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3 1293 0142

This is to certify that the
dissertation entitled

Physiology of Carbon Tetrachloride Transformation

by Pseudomonas stutzeri KC

 

presented by

Gregory M. Tatara

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Microbiology

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I Major professor

Date gl/Ij/Qa

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

LIERARY
Mlchigan State
University

 

 

 

PLACE N RETURN BOX to remove thte checkout from your record.
TO AVOID FINES return on or betore dete due.

DATE DUE DATE DUE DATE DUE

     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PHY

 

 

 

 

PHYSIOLOGY OF CARBON TETRACHLORIDE TRANSFORMATION BY
PSEUDOMONAS STUTZERI KC

By

Gregory Michael Tatara

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology

1996

PHYS

Pseudomc

condition:

    

iron plays

 

 

ABSTRACT

PHYSIOLOGY OF CARBON TETRACHLORIDE TRANSFORMATION BY
PSEUDOMONAS S TUT ZERI KC

By
Gregory M. Tatara

Pseudomonas stutzeri KC transforms carbon tetrachloride (CCl4) under denitrifying
conditions without the production of chloroform. Trace metal studies established that
iron played an inhibitory role in the transformation, leading to the development of a
hypothesis which stated an iron scavenging agent is responsible for the cometabolic
transformation of CCl4. Localization experiments established that both a small (< 500
dalton) secreted factor and cells of P. stutzeri KC were required to transform CCl4,
Initial purification experiments determined that the secreted factor was extractable with
acetone, and further purification of this factor revealed that activity was recoverable
following semi-preparative HPLC analysis. Based on published studies for iron
scavenging systems, it was hypothesized that a diverse range of other microbial species
and genera can be used in combination with the secreted factor to achieve CCl4
transformation. This hypothesis was proven true and lead to the development a bioassay
for the secreted factor using Pseudomonas fluorescens. The bioassay established that in
addition to P. stutzeri KC cells grown under denitrifying conditions, aerobically-grown
strain KC produces the factor, however oxygen reversibly inhibits CCl4 transformation.
The bioassay was also used to establish that live cells are needed for CCl4 transformation,
and the factor is stable indefinitely after lyophilization to powder. Preparation of crude
cell membranes demonstrated that membranes in combination with the secreted factor
rapidly transformed CCl4 when NADH was added as a source of reducing equivalents.

Inhibitor studies established that the respiratory electron-transport chain was not involved

mccu

ausxnau
combine
which pi
produce-t
ohdfled

roductior

l
I
l
I

 

in CC14 transformation. Lactobacillus acidophilus, an organism lacking any membrane-
associated electron-transfer-proteins, did not result in CCl4 transformation when
combined with the secreted factor. These studies lead to the development of a new model
which posits that CCl4 accepts an electron(s) from a reduced form of the secreted factor
produced during growth in medium that contains copper and is iron limited. The
oxidized form of the secreted factor is then capable of further transformations only after

reduction at the cell-membrane by a non-respiratory enzyme .

I humbly dedicate this dissertation to my loving parents

iv

the com

that has
compcii
accompl
Ciperirn
fnendl)

friend

filling m
and to [a
back IO 9'
Students

I feel Yer;

 

 

ACKNOWLEDGMENTS

It is my desire to show appreciation and give due credit to the people that made

the completion of this research and dissertation possible.

A special thanks goes to Dr. Mike Dybas, without whom the amount of progress
that has been made on Pseudomonas KC would not have been possible. The friendly
competition for success and knowledge that existed between Mike and me, helped me
accomplish more than I would have on my own. I thank Mike for teaching me many
experimental techniques, and for always questioning my results and methods. His
friendly criticism definitely made me a better scientist, and also made him a very good

friend

Dr. Criddle has a very special gift for advising students. I thank him for always
giving me the opportunity and encouragement to develop my own experimental design
and to take the project in a direction I felt was best. However, he always brought me
back to earth if I fell astray from the proper task at hand. Dr. Criddle always places his
students first, and has encouragement, patience, and a great deal of concern for all of us.

I feel very rewarded for having the opportunity to work with Dr. Criddle.

I wish to thank the Center for Microbial Ecology for enabling me to have the
unique opportunity to perform cross disciplinary research between Microbiology and

Engineering.

A special thanks goes to Dr. Breznak, Dr. Forney, Dr. Hausinger, and Dr. Oriel

for always being willing to discuss my results with me and providing me with direction

forf

man.

com;
auton
Mark
is so f.

[maker

great (it

student,

my Phi

 

 

 

for future studies. Without their help and guidance, I never would have accomplished

many of the experiments that are described in this thesis.

Many of my colleagues and friends deserve thanks for helping to make
completion of this dissertation possible. I appreciate Mike Szafranski's help with the
automated GC and for teaching me Turbochrome. I wish to thank Blake Key, Mike Witt,
Mark Sneathen, Fred Knoll, and Randy Brown for providing me with that extra hand that
is so frequently required during experimentation, and more importantly for helping to

make our lab a fun and enjoyable place to work.

I wish to show appreciation to our secretary Linda Steinman for helping me a
great deal during my time in Dr. Criddle's lab. Even though I was not an engineering
student, Linda always helped me with all of the administrative problems that arose during

my PhD. career.

I want to thank Dr. Rod Skeen of the Battelle Pacific Northwest Laboratories for
providing us with Hanford Consortium HC-14, the recipe for SGW medium, and aquifer
material from the Hanford site. I would also like to thank Dr. Harry Ridgway of the
Orange County Water District for providing us with Pseudomonas stutzeri strain EPB-
071388 116. Finally, I thank Timothy J. Mayotte and Golder Associates (East Lansing,

MI) for providing us with aquifer solids and groundwater from the Schoolcraft site.

vi

LIST

LIST
LIST
CHAl

CHAPT

 

 

 

TABLE OF CONTENTS

Page
LIST OF TABLES .................................................................................................... ix
LIST OF FIGURES ................................................................................................... x
LIST OF SYMBOLS ................................................................................................. xv
CHAPTER 1— INTRODUCTION:
TRANSFORMATIONS OF CARBON TETRACHLORIDE. l
Mechanisms and pathways .................................................... 3
Literature Summary ............................................................... 16
Pseudomonas stutzeri KC ...................................................... l7
Outline of this thesis ............................................................... 23
References .............................................................................. 24
CHAPTER 2— EFFECTS OF MEDIUM AND TRACE METALS ON
KINETICS OF CARBON TETRACHLORIDE
TRANSFORMATION BY PSEUDOMONAS STUYZERI.
KC ......................................................................................... 28
Abstract ................................................................................. 29
Introduction ........................................................................... 30
Materials and Methods ......................................................... 31
Results .................................................................................. 37
Discussion ............................................................................ 47
References ............................................................................ 49
CHAPTER 3 -— LOCALIZATION OF THE CARBON TETRACHLORIDE

TRANSFORMATION ACTIVITY OF PSEUDOMONAS

STUYZERI KC ...................................................................... 50
Abstract. ................................................................................ 51
Introduction ........................................................................... 52
Materials and Methods ......................................................... 53
Results .................................................................................. 57

vii

CHAPTER4 _

CHAPTERS—

CHAPTER 6—.

, Discussion ............................................................................ 68

References ............................................................................ 72

BIOFACI‘OR-MEDIATED TRANSFORMATION OF
CARBON TETRACHLORIDE BY DIVERSE CELL

TYPES .................................................................................. 73
Abstract. ................................................................................ 74
Introduction ........................................................................... 75
Materials and Methods ......................................................... 77
Results .................................................................................. 85
Discussion ............................................................................ 100
References ............................................................................ 105

ROLE OF CELL MEMBRANES IN THE TRANSFORMATION
OF CARBON TETRACHLORIDE BY PSEUDOMONAS

STUIZERI KC ...................................................................... 107

Abstract ................................................................................. 108
Introduction ........................................................................... 109
Materials and Methods ......................................................... 110
Results .................................................................................. 114
Discussion ............................................................................ 120
References ............................................................................ 125

THE USE OF VEGETABLE OILS FOR THE ENGINEERED

APPLICATION OF PSEUDOMONAS STUTZERI KC ...... 126
Abstract ................................................................................. 127
Introduction ........................................................................... 128
Materials and Methods ......................................................... 129
Results .................................................................................. 133
Discussion ............................................................................ 141
References ............................................................................ 145

viii

CHAPTER 7 — CONCLUSIONS AND FUTURE INVESTIGATIONS ...... 146

ix

Table 1.

Table 2.
transfor

Table 3.

Table 3.
produce.

Table 3..
Table 4.

Table 4. ;
Order rai

Table 4.3
P-fluore.

Table 4.;
Table 5.1

Table 5.:
”“6 COef i

Table 6.1I
{01m

 

 

LIST OF TABLES

Table 1.1. Kinetics and conditions of CCl4 transformation

Table 2.1. Second-order rate coefficients (:r: one standard deviation) for CCl4
transformation by P. stutzeri KC: effects of iron limitation and culture age.

