87
61 706 8.08 185 518 6.67
64 518 6.67 218 424 5.45
65 842 12.12 245 1,602 20.61
68 7,775 100.00 281 1,036 13.33
70 328 4.24
71 1,036 13 33
75 706 8 08
77 1,460 18 78
88 754 8 70
81 1,508 18 38
83 471 6 06
85 565 7 27

 

 

 

 

 

Table A7. GC/MS listing for peak #7 of strain D2 incubated with glucose and H-
PFOSA.

r r‘r 7"

 

131

averageoi‘202610830 Hims261800623 100$=1H

 

 

 

 

1332 57 _
i
1
3112; .
01181
. 231 t
77 109 139 . ' _
13217518 237 233 265 333 336
50 100 150 2M 250

Figure A.9. GC/MS spectrum for peak #8 of strain D2 incubated with glucose and H-
PFOSA.

 

Hass Intensity 20352 11355 Intensity 20352 11355 Intensity 28352
50 672 3.48 1,185 6.20 308 672 3.48
51 3,52 1S.I I 672 3.48 308 448 2.33
56 7% 3.88 81 587 3.10 336 522 2.71
57 18,268 133.33 100 1.269 6.58 337 522 2.71
58 870 5.04 107 1,418 7.36 348 448 2.33
58 3,0I18.77 1I1,841 10.08
64 886 4.65 118 1,06 5.43
65 886 4.65 126 448 2.33
66 373 1.84 131 870 5.04
8,365 43.41 138 1.568 8.14
75 1,344 6.88 181 587 3.10
76 7% 3.88 185 821 4.26
77 1,782 8.30 207 522 2.71
78 1,06 5.43 252 522 2.71
87 587 3.10 255 522 2.71
88 672 3.48 257 746 3.88

 

 

 

 

 

 

 

Table A8. GC/MS listing for peak #8 of strain D2 incubated with glucose and H-
PFOSA.

132

 

 

 

 

 

 

 

 

 

8057392011 334111333 "1711152710801 1332=5572
133; 153 _
, 59 117
337, -
3113.
‘ so
4 144
. 100 131 3‘7
1.. . .J l 175 251275 332 l 3i!
'1‘ 'I'l'l'l T'l'l 'I'I'F'I'ITI'I‘I‘I'l‘l‘l'l'l‘l"I'l'
so 133 153 233 253 333

Figure A.10. GC/MS spectrum for peak #9 of strain D2 incubated with glucose and H-
PFOSA.

 

 

Hass Intensity 2 Base 0355 Intensity 2 Base
50 784 7.74 275 463 4.52
51 827 8.03 317 1.182 11.61
57 463 4.52 331 528 5.16
68 8,080 78.71
88 3,377 32.80
83 662 6.45
88 528 5.16

100 883 8.68
116 1,125 10.87
117 6,480 63.23
118 1,7881 17.42
131 1,457 14.18
143 387 3.87
144 2,648 25.81
145 528 5.16
158 10,266 100.00

 

 

 

 

 

Table A9. GC/MS listing for.peak #9 of strain D2 incubated with glucose and H-
PFOSA.

133

 

 

 

 

 

 

 

liver-age 01‘! 858 to $2 Minus: 87310 877 1002 = 60345
133255 -
i
‘ i
68 ’
8111’. 83 _
3x9, 87
111
125 154 253 237
T'rTI'I'l'I'I’iWTWI'I'r'r‘r'
50 1M 150 233 250 333

Figure A.11. GC/MS spectrum for peak #10 of strain D2 incubated with glucose and
H-PFOSA.

 

Hass Intensity 2 Base Hass lntensrtg 2 Base
51 6,670 5.48 81 20,012 16.43
52 4,213 3.46 82 16.501 13.54
53 20,012 16.43 83 84,262 03.16
54 16,501 13.54 84 14.384 11.82
55 121,828 100.00 85 16,852 13.83
56 73.378 60.23 85 12,288 10.08
57 -87,071 71.47 86 11,837 8.80
58 8,830 8.07 87 70,568 57.83
65 4,213 3.46 88 6,318 5.18
66 3,862 3.17 110 4,564 3.75
67 37,215 30.55 111 12,880 10.66
68 15.788 12.87 125 4,815 4.03
68 833% 76.66 154 1,755 1.44
70 62,143 51.01
71 36,162 28.68
78 4,815 4.03

 

 

 

 

 

Table A.10. GC/MS listing for peak #10 of strain D2 incubated with glucose and H-
PFOSA.

134

GC/MS DATA USING ELECTRON IMPACT WITH QUADRAPOLE

DETECTOR

GC/MS (Ouadrapole) analysis:

 

GC:

Column:

Operation:

Injector:

Transfer Line:

Sample:

Hewlett-Packard 5995 Series II GC/MS

DB624 capillary column (30 m x 0.25 mm xl.4 pm) from
J & W Scientific (Folsom, CA)

40°C for 4 minutes, 10°C/minute ramp to 200°C

250°C

225°C

Headspace for 15-30 minutes with a solid phase
microextraction (SPME) fiber assembly with a 100 uM
polydimethylsiloxane coating (Supelco, Inc.,

Bellefonte, PA)

135

 

soooof D
52333;
48333;
443337
43333;
36000:
323331
28333:
24333;
23333: ,1
16000: B
1233 1

0 A F I

33333 n\
43331 l
l-%-- 24C-
4 6

 

 

 

'I'I'T'I' ITWVI'I'I'I'I'I'

. PH-
8 10 12 14 16 18 20 22 24
Time (minutes)

Figure A.12. GC/MS chromatogram for strain D2 incubated with glucose and H-
PFOSA.

 

100

136

 

 

69

 

95

 

§

125

ISO

175

103% =26528

275

300

 

325

341

 

 

350

Figure A.13. GC/MS spectrum for peak A of strain D2 incubated with glucose and H-

PFOSA.

137

 

 

mil intensity _1T_1/_§ intensity
31 8.919 122 31.416
32 0.516 123 6.472
40 0.345 124 0.833
42 0.456 125 3.675
43 0.422 126 1.421
44 2.337 131 8.617
45 5.259 137 2.119
47 0.75 140 0.456
50 1.76 141 0.818
51 3.966 145 1.764
53 1.172 153 1.218
55 0.95 157 1.12
56 3.012 163 3.061
57 3.679 169 4.471
68 0.912 172 6.416
69 71.592 173 0.426
70 10.63 175 3.023
71 2.198 176 0.539
72 1.022 181 2.247
73 2.077 187 1.598
74 1.685 195 1.116
75 22.542 207 1.138
76 2.284 213 1.239
87 0.841 225 5.602
88 0.494 231 4.821
91 3.613 242 0.66
93 3.837 243 0.622
94 9.993 245 0.988
95 78.04 275 4.667
96 2.94 291 14.438
100 7.641 292 1.225
104 1.037 341 100
106 2.258 342 49.389
113 2.36 343 4.412
119 8.041

121 0.471

 

 

Table A.11. GC/MS listing for peak A of strain D2 incubated with glucose and H-
PFOSA.

100

138

 

93 ..
33 .5
73 +
60 ..
50 3'-
43 1»
30 .1-
23 .L
13 .,

 

 

31
100% =62206

‘59 95

J1 - .131 169 213 231 295 34,4 333

‘4 A I l l .;
UV'F'TI'I'YIIITT'YYUTjYIIIIITYTTWTIV"YII'II’V'YYT'YUUIUV'UTTTY'lIII

 

 

Beggggggassaa

N N (V) ("I 4")

Figure A.14. GC/MS spectrum for peak 18 of strain D2 incubated with glucose and H-

PFOSA.