Table 3.1. Transformation kinetics for fractionated cell-free activity.

Table 3.2. Iron binding properities of ultra-filtration fractions from culture supernatant
produced by P. stutzeri KC.

Table 33. Results of acetone precipitation.
Table 4.1 First-order rates coefficients and half-lives of CCl4 transformation .

Table 4.2. Stability of the secreted factor(s) as indicated by changes in the apparent first-
order rate constants (k') under varied storage conditions.

Table 4.3. Concentration dependence of the secreted factor on transformation of CCl4 by
P. fluorescens cells.

Table 4.4.. Transformation of CCl4 in Hanford aquifer solid slurries.
Table 5.1. Summary of inhibitors used.

Table 5.2. Table 5.2. Effect of electron transport inhibitors as measured by first-order
rate coefficients.

Table 6.1. Growth of P. stutzeri KC on various vegetable oils and subsequent CCl4
transformation.

LIST OF FIGURES

Figure 1.1. Known transformation pathways of CCl4.
Figure 1.2. Dichlorocarbene intermediate pathway.
Figure 1.3 Condensation products observed by Lewis and Crawford.

Figure 1.4. Proposed phosgene and thiophosgene intermediate pathway resulting from a
dichlorocarbene radical.

Figure 2.1. Kinetics of CCl4 loss by strain KC.
Figure 2.2. Dependence of first-order rate coefficient on total protein concentration.
Figure 2.3. Fate of transformed radiolabeled CCl4 by P. stutzeri KC.

Figure 2.4. Growth of P. stutzeri KC in medium D and precipitate-free medium D in the
presence or absence of added iron.

Figure 2.5. CCl4 transformation capacity for cultures of P. stutzeri KC grown in medium
D.

Figure 2.6. CCl4 transformation capacity for cultures of P. slutzeri KC grown in
precipitate-free medium D.

Figure 2.7. Inhibition of CCl4 transformation by ferric iron.

Figure 2.8. Trace levels of copper affect CCl4 transformation activity.

Figure 3.1. P. stutzeri KC cells combined with 500 MW filtrate from strain KC.
Figure 3.2. Effect on iron on transformation with 10,000 MW filtrate from strain KC.

Figure 3.3. Semi-preparative HPLC chromatogram of a primary run of a sample
containing the secreted factor involved in CCl4 transformation.

 

 

 

Fig
foil

Figi
CCL

Figu
Willi

 

 

Figure 3.4. Mass of CCl4 removed by fractions combined in a P. fluorescens bioassay
following HPLC purification of acetone extracted CCl4 transformation activity.

Figure 3.5. A semi-preparative HPLC chromatogram illustrating the initial separation of
CCl4 transforming activity.

Figure 4.1. Rapid CCl4 transformation results from the combination of P. fluorescens
with 500 MW filtrate.

Figure 4.2. Secreted factor production during the growth of P. stutzeri KC.
Figure 43. Incubation under aerobic conditions inhibits CCl4 transformation.
Figure 4.4. Effect of cell viability on CCl4 transformation.

Figure 4.5. The effect of cell density on transformation rates.

Figure 4.6. pH optimum of CCl4 transformation.

Figure 4.7a. Breakthrough profile for tritiated water and the secreted factor in a column
packed with Schoolcraft, MI, aquifer material.

Figure 4.7b. Breakthrough profile for tritiated water and the secreted factor in a column
packed with Hanford, WA, aquifer material.

Figure 5.1. Transformation of CCl4 with crude cell membrane preparations from P.
stutzeri KC.

Figure 5.2. Effect of lNT-Formazan on CCl4 transformation by P. stutzeri KC.

Figure 5.3. Comparison of CCl4 transformation with B. subtilus and L. acidophilus when
combined with the secreted factor from P. slutzeri KC.

Figure 5.4. Transformation of CCl4 by strictly fermenting E. coli combined with the
secreted factor from P. stutzeri KC.

Figure 6.1. The cumulative mass of CCl4 removed and chloroform produced by P.
stutzeri KC.

xii

 

 

 

 

figure
enrichi

Figure

Figure
of P. 51

Figure
SChOOli

Figurei
andP.i

Figure i
a lipid r

Figure 7

 

Figure 6.2. The cumulative mass of CCl4 removed and chloroform produced by an
enrichment of organisms from Schoolcraft aquifer solids.

Figure 6.3. Abiotic loss of CCl4.

Figure 6.4. The cumulative mass of CCl4 removed and chloroform produced by a mixture
of P. stutzeri KC and an enrichment of organisms from Schoolcraf t aquifer solids.

Figure 6.5. Growth of P. stutzeri KC, Schoolcraf t consortium, and P. stutzeri KC +
Schoolcraft consortium.

Figure 6.6. Mass of N03 remaining in cultures of P. stutzeri KC, Schoolcraf t consortia,
and P. stutzeri KC + Schoolcraf t consortia.

Figure 6.7. Production of chloroform from the reaction of a trichloromethyl radical with
a lipid molecule.

Figure 7.1 Model of proposed CCl4 transformation process by P. stutzeri KC.

xiii

CHAPTER 1

INTRODUCTION:
TRANSFORMATIONS OF CARBON TETRACHLORIDE

Halogenated aliphatic compounds that contaminate groundwater pose a hazard to human
health, and thus their degradation to innocuous products by biotic and abiotic systems is
of interest. Carbon tetrachloride (CCl4) degradation is of particular interest because of its
toxicity and carcinogenicity. CCl4 adversely affects the eyes, liver, kidneys, central
nervous system, and skin of humans. Excessive exposure can result in central nervous
system depression and gastrointestinitis, while acute exposure can result in the condition

of toxic hepatitis [48].

The non-polar and nonflammable character of CCl4 made this molecule a very useful
solvent. CCl4 has been used in the manufacture of fire extinguishers, refrigerators,
aerosols, and chlorinating organic compounds including chlorofluoromethanes [51]. It
has also been used as a degreasing agent for metals, an agricultural fumigant, an
extractant, and a solvent for oils, fats, lacquers, vamishes, rubber, waxes, and resins [48,
51]. An estimated 5 million lbs/year of CCl4 were emitted during manufacture and
processing, and approximately 60 million lbs/year were released as solvent emissions
[48]. Because of its widespread use and improper disposal, CCl4 has become a
significant pollutant of soil and groundwater. In 1985, CCl4 was reported in 10% of 113
public water supplies surveyed, at mean concentrations of 2.4 to 6.4 pg/L, and in 25% of
groundwater supplies at concentrations of 1 to 400 rig/L [48]. Estimates indicate that 19
million people are exposed to CCl4 through ambient air, 20 million through contaminated
drinking water, and 2 million through contaminated soils and landfills at levels greater
than the EPA exposure standard of 5 rig/L [48]. Thus, the environmental remediation of

CCl4 contamination is of scientific and practical significance.

 

 

 

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Mechanisms and pathways of CCl4 transformation

Known pathways for CCl4 are shown in Figure 1.1. Although CCl4 may theoretically
undergo direct hydrolysis, it is generally believed that the first step of this mechanism is
the one-electron reduction of CCl4 to give a trichloromethyl radical and a chloride ion ( l)
[13]. Depending on the environmental or experimental conditions, the trichloromethyl
radical can undergo one of several possible reactions: dimerization of the trichloromethyl
radical to give hexachloroethane (2), interaction with a lipid to form chloroform(3);
sequential reduction first to chloroform and subsequently, in sufficiently reduced
environments to dichloromethane (4), a second one-electron reduction to form a
dichlorocarbene radical which can undergo hydrolysis to carbon monoxide(CO) and
forrnate (5); reaction with HS- to form thiophosgene which can subsequently react to
form C02 (6), addition of molecular oxygen to form phos gene which can hydrolyze to
C02 (7); and lastly, covalent bonding to cell material (8). Typically, several pathways
operate simultaneously and competitively in both biotic and abiotic systems. The
numbers shown next to the pathways in Figure 1.1 correspond to numbering of the text in
the following sections. The text provides documentation and a discussion of each
pathway, followed by a section discussing the relationship between the known CCl4

transformation pathways and transformation by P. stutzeri KC.

 

 

 

ZHCI

 

 

 

 

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Ci Cl
\C/ (In Ci
Cl/ \C' Cl-(lZ-(llZ-Cl
carbon tetrachloride CI Cl

   
 

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hexachloroethane

(minor)

 

 

 

 

  
 
  

 

 

 

 

 

 

 

 

 

 

 

 

 

= ' Li id R- C. H
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Figure 1.1. Known transformation pathways of CCl4. Individual pathways are indicated
by the Circled numbers. Transformation products produced by P. slutzeri KC are shown
in boxes. Adapted from Criddle and McCarty [26], Kreigman-King and Reinhard[33],
Jain and Criddle [27], and Lewis and Crawford [39].