138

100

 

31
90 ‘5 1005:62206

80 «-

 

60 4.
50 1-
40 .1.
30 1*
20 4c

69
'° " 131 344
. 169 213 231 295 363

l
U

A

 

 

 

 

A I
IVY TYVTTIVYUVYUTVVVVVV'VUUIUUUUUUUIUU UTTTUVVVU'V‘I

33-333335333

N N N t"!

Figure A.14. GC/MS spectrum for peak B of strain D2 incubated with glucose and H-
PFOSA.

139

 

 

M intensity M intensity
31 100 106 0.254
39 0.186 107 0.305
40 0.164 112 0.233
42 0.524 113 0.725
43 1.163 119 2.249
44 1.088 124 0.296
45 1.629 127 3.207
46 0.412 131 3.559
47 1.008 137 0.268
49 4.6004 145 0.416
50 0.593 157 0.296
51 2.677 169 0.897
57 0.995 207 0.252
64 2.514 213 0.362
65 6.884 219 0.35
67 0.858 231 0.436
68 0.262 295 1.27
69 14.741 296 1.174
71 0.217 305 0.453
73 0.95 314 1.101
74 0.354 315 0.497
75 2.762 344 2.516
76 0.379 345 0.701
77 2.99 363 0.701
80 0.24
81 0.256
83 0.212
88 0.166
93 1.534
95 12.926
96 0.423
100 2.262
105 0.198

 

 

Table A.12. GC/MS listing for peak B of strain D2 incubated with glucose and H-
PFOSA.

140

 

100
90 ._ 75 1335:1935
80 ..
7o "31 69
60 ,_.
so ~-

40 7r 127
51 157
137

276

30 ‘1-
20 «-

 

 

 

 

 

 

 

101-
.11

 

 

25
53
75
33

125

153

17s

233

225 «
so

275

3
25

Figure A.15. GC/MS spectrum for peak C of strain D2 incubated with glucose and H-
PFOSA.

 

141

 

 

M intensity
31 64.91
34 1.525
36 10.956
40 6.718
42 6.305
44 14.522
45 9.612
49 14.884
51 29.147
55 11.473
57 13.54
58 7.907
59 8.217
61 6.615
69 65.426
73 6.822
74 100
75 32.558
100 6.357
103 7.183
107 21.499
124 12.765
127 35.711
131 10.853
157 28.217
207 8.269
276 38.76

 

 

Table A.13. GC/MS listing for peak C of strain D2 incubated with glucose and H-
PFOSA.

142

 

100 ‘1-
93 --
33 ..
73 ~-
60 «31
so ..

30 .1.-
20 ..
10 4b

 

 

51

69

 

 

 

 

1113‘ = 123862
276
127
157
137
343
169 237 213 257 296 32.,
+' ' 'l'JEA?‘Ul‘LI:U Ul'li I :AU‘IL' :I 0“. I IA. '1‘ ULTL‘ 1A UUUUUUUUU d‘

Figure A.16. GC/MS spectrum for peak D of strain D2 incubated with glucose and H-

PFOSA.

 

 

 

143

 

 

m/z intensity m/z intensity m/z intensity m/z intensity

29 56.226 83 0.314 145 2.055 291 0.782
31 58.158 86 0.655 146 0.109 293 1.998
33 1.515 87 0.806 150 0.352 294 0.146
37 0.678 88 2.274 154 2.189 296 4.308
38 1.62 89 1.891 155 1.035 297 0.389
39 5.529 91 0.808 156 1.02 308 0.235

40 0.318 92 0.177 157 26.085 323 0.188
42 1.106 93 4.683 158 1.931 327 0.775
43 2.238 94 2.895 163 1.648 341 4.622
44 2.235 95 12.162 168 0.304 342 1.248

45 3.008 96 0.77 169 3.924 343 7.364
46 1.577 97 0.75 170 0.154 344 0.61
47 4.797 99 0.802 172 0.477

49 14.25 100 7.814 173 0.412

50 2.388 101 0.528 174 0.134
51 21.958 104 3.132 175 0.683
52 0.357 105 3.323 176 0.201
53 0.758 106 3.354 177 0.566
55 3.067 107 17.056 181 1.476
56 2.467 108 6.964 187 0.877
57 12.894 109 0.554 188 0.325
58 2.149 112 0.559 195 0.677
59 0.58 113 4.28 205 0.142
62 0.558 117 0.359 207 4.068
63 0.309 119 10.463 208 0.363
64 1.117 120 0.501 213 4.802
65 7.982 122 2.962 219 0.433
66 0.258 123 2.674 225 1.382
67 0.703 124 11.42 227 0.412
69 61.566 125 3.099 231 1.389
70 0.936 126 1.547 237 0.899

71 0.603 127 34.241 239 0.121
72 0.22 128 1.547 243 0.315
73 3.354 131 8.779 245 0.323
75 100 132 0.287 257 2.793
76 7.297 135 0.828 258 0.16
77 30.108 136 0.323 263 0.3

78 1.611 137 3.776 273 0.416
79 0.15 138 1.333 275 0.948

80 0.273 139 1.867 276 46.02
81 0.902 143 0.437 277 3.567
82 0.665 144 0.277 281 1.786

 

 

Table A.14. GC/MS listing for peak D of strain D2 incubated with glucose and H-
PFOSA.

100

144

 

33 ..
73 4.
6O ,.
so .1-

30 3-
20 ..-
IO .1-

 

 

31

47

 

11

8

69
77 91

19

.8.

109

131

3

169

195

§

219
18
N

245

8

259

lCDi=Il3136

291 337 328
§ §

 

350 .

Figure A.17. GC/MS spectrum for peak E of strain D2 incubated with glucose and H-

PFOSA.

 

145

 

M intensity M intensity g1/_z intensity
30 0.59 78 0.725 201 0.479
31 100 88 0.486 213 0.411
32 0.402 89 1.849 219 0.681
33 0.814 90 0.698 221 0.271
37 0.025 91 3.643 225 0.218
38 0.369 92 0.285 227 0.51
39 2.139 93 1.003 239 0.688
40 0.246 95 1.115 241 0.253
41 3.042 97 0.13 245 4.596
42 0.821 100 1.863 246 0.301
43 2.097 101 0.468 258 0.32
44 1.652 106 0.182 259 0.462
45 2.181 107 0.21 278 0.209
46 0.27 108 0.371 291 0.789
47 6.299 109 2.586 307 0.944
49 0.633 113 0.742 308 0.105
50 0.416 115 0.171 327 0.737
51 4.173 119 1.813 328 0.41
52 0.262 120 0.209
55 0.366 121 0.381
56 0.133 122 0.364
57 1.536 123 0.122
59 3.707 129 0.313
60 0.354 131 2.3
61 1.424 132 0.226
64 0.811 139 0.468
65 1.707 145 0.619
67 0.392 152 0.195
69 10.311 159 0.108
71 1.732 163 0.51
72 0.077 169 0.655
75 0.701 177 0.252
77 4.02 195 1.39

 

 

Table A.15. GC/MS listing for peak E of strain D2 incubated with glucose and H-
PFOSA.

146

100

 

90 w 339

100% :26924
80 41-
73 3-
60 4»-

50 <1-

272 31 l

232 232 252 296 320
l.

I I T11 null !

 

 

 

 

A l
'IYTrTYIIY T'IITW'VI’T

§ 3 6 E 3 6

 

350

Figure A.18. GC/MS spectrum for peak F of strain D2 incubated with glucose and H-
PFOSA.