Pathway 1: Trichloromethyl radical f ormation

Transformation of CCl4 to the trichloromethyl radical proceeds by the following reaction:

CCl4 + e- —> ° CCl3 + CI' (1.1)

The formation of the trichloromethyl radical is generally accepted as the rate limiting step
in reductions of alkyl halides [6,54]. The electron can come from one of several reduced
species, including certain transition metals, organics, enzymes and co-factors, or it can be
omitted by a cathode. Direct evidence of trichloromethyl radical formation comes from
time resolved pulse radiolysis conductivity experiments and chloride-ion product analysis
[32]. Trichloromethyl radical formation has also been detected using spin trapping
techniques [37]. The trichloromethyl radical formed from this step is extremely reactive,
giving rise to the broad product distributions observed for CCl4 transformations

proceeding through this step (Figure 1.1).

Pathway 2: Dimerization of the trichloromethyl radical to give hex_achloroethane
Evidence for hexachloromethane production from CCl4 comes from observations that
rabbits which breathed CCl4 exhaled small amounts of hexachloroethane [22]. The

production of hexachloroethane also provides indirect evidence of radical formation.

2[°CCl3] —> C2Cl6 (1.2)

Pathway (3): Interaction with a lipid to form chloroform

The formation of chloroform from CCl4 was first reported by Butler [9], who observed
the production of chloroform in animals given CCl4. Later studies showed that this
biotransformation involved microsomal P-450 dependent enzymes. Several researchers
have suggested that the conversion of CCl4 to chloroform in microsomes requires the
intermediate formation of the trichloromethyl radical via a one-electron process. [50,22].
Furthermore, abstraction of a methylene hydrogen by the trichloromethyl radical would
account for both the formation of chloroform and the initiation of lipid peroxidation. In
order to determine the source of the hydrogen atom in chloroform, Kubic and Anders [36]
studied the enzymatic conversion of CCl4 to chloroform by microsomal P-450 dependent
enzymes in the presence of deuterium oxide. They observed no enrichment in deuterium
when microsomes from rats were employed, indicating that the source of hydrogen was
from lipids and proteins. Luke et al. [40], observed that the trichloromethyl radical could
abstract hydrogen if the carbon-hydrogen bond strength was less than 9.2 kcal/mole,
which implied that in microsomal systems, the hydrogen would have to come from the
unsaturated region of lipid molecules or from localized environments where the resulting
carbon radical is stabilized. Therefore, the production of chloroform involves

hydrogenation of the trichloromethyl radical by reaction 1.3.

0CCI3 + RH—> CHCl3 + R0 (1.3)

Pathway (4): Seguential reduction to chloroform and dichloromethane

Chloroforrn is one of the most recalcitrant one-carbon alkyl halide, and because of its
persistence, (hydrolysis half life of 1850 years at 25 0C; [29]), toxicity, and

carcinogenicity, it is one of the least desirable metabolites of CCl4 transformations.

 

 

 

Chloro
WhICh
the firs

trichlor

Table 1
microbi

i
environ

methani

Chloroforrn is produced from CCl4 by hydrogenolysis» a reductive dehalogenation in
which hydrogen replaces chlorine. The hydrogenolysis of CCl4 is a two-step process: in
the first step, a chlorine is removed by reaction 1.1; in the second step, the
trichloromethyl radical is hydrogenated by reaction 1.4.

0CCl3 + H1' + e- > CHCI3 (1.4)

 

Table 1.1 summarizes the available information on the hydrogenolysis of CCl4 in
microbial systems. Generally, faster and more extensive hydrogenolysis occurs as the
environment becomes more reducing--- the highest rate is observed under a
methanogenic condition, the next highest under sulfate respiring, and lower rates are

observed for f umaiate respiring and fermenting conditions.

Table 1.1. Kinetics and conditions of CCl4 transformation

 

 

 

 

 

 

 

 

 

 

 

 

 

WM 1' W ucts W
(1ng protein/day) By
Fe 3+ 0. 177:1: 0.057 anaerobic CHCI3,
Shewanellaputrefaciens Nitrate 0.105 :i: 0.039 respiratory (1)2, 45
MR-l TMAO 0.053 :I: 0.018 components, cell bound
Fumarate 0.029 :I: 0.004 cytochrome c mamfiaL
unknowns
Desulfiibacterium acetyl CoA C102, CHCI3.
araotrophicum 039 pathway (11202, cell 20.21
material,
unknowns
Methanobacterium acetyl CoA (1)2, CHCI3.
thermoautotrophicwn 0.019 pathway CH 202, CH4, 1 1, 19
cell material
acetyl CoA CHCI3.
Methanosarcina barkeri 0-873 pathway CHzCIz,CO 2, 34
F430 014.
unknowns
acetyl CoA CHCI3.
pathway (112C112.
Acetobacterium woodr'i 0.24 (102.ccll 21
material
unknowns
acetyl CoA CHCI3,
Clostridiwn Not Determined“ pathway (11202.
thermoaceu'cwn (1.130. 21
unknowns
Closm'dr'wn sp. strain C1103,
TCAIIB 0.181 “mm CH 202 23
unknowns
Escherichia coli' K12 0.0041 fermenting unknown, CHCI3,
0.0025 fumarate possibly cell material 14
respiring cytochromes unknowns

 

 

 

As shown in Figure 1.1, dichloromethane is produced by the further reduction of

chloroform. Numerous isolates have been obtained which are capable of this reduction

(Table 1.1). Gossett [25] reported that chloroform disappeared in methanogenic mixed

cultures: 31% of the chloroform was converted to dichloromethane, and a small fraction

was further converted to chloromethane; 32-34% was further converted to C02. As the

degree of chlorination decreases in the series from CCl4 to chloromethane, removal of

chlorine substituents by reduction becomes energetically and kinetically more difficult.
This is true of alkyl halides in general [52]. In the case of microbial hydrogenolysis,
experimental observations tend to corroborate theoretical predictions; i.e. chloroform
reduction to dichloromethane and dichloromethane reduction to chloromethane are only

known to occur in highly reduced environments [53].

The frequent persistence of CCl4 in microbial systems should also be emphasized. Egli
et al. [21] reported that neither Desulfobacter hydrogenophilus nor a hydrogen-oxidizing,
nitrate-reducing autotrophic bacterium from a groundwater treatment plant could degrade
CCl4. Several denitrifying enrichments from Moffett Field, California, were incapable of
CCl4 transformation as well [28]. Additionally, Bouwer and McCarty [7] reported no

CCl4 transformation for an aerobic mixed culture derived from a sewage inoculum.

In addition to the microbial transformations listed above, several in vitro transformation
systems have been described. Butler [9] reported that high concentrations (50 mM) of
glutathione, cysteine, and ascorbic acid were all capable of reducing CCl4 to chloroform.
Yeast extract with sulfide has been shown to reduce CCl4 to chloroform [24]. A
combination of cytochrome c (0.8 mM) and ascorbic acid (5 mM) also brought about
conversion of CCl4 to chloroform. In addition, the reduction of CCl4 to chloroform is
observed in many complex mammalian systems [9, 22, 41], iron porphyiin-sulfide
systems [30], and electrolytic systems [13]. Gantzer and Wackett [23] demonstrated the
reductive dechlorination of CCl4 by bacterial transition-metal coenzymes vitamin 812
(C0), coenzyme F430 (Ni), and hematin (Fe). Rates of reductive dechlorination catalyzed
by the co-enzymes decreased markedly with decreasing chlorine content of the alkyl

halide.

10

In summary, reduction of CCl4 to chloroform occurs when reductants of sufficient
reducing power are present and competitive oxidants are not. As the environment
becomes more reduced, the rate of chloroform production increases, as does the
likelihood of further reduction to dichloromethane. Under highly reducing conditions (as
in methanogenic cultures) it may be possible to drive the reductive dehalogenation of
CCl4 past chloroform to dichloromethane, chloromethane, CH4, and C02.

PathwatMS): Second one-elect_ron redflion to form fiichlorocafiene radical

Dichlorocarbene (:CClz) is produced from chloroform under alkaline conditions by the
reactions 1.5a and 1.5b [26]. In theory, it is also possible for dichlorocarbene to be
produced by a two-electron reduction of CCl4, as shown in reaction 1.5c [3].
Dichlorocarbene is known to hydrolyze to give carbon monoxide (CO) and/or formic acid

by reactions 1.5d and 1.5e [30; 43].

CHCI3 + OH‘ —> CCl3’ + H20 (1.5a)
CCl3’ —> :CClz +Cl' (1.5b)
CCl4 + 2e- —> :CClz + 2Cl' (1.50)
:CClz + H20 —> C0 + 2HCl (1.5d)
:CC12 + 2H20 -—> HCOOH + 2HCI (1.56)

The observed formation of C02 under anaerobic conditions may be explained by the
oxidation of a carbenoid or by the oxidation of CO or forrnate. The term ”carbenoid” is
used when the existence of free carbenes is uncertain [43]. Thus, carbenoids may be free
or bound in a chloromethyl complex. Carbenoids or chloromethyl complexes that are
known or believed to participate in CCl4 transformations are: chromium carbenoids, iron-

porphyrin complexes, cytochrome P-450 complexes, and cobalt coninoids. An

ll

explanation and evidence for each of these species is described in the following

paragraphs-

Castro and Kiay [10] reported the Cr(II) sulfate converts CCl4 to CO (75%) and CH4
(25%) in a 1:1 dimethylformamidezwater solution. They hypothesized that this

transformation proceeded via a sequence of reactive halocarbene intermediates.