147

 

 

M intensity M intensity M intensity
31 4.513 97 0.416 273 1.72
32 1.549 100 5.59 291 0.917
39 1.192 101 5.649 292 1.252
40 4.598 102 1.601 293 0.813
41 12.073 103 2.002 296 3.87
42 31.411 106 0.94 300 2.722
43 4.895 113 0.579 311 21.579
44 1.445 117 0.379 312 1.612
45 0.42 118 0.602 320 3.759
50 1.753 119 26.326 338 37.695
51 1.319 120 1.909 339 100
52 3.283 121 1.645 340 8.799
53 2.429 122 1.801 341 0.602
54 1.419 131 6.47
57 0.758 137 0.773
60 0.977 141 2.254
66 3.124 149 0.825
68 5.868 150 0.813
69 26.122 152 0.984
70 36.272 153 1.553
71 1.606 169 1.772
72 4.3347 182 1.062
73 2.563 202 2.897
74 0.739 203 1.255
75 6.47 216 0.728
76 1.616 222 1.701
77 1.679 225 1.189
78 2.046 232 2.403
79 1.437 242 1.4
80 0.49 250 0.895
90 4.085 252 1.994
91 2.414 267 0.661
92 8.877 272 19.009

 

 

Table A.16. GC/MS listing for peak F of strain D2 incubated with glucose and H-
PFOSA.

148

100

 

57 11131::12228

80 ‘1‘
70 <1-
60 «p-
50 ..

30‘
20<

 

10‘

 

 

 

 

 

 

x: a to g x: a :2 g g

250
275
300
325

Figure A.19. GC/MS spectrum for peak G of strain D2 incubated with glucose and H-
PFOSA.

 

149

 

g; intensity mi; intensity
31 20.527 108 1.382
38 2.535 109 17.722
39 11.833 113 1.497
41 2.077 119 5.757
42 1.047 127 1.914
43 1.922 131 5.904
47 1.652 137 1.398
51 15.289 139 8.096
52 0.131 145 2.412
55 3.439 146 1.333
56 1.865 150 1.079
57 100 169 2.543
58 4.899 189 3.304
59 15.718 195 3.688
64 2.584 219 2.355
67 2.486 239 2.241
69 29.31 258 7.924
75 4.342 278 1.717
76 1.832 325 0.99
77 13.036 -

78 1.088

79 6.608

81 1.317

87 1.946

89 4.236

90 3.042

93 1.807

95 2.453

100 5.684

101 2.633

104 2.085

106 5.806

107 5.397

 

 

Table A.17. GC/MS listing for peak G of strain D2 incubated with glucose and H-
PFOSA.

100

150

 

so ..
7o ‘-
60 ..
so --
4o ._
so 4-
2o ._

 

240

100% = 56744

317
267 1 I.

A

363

378

 

1

 

k
I 'TTTWrT'T'TTTTT

' a

fi

V‘TVT

rsé's

 

A
‘VIT‘I II

Figure A20. GC/MS spectrum for peak H of strain D2 incubated with glucose and H-

PFOSA.

 

 

151

 

 

gill intensity ml; intensity
31 0.511 119 2.192
38 2.226 120 0.289
39 1.353 130 2.132
41 0.906 131 1.165
42 1.269 138 0.296
43 9.821 144 8.319
44 0.236 145 0.737
49 0.303 148 1.163
50 0.684 158 0.34
51 0.927 159 24.129
52 0.633 160 1.914
53 0.687 169 0.624
56 0.465 175 0.888
57 0.732 190 0.497
62 0.46 198 0.62
63 0.405 225 0.293
69 4.786 240 0.793
72 0.375 267 0.936
77 0.363 309 3.221
79 0.43 310 0.226
81 0.513 317 5.072
83 0.189 318 0.522
86 0.384 349 0.809
87 0.33 359 10.177
88 4.504 360 1.348
89 0.441 363 100
99 0.342 364 11.192
100 0.691 365 0.895
105 0.264 378 39.053
115 0.208 379 5.139
116 5.694 380 0.4
117 8.154
118 0.541

 

 

Table A.18. GC/MS listing for peak H of strain D2 incubated with glucose and H-
PFOSA.