The formation of carbenoids by iron center species, such as iron-porphyiins and
cytochrome P-450 is debated. Brault et al. [8] observed the formation of a
dichlorocarbene complex by reacting CCl4 with reduced deuterohemin. Subsequent
oxidation of the complex by molecular oxygen gave C02 and small amounts of CO.
Mansuy [42] reported a method for preparation of iron-porphyrin carbene complexes, and
hypothesized the formation of cytochrome p-450 carbene complexes in the metabolism of
polyhalogenated compounds. Klecka and Gonsior [31], in their study of iron-porphyrin
reactions with CCl4 and other alkyl halides, could account for only 31% of the initial
CCl4 added as chloroform produced, and postulated that the missing CCl4 may have

formed a dichlorocarbene complex.

Wolf et al. [55] reported CO formation by rat liver microsomes. They postulated that a
carbene or carbenoid species was an intermediate in this reaction. Ahr et al. [2] also
reported CO production in microsomal systems. They reported that the reaction was
dependent upon cytochrome P-450 and a source of reducing equivalents (NADH or
sodium dithionite). DeGroot and Has [16] reported formation of limited CO in rat liver
microsomes, and found that microsomal CO production was inhibited by oxygen. They
found evidence to suggest that CCl4 conversion to CO in microsomes was destructive
process in which CCl4 metabolites inactivated the cytochrome P-450. However, Castro
et al. [12] argued that chloroform was the only major product of cytochrome P-450. They

12

attributed observations of CO production by cytochrome P450 to the incorrect use of
alkaline conditions, resulting in the hydrolysis of CF to CO by reactions 1.5 ab and (1.
Thus, the direct conversion of CCl4 to a dichlorocarbene radical in biotic systems remains

controversial, and can only theorized to occur based on observed end-products.

Pathway (6): Reaction of trichloromethyl radical with H8-

The first report of C82 as a CCl4 transformation product was in systems with f umarate-
respiring and fermenting Escherichia coli [14]. In the fumarate respiring and fermenting
E. coli systems, C82 was a minor product (4.3% and 1.6% of the added CCl4
respectively). Subsequently, Kreigman-King and Reinhard [33] reported the
disappearance of CCl4 and the appearance of products in an abiotic heterogeneous system
containing biotite and 1 mM HS-. Carbon disulfide (CS2) was identified as a major
intermediate of this transformation. Studies on the hydrolysis and oxidation of CS2 show
that C82 is hydrolyzed to C02 by hydroxide ion (OH'). Adewuyi and Carmichael [1]
proposed the following steps for CS2 hydrolysis:

C82 + OH' —> CSzOH’ (slow) (1.6a)
C820H' + OH“ —> CSOzH‘ + HS‘ (1.6b)
CSOzH‘ + OH' > CO3H' + H8" (1.6c)

 

where the hydrolysis of CS2 to dithiocarbonate (CS20H‘) is the rate limiting step.
Assuming C82 is stoichiometrically converted to C02, ~85% of the CCl4 is ultimately

transformed to C02 in these systems [1].

The only pathway previously hypothesized to form C02 in anaerobic systems is direct

hydrolysis, however, there has been no evidence that CCl4 can undergo direct hydrolysis

13

at appreciable rates [29]. Kriegman -King [33] reported that C82 appears to be a major
intermediate which is transformed to C02. They hypothesized that C82 may form via
nucleophilic substitution of CCl4 by aqueous or absorbed HS' or by S3: The SN2
reactivity of a compound with four identical leaving groups on the same carbon is quite
low. Therefore, it is conceivable that CCl4 accepts one electron to form a trichloromethyl
radical, which can react in one of the following ways: (1) it can react with HS‘ and
release H0; (2) it can first accept another electron to form the trichloromethyl anion which
then reacts with S8: or S2032" to release 8,.12' or SO32; respectively; or; (3) it can first
react with 8x2: or S2032' producing S,.,2= or 8032' respectively, and a trichloromethyl
radical which then accepts an electron. These proposed pathways are illustrated below as
reactions 1.6d, e and f. These three proposed pathways all result in the formation of
trichloromethanethiolate (CC13S'). szn and 82032' are likely to be present in these
systems described by Kreigman -King and Reinhard [33] from the reaction of HS' and
fenic-ion in minerals. CClgS', the proposed intermediate that could form by either the
nucleophilic substitution pathway or the electron-transfer—pathway, should decompose to
form thiophosgene (C12C= S), which is transformed to C02. As discussed above, C82 is
ultimately hydrolyzed to C02 by OH—. In addition, it is possible that rather than by
reacting with OH" to form C02, thiophosgene could react with water to form C02 via a
carbonyl sulfide (COS) intermediate. However, this intermediate was not observed in the

abiotic transformation system described by Kreigman-King and Reinhard [33].

0CCl3 + HS- __.> CClgS' + H0 (1.6d)
0CCI3 + $2032’ + e- —> CClgS' + 8032' (1.6 e)
0CCl3 + 83‘ + e- -——> CClgS‘ + SHZ‘ (1.61)

l4

Pathway (1) Addition of oxygen to the trichloromethyl radical

Molecular oxygen (02) adds to the trichloromethyl radical to give a peroxy radical by the
reaction:

0 CCI3 + 02 —> CCIBOO- (1.73)

The mechanism by which peroxy radicals (as shown in the above reaction) are converted
to CO2 is uncertain. In one proposed pathway, peroxy radicals abstract hydrogen from an
organic compound in the environment, leading to the production of trichlorohydro-
peroxide (1.7b). Trichlorohydroperoxide hydrolyzes releasing H202 and phos gene (1.7c-
[4]). In the second pathway, two peroxy radicals combine, releasing molecular oxygen
and two alkoxy radicals ( 1.7d). The alkoxy radicals cleave homolytically to give a
chlorine radical and phosgene (1.7e) [44], or they are reduced to give trichloromethanol
(1.71) [5], which decomposes to phosgene (1.7g) Phosgene formed by 1.7 c,e, or g
hydrolyzes to CO2 as shown in reaction 1.7 h.

CCl3OO° + RH —-—> CCI3OOH + R0 (1.7b)
CCI3OOH + H20 —> O=CC12 + H202 + HCI (1.7c)
2[CCI3OO°] —> 2CCI3O° + 02 (1.7d)
CCl3O' ———-> O=CC12 + C10 (1.7e)
CCI30° +H+ + c- —> CCI3OH (1.71)
CCI3OH —> O=CC12 + HCl (1.7g)
O=CC12 + H2O ————> C02 + 2HCl (1.711)

In the above reactions, phosgene (O=CCl2) is an expected intermediate in the aerobic
transformation of CCl4 to C02. Shah et al. [46] and Kubik and Anders [35] reported the

15

formation of phosgene from CCl4. Kubik and Anders [35] used 180 labeled O2 to
demonstrate that oxygen incorporated into phosgene was derived from 02 and not from

water.

Phosgene acylates amines, including amino acids such as cysteine as shown in reaction
1.7i. The reactivity of phos gene with cysteine led to the development of means trap
phosgene, thereby proving its role as an intermediate in the aerobic transformation of

CCl4 to C02 in rat liver homogenate [46].

O=CCl2 + R-NH2 —> O=CCINH-R (1.71)

Given the transformation of CCl4 in aerobic mammalian and abiotic systems, it is
puzzling that aerobic transformations of CCl4 by microbial systems are not well
documented. One reason aerobic transformations may not occur is that oxygen can act as
a competitive inhibitor with CCl4 for electrons, thus blocking the fist step of the reaction
which is the formation of a trichloromethyl radical. Competition for elections may

explain the lack of CCl4 transformation in aerobic mixed cultures observed by Bouwer

and McCarty [7].

Pathway 8: Covalent binding of the trichloromethyl radical to cell material

The binding of trichloromethyl radicals to cell material is well documented, and
represents a significant portion of transformed CCl4 products in many systems. Trudell
et al. [49] demonstrated that the trichloromethyl radical reacts with the double-bonds of
phospholipids. Trichloromethyl radicals also react with other double bonds--typically
attaching to the carbon with the most hydrogen substituents [43]. Covalently-bound
CCl4-metabolites were reported by Ahr et al. [2] for microsomal systems. Most of the

16

reported metabolites bound to lipids rather than proteins. DiRenzo et al. [17] reported
that covalent binding of trichloromethyl radicals to the lipids and proteins of rat
hepatocytes was greater under anoxic conditions. Sipes et al. [47] found that the binding
of trichloromethyl radicals to rat liver microsomes was three times greater under a
nitrogen atmospheres rather than under an atmosphere of air. This increase in cell bound
material under anoxic conditions is likely due to the competition between 02 and cell

material for trichloromethyl radicals.

Literature Summary

From the above literature summary, the unifying aspect of CCl4 transformation is the
initial generation of a trichloromethyl radical; subsequent transformations occur by
competing pathways in both biotic and abiotic systems. However, a few general
conclusions can be made about these pathways. In anoxic systems, faster and more
extensive reductive dechlorination generally occurs as the environment becomes more
reducing--highest rates are generally obtained under methanogenic conditions, next
highest under sulfate-respiring, and slower rates observed under fumarate respiring and
fermenting conditions. If the environment is sufficiently reducing, the chloroform
produced can be further reduced to dichloromethane or chloromethane. Transformations
in the presence of HS- or RS- holds promise for abiotic transformations that do not
produce chloroform. However, these transformations are typically slow. Alternately, the
competition for electrons by other oxidants such as molecular oxygen may reduce the
efficiency of electron transfer to CCl4, thus blocking the fist step of the reaction which is

the formation of a trichloromethyl radical.

17

Pseudomonas stutzen’ KC

In 1988, Criddle et al. obtained a natural aquifer isolate that was capable of rapidly
transforming CCl4 to C02 (45-55%), a cell-associated fraction (5%), and a remaining
unidentified non-volatile fraction (40-50%) under denitrifying conditions [15].
Immediately, it was apparent that the mechanism of transformation by this organism was
not easily understood in the conth of previously characterized transformations. This
was the first report of a microbial transformation of CCl4 that did not result in appreciable
chloroform production under anoxic conditions. The rates observed for CCl4
transformation under denitrifying conditions by P. stutzeri KC [15] were greater than
those previously observed under methanogenic conditions [34], thus disputing the
original conclusion that rates of CCl4 transformation increase as the environment
becomes more reduced. The inhibition of CCl4 transformation under aerobic conditions
argued against the formation of C02 by a phosgene intermediate. Initially, it was not
even possible to conclude that the transformation occurred by the generation of a
trichloromethyl radical.

Since the original report by Criddle et al. [15], much work has focused on the elucidation
of the mechanism of CCl4 transformation by P. stutzeri KC. In Figure 1.1, the
determined products and intermediates of CCl4 transformation by P. stutzeri KC are
boxed. Originally, Criddle et al.[15] determined that CO2, unidentified non-volatiles, and
cell-bound material were produced from the anoxic transformation with little or no
chloroform production. Lewis and Crawford [38], reported that approximately 5% of the
originally added CCl4 was converted to chloroform by anoxic cultures of strain KC.
Dybas et al [18] determined that 20% of the initially added CCl4 was converted to

f ormate. More recently, Lewis and Crawford [39] identified thiophosgene and phosgene

18

condensation products, suggesting that these compounds are intermediates in the

transformation of CCl4 by P. stutzeri KC.

The transformation products and reaction intermediates identified suggests a very
complex picture for CCl4 transformation by P. stutzeri KC. The production of formate
requires a two-electron transfer to the CCl4 molecule, resulting in the formation of a
dichlorocarbene radical as illustrated in Figure 1.2. This radical can subsequently be
hydrolyzed to formate and CO. In the report by Dybas et al. [18], CO was not tested as a
product of transformation. Additionally, the observation of f orrnate production by Dybas
et al. does not address the large proportion of CCl4 that is converted to C02 by strain KC.
Alternatively, Lewis and Crawford identified condensation products that are typically
produced following the reaction of either thiophosgene with DMED or phos gene with
cysteine (Figure 1.3). Additionally, a HEPES condensation product was identified as
resulting from the interaction of the buffer with thiophosgene. The identification of these
products led Lewis and Crawford to conclude that phos gene and thiophosgene are
intermediates in the transformation, and that the transformation occurs via a one-electron
reduction pathway. However, their conclusions leave many questions unanswered. The
production of phosgene requires the addition of 02in the system. Therefore, it is very
difficult to explain CO2 production by this pathway under anoxic conditions. Secondly,
the conversion of thiophosgene to C02 is very slow, as previously discussed in section 6
of this chapter. Additionally, these pathways proposed by Lewis and Crawford do not
take into account the production of f ormate reported by Dybas et al.

It may be possible to bring some unity to this transformation mechanism if phosgene and
thiophosgene are formed from the dichlorocarbene radical as shown in Figure 1.4. There
currently exist no direct experimental evidence for this pathway, but there also exists no

evidence to suggest that this pathway is n_ot theoretically possible. A thermodynamic

l9

analysis indicates that reduction of water by dichlorocarbene to f orrn phosgene and H2
has an E°' value of +0.66 V. Although this reaction is thermodynamically favorable,
further evidence is required to either prove or disprove this pathway, as currently there is
not enough data to support either a one- or two-electron reduction pathway for CC] 4

transformation by P. stutzeri KC.

2111) HCOOH

a e" C" e" Formate
Ci—C—Ci .c—Ci C’
l / \ 211$ 211C]
(1 G G G
(1 ' . Cl '
(I)
we] Carbonmonoxide

Figure 1.2. Dichlorocarbene intermediate pathway

.4._

20

C?
CI—(E—a
(:1
,3.
Cl-
Ci‘
eCI—a
HS-
Ci
33$“
W. ZHCI
o ' a
r “f . H- ‘v
\ oo \
C‘/ a 2 Cl/ 0
phosgene ZHCI ba-
+ S
”F3 g
. jig—H o/ \a CI-lz-NH-CI-I,-CH;NH-CH3

thio e DMED
’I-ICI 5" 233° phosgen
‘ Cysteine Slow
HS 5

00 OTZ
C l .3-dimethyl -2-thioxoirnidizole

0
'oc c VnN CH C Isl-o-
J “3' “Tu ' 2' H7n

'/ HEPES °

ZHCI '

o o

0)\i~f+-\~i-Cii -Cri .ii-o-
\_/ L1 3 2 u
o

HEPES Condensation Product

 

Figure 1.3 Condensation products observed by Lewis and Crawford[38]

21

‘i’
O — ('3- Cl
(1
2 e'
2 Cl-
/C \
1120 a a
H28 1120 21120
2HCI
Hz
(I) m 2HCi HCOOH
C S
a/ \a Cl 00
/ \
phosgene CI CI
thiophosgene
H1)
ZHCI ”“0
ZHCI
CO
2 002

Figure 1.4. Proposed phosgene and thiophosgene intermediate pathway resulting from a
dichlorocarbene radical.

Criddle et al. [15] reported that the mechanism for transformation by P. stutzeri KC
appeared to be linked to the trace metal- or iron-scavenging functions of the cell. This
observation provided the underlying hypothesis for the studies presented in this thesis
which states that the iron scavenging or siderophore system of P. stutzeri is responsible
for the cometabolism of CCl4. It is my rational that identification of the biochemical

components and processes responsible for CCl4 transformation provides the most

22

appropriate means to elucidate the mechanism(s) and pathway(s) of CCl4 transformation.

by P. stutzeri KC.

OUTLINE OF THIS THESIS

The following chapters describe my studies on the CCl4 transformation system of P.
stutzeri KC . In Chapter 2, I examine CCl4 transformation kinetics with respect to CCl4
concentration, determine the effects of iron and copper on CCl4 transformation, and
determine the fate of l“C-CC14 in cultures of P. stutzeri KC. These studies were
combined with transformations of CCl4 in groundwater and soil systems conducted by
Michael Dybas, and published in Appl. Env. Microb. 59: 2126-2131 1993. In chapter 3, I
present the discovery that both extracellular and cellular factors from P. stutzeri KC are
required for CCl4 transformation. The extracellular factor is shown to be less than 500
MW, and preliminary purification by acetone extraction and HPLC is also described.
These results were combined with product identification studies performed by Michael
Dybas and published in Appl. Environ. Microbiol. 61: 758-762, 1995. The combination
of the secreted factor(s) produced by strain KC with a diverse range of cell types to result
in CCl4 transformation is described in chapter 4. This discovery led to the development
of a bioassay which enabled me to determine: (1) the pH optimum of CCl4
transformation, (2) the reversible inhibition of CCl4 transformation by 02, and (3) the
transformation of CCl4 by indigenous microorganisms in soil slurries amended with a
secreted factor preparations. Portions of this chapter were published in Bioremediation
Series, 3(3) Bioremediation of Chlorinated Solvents, p. 69-77. In chapter 5, I describe
the role of membranes in CCl4 transformation. The possible engineered application of P.
stutzeri KC using vegetable oils as a growth substrate is described in chapter 6 and the
production of chloroform as an end product of CCl4 transformation is demonstrated under
these growth conditions. Finally, in chapter 7, I summarize my results on CCl4
transformation and present a model that summarizes my current understanding of CCl4

transformation by P. slutzeri KC and aids in facilitating future studies.