100

152

 

90 ..
80 -
70 4

 

 

 

 

103%:8210

 

 

I Y F T I I V j I TTTTT

Figure A.21. GC/MS spectrum for peak I of strain D2 incubated with glucose and H-

PFOSA.

153

 

 

g1/_z intensity ml; intensity
32 1.23 111 6.164
38 1.309 112 3.63
39 40.977 115 2.473
40 7.759 121 1.34
41 91.711 125 3.435
42 34.728 126 3.131
43 100 154 2.631
44 5.019

50 1.961

51 2.351

53 9.733

54 16.725

55 83.044

56 72.587

57 44.095

63 2.217

65 3.386

67 13.557

68 11.304

69 59.005

70 54.875

71 14.483

74 1.3852

77 1.376

81 5.652

82 11.974

83 33.973

84 23.881

85 4.726

95 1.571

96 4.032

97 16.773

98 6.042

 

 

Table A.19. GC/MS listing for peak I of strain D2 incubated with glucose and H-
PFOSA.

APPENDIX B

APPENDIX B

GC/MS DATA USING CHEMICAL IONIZATION WITH QUADRAPOLE
DETECTOR

GC/MS (Chemical Ionization) analysis:

GC: Varian 3400

MS: Axtrel ELQ 400 (Methane as ionization agent)

Column: Hewlett-Packard HP-FFAP (25 m x 0.32 mm x 0.52 pm)

Operation: -20°C for 4 minutes, 25°C/minute ramp to 225°C, hold
10minutes

Injector: 250°C

Transfer Line: 225°C

Sample: Headspace for 15-30 minutes with a solid phase

microextraction (SPME) fiber assembly with a 100 uM
polydimethylsiloxane coating (Supelco, Inc.,

Bellefonte, PA)

03 TIC. Sns: 1-2127. Max: 231922. C Max: 231922. RT: 0.0. 19.9
5

 

 

3 s
,_ a L 2E 31:1]? 17. at _ -
I I

Fl 1 l 1 ‘I ‘I I 1‘1 1 I
100 300 500 700 900 1100 1300 1500 1700 1900

Figure 3.1. GC/MS chromatogram for strain D2 incubated with glucose and H-
PFOSA.

154

 

155

ID

3 M55: 255.0-380.0. Sns: 627-632. Max: 68165. ART: 5.9
343

 

 

 

A
Ivvvv'vVvvlvvvafivvleYVV'vvvv vvvv vvvv VVVY Vvvv vvvr rwvr'

' T
960 270 290 900 300 310 390 330 940 QSIO 280 370

Figure 8.2. GC/MS spectrum for peak #1 strain D2 incubated with glucose and H-
PFOSA.

 

 

 

 

Q3 Mss: 240.0—675.0. ire: 992—999. B: 1.0 993.997.999. Max: 1638. ART: 9.3
3 5
j 327
I 393
. 295 l 431
300 - - '400 vvvvvvv - '560' - vvvvvvv sr'm' I - i -

Figure 8.3. GC/MS spectrum for peak #2 strain D2 incubated with glucose and H-
PFOSA.

ID

3 M55: 274.0-436.0. Sns: 1001-1003. Max: 2735 TART: 9.4
319

359

341

114141

395 295 303 316 327

I.:Ij".VIIII"I‘_'III...1.4'11"rI u :II'JII‘ V'J'II'A' 393

VI: A vjll' 1!. A vi. A

vvvv

 

 

 

Figure 8.4. GC/MS spectrum for peak #3 strain D2 incubated with glucose and H-
PFOSA.

156

3 M55: 26290-4730. Sns: 1031-1034. Max: 3156. ART: 9.7
2 1

 

 

 

Figure 8.5. GC/MS spectrum for peak #4 strain D2 incubated with glucose and H-
PFOSA.