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45. Petrovski, E.A., T.M. Vogel, and P. Adriens. 1994. Effects of electron acceptors
and donors on transformation of tetrachloromethane by Shewanella putrefaciens MR-l.
FEMS Microbiol. Lett. 121: 357-364.

46. Shah, H., S.P. Hartman, and S. Weinhous. 1979. Formation of carbonyl chloride in
carbon tetrachloride metabolism by rat liver in vitro. Cancer Research 39: 3942-3947.

47. Sipes, I.G., G. Krishna, and J.R. Gillette. 1977. Bioactivaton of carbon
tetrachloride, chloroform and bromotrichloromethane: role of cytochrome p—450. Life
Sciences 20: 1541-1548.

48. Sittig, M. (ed.) 1985. Handbook of Toxic and Hazardous Chemicals and
Carcinogens, Second Edition, pp. 194-196, Noyes Publications, New York.

49. Trudell, J. R., B. Bosterling, and A.,]. Trevor. 1982. Reductive metabolism of
carbon tetrachloride by human cytochromes p—450 reconstituted in phospholipid
vvescicles: mass spectral identification of trichloromethyl radical bound to dioleoyl
phosphatidylcholine. Proc. Natl. Acad. Sci. 79: 2678-2682.

50. Uehleke, H., K.H. Hellmer, S. Tabarelli. 1973. Binding of 1‘iC-carbon tetrachloride
to microsomal proteins in vitro and formation of CHCl3 by reduced liver microsomes,
Xenobiotica, 3: l

51. Verschueren K. (ed) 1983. Handbook of Toxic and Hazardous Chemicals and
Carcinogens, Second Edition, pp. 166-168, Van Nostrand Reinhold Company, New
York.

52. Vogel, T.M. and P.L. McCarty. 1987. Abiotic and biotic transformations of 1,1,1-
trichloroethane under methanogenic conditions. Env. Sci. Tech. 21: 1208-1213.

53. Vogel, T.M., C.S. Criddle, and P.L. McCarty. 1987. Transformations of
halogenated aliphatic compounds, Env. Sci. Tech. 21: 722-736.

54. Wade, R.S. and CE. Castro. 1973. Oxidation of iron (11 ) porpyrins by alkyl
halides. J. Am. Chem. Soc. 95 (1): 226-230.

55. Wolf, C.R., D. Mansuy, W. Nastainczyk, G. Deutschmann, and V. Ullrich. 1977.
The reduction of polyhalogenated methanes by liver microsomal cytochrome p—450.
Molecular Pharm. 13: 698-705.

CHAPTER 2

EFFECTS OF MEDIUM AND TRACE METALS ON KINETICS OF CARBON
TETRACHLORIDE TRANSFORMATION BY PSEUDOMONAS STUTZERI KC

These studies were combined with transformations of CCl4 in groundwater and soil systems performed by
Dr. Michael J. Dybas and published in
Applied and Environmental Microbiology, 59: 2126-2131, 1993.

ABSTRACT

Under denitrifying conditions, Pseudamonas stutzeri KC transforms CCl4 to C02 via an
undetermined mechanism. P. stutzeri KC converted l4C-labeled CCl4 to 1“C02 (37%), a
non-purgeable filterable fraction (20%), and a non-purgeable non-filterable fraction
(34%), with no evidence of interconversion of products. Transformation rates were first-
order with respect to CCl4 concentration over the range examined (0-100 rig/L), and
proportional to protein concentration, giving pseudo-second-order kinetics overall.
Addition of ferric ion (1-20 pM) to an actively transforming culture inhibited CCl4
transformation, and the degree of inhibition increased with increasing iron concentration.
By removing iron from the trace metals solution or by removing iron-containing
precipitate from the growth medium, higher second-order rate coefficients were obtained.
Copper also plays a role in CCl4 transformation. Copper (1}4M) was toxic to strain KC
cells at neutral pH. By adjusting media pH to 8.2, soluble iron and copper levels
decreased as a precipitate formed, and CCl4 transformation rates increased. However,
cultures grown at pH 8.0 without 1 yM copper added to the growth medium exhibited
slower growth rates and greatly reduced rates of CCl4 transformation, indicating that

copper is required for CCl4 transformation.

29

INTRODUCTION

In recent years, considerable interest has surrounded prospects for degrading hazardous
contaminants by stimulating selected bacterial populations (biostimulation) or by addition
of novel organisms to contaminated sites (bioaugmentation). Stimulation of an
indigenous population is likely to yield an enrichment that is well adapted to its
environment, whereas foreign organisms introduced into such an environment may be
unable to compete. However, introduced organisms do offer certain advantages provided
they can compete with the indigenous microorganisms. Unlike indigenous organisms,
introduced organisms can be extensively studied and understood in the laboratory,
improving prospects for control of their activity in the field. Among other reasons,
control of activity is needed to avoid the production of unwanted by-products.
Chloroforrn, for example, is a common end product of CCl4 transformation in both
laboratory and field environments [2, 4, 5, 7]. chloroform is more persistent than CCl4 in
many environments, and it is also a suspected carcinogen. Consequently, metabolic
pathways that do not produce chloroform are of interest. Pseudomonas stutzeri KC is an
aquifer-derived organism that transforms CCl4 to C02 and unidentified non-volatile
products without chloroform production under denitrifying conditions [3]. Related
Pseudamonas species grown and assayed under the same conditions do not transform
CCl4 In the following sections I report on: (1)the quantification of end products of CCl4
transformation, (2) the kinetics of CCl4 transformation by P. stutzeri KC, and (3) the role

of iron and copper in CCl4 transformation kinetics.

30

MATERIALS AND METHODS

Chemicals. CCl4 (99+% purity) was obtained from Aldrich Chemical Co., Milwaukee,
Wisconsin. l4C-labeled CCl4 (250 pCi @ 99% purity) with a specific activity of 4.3
mCi/mmol was obtained from NEN Research Products. A l“C-labeled CCl4 stock
solution in isooctane was prepared as per Criddle et al. (1990). All chemicals for media
preparation were ACS reagent grade (Aldrich or Sigma Chem. Co.), and all water used

was 18 megaohm resistance or greater.

Media preparation and growth conditions. Medium D [3] contained (per liter of de-
ionized water) 2.0 g of KHzPO4, 3.5 g KzHPO4, 1.0 g of (NH4)2804, 0.5 g of MgSO4-
7I-IzO, 1 milliliter of trace nutrient stock TN2, l milliliter of 0.15M Ca(NO3)2 , 3.0 g of
sodium acetate, and 2.0 g of sodium nitrate. Stock solution TN2 contained (per liter of
deionized water) 1.36 g of R804 - 7H20, 0.24 g of Na2M004 - 2H20, 0.25 g of
CuSO4 - 5H20, 0.58 g of ZnSO4 - 7H20, 0.29 g of Co(NO3)2 - 6H20, 0.11 g of NiSO4 -
6120, 35 mg of Na28e03, 62 mg of H3BO3, 0.12 g of NH4VO3, 1.01 g of MnSO4 -
H20, and 1 ml of H2804 (concentrated). Some experiments used different trace metal
preparations to study their effects on CCl4 transformation. TN2—Cu and TN2-Fe stock
solutions lacked CuSO4 and FeSO4 respectively, but were otherwise identical to TN2.
After addition of all essential media components, medium D was adjusted to a desired
initial pH of 8.0 or 8.2 with 3 N KOH. This final adjustment in pH resulted in the
formation of a white precipitate. The resulting medium was autoclaved at 121 °C for 30

minutes and transferred to an anaerobic glove box (Coy Laboratories, Ann Arbor, Mich).
Precipitate-free medium D was prepared as follows: medium D (prepared as previously

described) was transferred to an anaerobic glove box for quiescent settling of precipitate,

and decanted after 24 hours. The precipitate and oxygen-free decanted medium was re-

31

32

autoclaved to assure sterility and cooled before use. Precipitate-free medium D contained
24 mM acetate, 25 mM PO43', 19 mM NO3', determined using a Dionex model 2000i-
SP ion-chromatography system, and 3.8 nM iron determined using a Perkin Elmer model

1100 graphite furnace atomic absorption spectroscopy system.

Cultures were grown under a N2 atmosphere in one of three different containers: (1) 28-
mL serum tubes (Bellco Glass No. 2048-00150), (2) a modified one-liter Wheaton Bottle
as described by Balch and Wolfe [1], and (3) 250-mL (8 oz.) bottles sealed with screw-
cap Mininert valves (Alltech catalog number 95326). Both the serum tubes and the
modified Wheaton bottles were sealed with teflon-faced butyl rubber septa (West Catalog
number 1014-4852) and aluminum crimp seals. All cultures were shaken at 100-150 rpm
at 20-23 0C. Strain KC did not transform CCl4 at temperatures above 25°C, and it did
not grow at temperatures above 30°C (data not shown). Culture manipulations were
typically performed in an anaerobic glove box under an atmosphere of 98% N2 and 2%
H2. Oxygen level was monitored continuously with a Coy gas detector model no. 10.

Hungate technique was used for anaerobic manipulations outside the glove box.