JD

3 M85: 248.0-386.0. Sns: 1034-1039. Max: 52900. ART: 9.7
3??

« _ 217 age 309

AA
vvvvr V‘vvv vvvw

-- -.c: -... .372 -:L.- h--‘T‘v.f -311 -.fif ...3 3-3
aéo 270' aéo 290 360 310 aéo 350 330 350 360 310 350

 

 

 

Figure 8.6. GC/MS spectrum for peak #5 strain D2 incubated with glucose and H-
PFOSA.

Q3 Mss: 276.0—414.0. Sns: 1061-1065. Max: 2166. ART: 10.0
300
1

A

324
281 I 304 3“ I
.gw'-. 1: 1 n

I- -II-. ‘
"T 'T"' -1---

- rt fi;‘;.‘r-‘-~!‘ fiat‘..v. ..4fi --c- v-
290 350 310 aéo 330 340 330' 360 310 350 390 460 410

41.1

 

l 368
I

.11- 1-

vv—v

 

firvw

 

Figure B.7. GC/MS spectrum for peak #6 strain D2 incubated with glucose and H-
PFOSA.

 

157

3 M33: 245.0-408.0. Sns: 1112-1116. Max: 352. ART: 10.4
3 7

291

279

‘_ A I A 14 I A l A In

 

 

 

 

254 265
1.1.11111

- :1_1'1' 1111 1 _____
20270 250 290 360 310 30 30 340 30 30 30 30 30 40

 

 

Figure 8.8. GC/MS spectrum for peak #7 strain D2 incubated with glucose and H-
PFOSA.

JO

3 M55: 352.0-465.0. Sns: 1353-1362. Max: 881. ART: 12.7
416

400

4441 A 1 A l L l A

38 80 339 428
II III III :1111i li‘- I I : : III! - -II I v '_I.'

36 o 390 400 410 420 450 430 830

 

 

 

 

 

”Fm
o _____

Figure 8.9. GC/MS spectrum for peak #8 strain D2 incubated with glucose and H-
PFOSA.

 

APPENDIX C

 

APPENDIX C

GC/ATOMIC EMISSION DETECTION

 

 

 

 

 

 

 

 

- 3
12B. ‘51
" ‘1'
180‘
80‘
68‘
L0
1 m U')
‘ m- tn SS 2
‘ m m L0.
20- 53 — v‘m
1 U” -
Ln
L—41 l L
a? . . . . . . . . . . . . .9 ,
B S 10 15
Time (min.)

 

 

Figure C.1. AED chromatogram for fluorine (at 690 nm) strain D2 incubated with
glucose and H-PFOSA.

158

159

 

 

4
4

B S 10 15 20
Time (min.)

 

 

 

 

Figure C.2. AED chromatogram for sulfur (at 181 nm) strain D2 incubated with
glucose and H-PFOSA.

160

 

 

 

 

 

 

 

. E
48: EE
3%;

28‘:

7‘1

mi

 

T V V V I V I I Y I

an
d

B 5 10 15 28

Time (min.)

 

 

 

Figure C.3. AED chromatogram for hydrogen (at 486 nm) strain D2 incubated with
glucose and H-PFOSA.

161

 

 

 

 

 

 

n14
m
. fl,
4%“ 1%
3B“
28‘
‘ v
CD CD
1\ <1-
m .
. , m
m c—o
10"
‘ v
. 0". cs.)
4 N 8
1| 8. 1:)
@—
I r T Y 1 T ' I f I I
U 13 15 28
Time (min.)

 

Figure C.4. AED chromatogram for carbon (at 496 nm) strain D2 incubated with

glucose and H-PFOSA.

 

162

 

147
I1.TWT

1A

 

 

 

 

 

 

 

 

8 S 18 15 28
Time (min.)

1
I
1

.4

 

 

 

Figure C.5. AED chromatogram for oxygen (at 777 nm) strain D2 incubated with
glucose and H-PFOSA.

 

 

TE UN V. IBRRRIES

1111111 ll um

815643343

MICHIGAN STR
11111111111111“
31293