Analytical methods. All bottles used to evaluate CCl4 transformation were sealed with
pressure tested screw-cap Mininert valves or Teflon-lined butyl rubber stoppers. CCl4
was assayed by removing 0.1 mL of headspace gas with a 0.25 or 0.5 mL Precision gas-
tight syringe (Alltech catalog no. 050032), and injecting the sample into the GC. For
yg/L concentrations, the GC was a Perkin Elmer model 8500 equipped with a 100/ 120
mesh column (10% Alltech CS- 10 on Chromsorb W-AW, Alltech Catalog # 12009 PC)
and an electron capture detector with nitrogen carrier (40 mIJmin) and nitrogen make-up
(27 mUmin). For mg/L concentrations, the GC was a Hewlett Packard 5890 gas

chromatograph operated isotherrnally at 150°C and equipped with a DO 624 column

33

(J&W Scientific Catalog # 125-1334) and a flame ionization detector (hydrogen flowrate

= 100 mL/min, air flowrate = 250 mUmin). The carrier gas was nitrogen (16 mIJmin).

External standard calibration curves were prepared by addition of a primary standard (7.8
ng CCl4 per yL methanol or 0.82 p g CCl4 per yL methanol) to secondary standard water
solutions having the same gas/water ratio, ionic strength, incubation temperature, and
speed of shaking as the assay samples. A four point calibration curve was prepared over
a concentration range bracketing that of the assay samples. Protein was stored by
freezing at -20°C, and assayed using the modified Lowry method, with bovine serum
albumin as the standard [6].

Mass transfer rates were estimated for 28 mL serum tubes containing 10 mL of medium
D and sealed with teflon-lined rubber septa. CCl4 was added to the aqueous phase, and
the tubes were shaken at 100 rpm on a shaker table. Headspace CCl4 was assayed every

60 seconds for 10 minutes by GC. The mass transfer rate kLa(h'1) was determined by

. C .
plotting In ‘1 - 6%} versus shaking time, where Cg = headspace CCl4 concentration and
8

g = headspace CCl4 concentration at equilibrium. The slope of the resulting plot is

C

V
-kLa ‘v—34- i), where Hc = dimensionless Henry's constant (1.0 for CCl4 at 20°C), Vaq
aq

= water volume and V8 = headspace volume.

Radioisotope experiments. To assess the time course of 14C-CCl4 transformation, a
method of quenching the reaction was needed. In separate experiments, hydrogen
peroxide was evaluated and found suitable for this purpose. Hydrogen peroxide halted
CCl4 transformation, did not itself transform CCl4, and did not alter the distribution of

CCl4 transformation products.

34

A time series experiment with radiolabeled CCl4 was conducted using a 2.18 L glass
reaction vessel. The vessel was modified so that access to its contents was only possible
through two threaded glass openings. These openings were sealed with screw-cap tefion
Mininert valves (individually pressure tested). The vessel was filled with 2.17 L of
medium D, then inoculated with a 0.15% solution of stationary phase P. stutzeri KC, and
incubated at room temperature for 120 hours. After the culture had grown to an OD660 of
0.75, the bottle was spiked with the l4C'CCl4 solution in isooctane, and transferred to a
rotary shaker for vigorous agitation (150 RPM). After 15 minutes and at regular intervals
thereafter, 5 mL of culture was withdrawn using a 5 mL Hamilton gas-tight syringe
(Alltech catalog no. 81530). One mL was injected into a scintillation vial containing 10
mg (23 FL) of hydrogen peroxide, 9 ml. scintillation cocktail was then added, and the
vial was sealed to prevent loss of volatile l4C-labeled CCl4. A second milliliter of
sample was injected into a scintillation vial containing 10 mg (23 pl.) of H202 and 35 yL
of 3 N KOH. A third milliliter was injected into a scintillation vial containing 10 mg
(23}:1.) of H202 and 30 pL of 6 N HCl. A final milliliter of sample was filtered through a
0.2 pm nylon filter into a scintillation vial containing 10 mg (23 FL) H202 and 30 FL (1'
6 N HCl. The acid and base samples were purged for 25 minutes with N2 gas at a flow
rate of 10 mUmin. After purging, 9 mL of scintillation cocktail (Beckrnan Cat. No.
158726) was added. All samples were counted for 5 minutes on a Packard tri-carb liquid
scintillation counter (Model 1500). Because 14C was added to the culture in isooctane, a
portion of the 14C added to the reaction flask was present in the isooctane phase during
the reaction. The partition coefficient for CCl4 in a water/isooctane system (i.e., dpm/mL
of the isooctane divided by dpm/mL of the equilibrated aqueous phase) was measured
separately, and a value of 1032398 was obtained.

Disappearance of CCl4 and appearance of products were quantified with a simple model

in which transformation activity is assumed to decay with time:

35

(18 -k r
—- -kSe d
dt (1)

where S: CCl4 concentration (mg/L), k=first order rate coefficient (h'l), kd = first-order

decay coefficient (h'l). Equation 1 is integrated to obtain S as a function of time:

S - S°exp[k£d(cxp(-kdt) - 1)] (2)

where 3°: initial CCl4 concentration(mg/L).
Formation of products (P) were modeled by using equation (2) assuming that a fraction of

S was converted to the products P.

P - as" — S) (3)

where h product concentration. Data were evaluated using a least squares fit of the data

to equations 1-3.

Determination of reaction rate order and transformation capacity. The dependence
of CCl4 transformation rate on CCl4 concentration was assessed with stationary phase
cultures grown 72 hours from a 1% inoculum in both medium D and precipitate-free
medium D. Cultures were dispensed into 28-mL serum tubes, and CCl4 was added (5-
100 flg/L). The tubes were then transferred to a shaker table, and headspace CCl4 was
periodically monitored by GC. Reaction rates were calculated using measurements taken
after 20 minutes had elapsed to allow sufficient time for equilibration of headspace CCl4
with the water phase CCl4. Under these conditions, the mass transfer rate (~25 h'l) was
much greater than the reaction rate (~l h'l). The observed rates were corrected for
equilibrium partitioning into the gas phase to obtain the true reaction rates (see

Modeling). To evaluate the dependence of CCl4 transformation rate on total culture

pt

“‘3

36

protein, a stationary phase culture was diluted 1:5, 1:3, and 1:2 with media, then

monitored for CCl4 removal by sampling of the gas phase.

To determine the transformation capacity of KC cultures grown under different
conditions, 100 mL of stationary phase culture (grown for 72 hours in 1 liter modified
Wheaton bottles in either medium D or precipitate-free medium D) was dispensed into
170-mL serum vials sealed with teflon-lined septa. The vials were then spiked with CCl4

to give initial concentrations of 1 mg/L or 5 mg/L, shaken at 100 rpm at 20°C, and

monitored for CCl4 removal by sampling of the gas phase.

Effects of trace metals. To assess the effects of trace copper, medium D was prepared
with either stock solution TN2 or TN2-Cu, transferred to 802 (250-mL) bottles, sealed,
autoclaved, cooled, and inoculated with a 1% (v/v) suspension of a stationary phase
culture of P. stutzeri KC. Cultures were grown to stationary phase, spiked with CCl4,

and assayed for CCl4 transformation.

To assess the effects of trace iron, medium D and precipitate-free medium D were
Prepared using trace metal stock solutions TN2 and TN2-Fe. Cultures were grown 48 or
72 hours, spiked with CCl4, and assayed for CCl4 transformation. To investigate iron
inhibition, 10 mL of early stationary phase culture (grown for 72 hours in precipitate-free
medium D) was transfened to 28 mL serum tubes in an anaerobic glove box, spiked with
020 FM ferric iron (as ferric ammonium sulfate), and equilibrated for 10 minutes. The
serum mbes were sealed with teflon-linai rubber stoppers, spiked with CCl4, shaken
throughout the experiment, and monitored by sampling of the gas phase.

Modeling. Separate experiments (see Results) established that transformation of CCl4

was first~<>rder with respect to CCl4 concentration over the concentration range examined

37

and first-order with respect to total protein concentration. Assuming a second-order
kinetic expression, a mass balance can be written for a closed batch system in which a

volatile aqueous phase substrate is in equilibrium with its gas phase:

———-dN’Cr: ' =—k—' 1
dt 1‘ C.,,xvu, V.,qmcvg MCTXV“! ( )

where: Mccj4 = total mass of CCl4 in the system (mg) = C1,.q(Vaq + HCVg), t = time (d), k'
= second-order rate coefficient (Umg protein-d), Caq = aqueous phase concentration of
CCl4 (mg/L), X = concentration of protein (mg/L). Separation of variables and

integration of equation 1 yields:

(as—4)

 

For a reaction that is first-order with respect to CCl4 concentration, a plot of the
logarithm of the mass of CCl4 in the bottle vs. time should give a straight line with slope
of k'XVaq/(V aq+HcV g).

RESULTS

Kinetics of CCl4 transformation. The dependence of CCl4 transformation rates on CCl4
concentration was evaluated by plotting the logarithm of mass of CCl4 versus time. As
shown in Figure 2.1, this plot was linear, and the slopes were essentially constant over the
concentration range examined, indicating that the reaction could be represented as first

order with respect to CCl4 concentration.

38

 

 

0 .-
l
_ 1 _, x 100 ug/L
In (CF 14g) 50 ug/L
- 2__
25 ug/L
— 3 . , . . - I . 1
20 40 60 80 100
Time (nrin)

Figure 2.1. Kinetics of CCl4 loss by strain KC. Slopes for CCl4 transformation were
calculated from natural logarithm of the concentrations 25, 50, and 100pg/L of CCl4
versus time. Slopes were determined to be 0.0096, 0.0094, and 0.0076 respectively.
Error bars representing the standard deviations of four independent samples are generally
less than the dimensions of symbols used.

To assess the dependence of the CCl4 transformation rate on protein concentration, first-
order rate coefficients of the culture were plotted against total protein concentration. As
shown in Figure 2.2, rates were linearly related to protein concentration range evaluated
(6.25-22 pg/mL). Thus, a pseudo-second-order rate expression (first-order in CCl4
concentration and pseudo-first order in protein concentration) was considered

appropriate. Pseudo-second-order rate coefficients were calculated from the slopes of the

logarithm of mass versus time plot.

39

 

 

0.2 -
a
3
E
.8
é a
g 0.1 - '3
3
'8
o
‘g a
h.
0.0 ' I fi I ' I
0 10 20 30
Protein concentration (14 g/mL)

Fr gure 2.2. Dependence of first-order rate coefficient on total protein concentration.

Fate of 1“C-Iabeled CCl4. Radiolabeled CCl4 was rapidly converted to 14C02 (37%), a
non-purgeable non-filterable fraction (34%), and a non-purgeable f ilterable fraction
(20%), with no apparent accumulation or interconversion of products (Figure 2.3). 14C-
labeled CCl4 was added as an isooctane stock so partitioning had to be considered.
Assuming equilibrium, approximately 17% of CCl4 was present in isooctane initially, but
this fraction dropped to 2% within four hours. Partitioning of CCl4 into the gas phase
was also considered (assuming a dimensionless Henry's constant of 1.0). As liquid
volume was removed by sampling, the gas phase volume increased from 0.5% of the
reactor volume initially to 6.4% by the end of the experiment (the gas phase was under
positive pressure so no vacuum formed during sampling). Assuming equilibrium, the
CCl4 present in the gas phase increased from about 1% of the total CCl4 present initially

to about 8% of the CCl4 present at the end of the experiment.

 

 

 

 

 

 

Figure 2.3. Fate of transformed radiolabeled CCl4 by P. stutzeri KC. Lines represent the
best fit to the data points as described in materials and methods. k: 0.82 M, 6,“, = 0.200,
5am = 0.341, 6(1), = 0.371, kd = 0.41 h'1 of equations 2 and 3

Effects of trace metals. Removal of the precipitate that formed during preparation of
medium D at pH 8.0 had a profound effect on the iron level of the medium. As
determined by atomic adsorption spectroscopy, precipitate-free medium D contained 3.8
nM iron. Removal of iron from the growth medium affected growth rate, protein levels,
CCl4 transformation rate, and CCl4 transformation capacity. As shown in Figure 2.4,
protein concentration at the end of the growth phase was greater for cells grown in
medium D (protein concentration = 350 yg/mL) than for cells grown in medium D
prepared with TN2-Fe ( protein concentration = 51 pg/mL) precipitate-free medium D
(protein concentration = 46 yg/mL) and precipitate-free medium D prepared with TN2-Fe

(protein concentration = 22.5 pg/mL). These observations indicate that diminished

41

growth was due to the removal of iron. Cells grown in precipitate-free medium D had
higher pseudo-second-order rate coefficients (Table 2.1), but lower 24-hour
transformation capacities (Figures 2.5 and 2.6) when compared to medium D. Pseudo-
second-order rate coefficients were lowest in the "high" iron media, and highest in the

"low" iron media (prepared with TN2-Fe or without precipitate).

 

 

 

‘ ':
1 _ Medium D
.1 1 , Medium D- ‘
: ' Ppt-free
3 edium D
OD :
660 Ppt-free
Medium D-Fe
.01 -:
~001 _‘ r ' T ' l r r
0 20 40 60 80 1 00

Time (hours)

Figure 2.4. Growth of P. stutzeri KC in medium D and precipitate-free medium D in the
presence or absence of added iron.

42

 

 

 

 

0.9
'1 5mg/L
control
‘ll
I
0.6 -
CI‘
(mg) 5 mg/L
+ KC
0.3 1
1 mg/L
control
1 mg/L
0.0 ' l V I v I v I I + KC
0 300 600 900 1 200 1 500

Time (min)

Figure 2.5. CCl4 transformation capacity for cultures of P. stutzeri KC grown in medium

D. Concentrations of CCl4 are given in mg/L are shown on the graph. Error bars represent
standard deviations on triplicate samples.

43

 

0.9
] 5mg/LCT
d conuol
i i i 5 LCT
7 mg,

+ KC
0.6 -

(mg)

0.3 .
1 mg/LCI‘
' 1r”- control

1 mg/l CT
0.0 ' I + KC

' r 1 r f r ' r
0 300 600 900 1 200 1 500

 

 

Time (min)

Figure 2.6. CCl4 transformation capacity for cultures of P. stutzeri KC grown in
precipitate-free medium D. Concentration of CCl4 are given in mg/L are shown on the
graph. Error bars represent standard deviations on triplicate samples.

As shown in Table 2.1, pseudo-second-order rate coefficients for CCl4 transformation
generally decreased as cultures aged from 48 to 72 hours, indicating decay of
transformation activity as cells entered the stationary phase. The exception was cultures
grown in medium D with TN2—Fe. These cultures continued to grow between 48 and 72
hours, and showed no decrease in the second order rate coefficient over this period.
Growth rates for these cultures were higher and less variable than those of cultures grown
in precipitate-free media (Figure 2.4). These observations suggest that, for this medium,
cell growth and production of CCl4 transformation activity may be controlled by the

solubilization of iron in the precipitate.

Table 2.1. Second-order rate coefficients (:1: one standard deviation) for CCl4
transformation by P. stutzeri KC: effects of iron limitation and culture age.

 

 
  

  
   
 

 

 

 

 

Medium Culture age k'
modification (Umg protein-d)
+ precipitate 48 0.893zt0.03
72 0.362:r:0.08
+ precipitate 48 3.93:1.48
- trace Fe
72 4.03:0.79
- precipitate 48 6.18:0.48
72 2.28¢0.45
- precipitate 48 91:12
- trace Fe
72 4.41:0.56

 

 

 

 

 

The effect of ferric iron addition to precipitate-free medium D is shown in Figure 2.7.
Addition of ferric ammonium sulfate (1, 5, 10, and 20 pM) to an actively transforming
stationary phase culture inhibited the rate of CCl4 transformation CCl4, and the degree of

inhibition increased as the concentration of iron increased.

45

 

 

 

 

 

 

0.20! ”.2 ..
‘a T “ 3 Control
«MN
i ~ ‘ ““W 20 yM Iron
0.19: .. 10 yM Iron
CI' 5yM Iron
(kg)
0.10-
0.05-
1 yM Iron
OyM Iron
0.0 I I j I ' I I I
0 50 100 150 200

Time (min)

Figure 2.7. Inhibition of CCl4 transformation by ferric iron. Error bars represent standard
devrations on triplicate samples.

 

’ - (lower

(#8)

 

 

 

 

 

 

. . , . , . a , . .
0 5 10 15 20 25 30
Time (Hours)

Figure 2.8. Trace levels of copper affect CCl4 transformation activity. Error bars
represent standard deviations on triplicate samples.

Criddle et al. [3] found that 1 14M copper prevented growth of P. stutzeri KC at neutral
pH. The present work confirmed this finding. In medium D adjusted to pH 8.0, however,
the maximum specific growth rate of strain KC decreased in the absence of copper,
dropping from 0.047 hr:l for TN2+Cu to 0.016 hr'1 for TN2-Cu. Final protein
concentration was not greatly affected by copper (185 jig/m1. for TN2+Cu compared with
173 pg/ml for TN2-Cu). As shown in Figure 2.8, rapid transformation of CCl4 was only
obtained with TN2+Cu, and little or no transformation of CCl4 was obtained with TN2-

Cu. Thus, omission of only 1 FM cOpper was sufficient to prevent CCl4 transformation.

DISCUSSION

My results indicate that transformation of CCl4 by P. stutzeri KC proceeds by a complex
mechanism. The transformation appears to be linked to the iron-scavenging functions of
the cell, as previously proposed [3]. Observations supporting this hypothesis include the
following: (1) KC grown in precipitate-free medium D does not transform CCl4 if the
growth medium is supplemented with trace iron before inoculation with strain KC [3], (2)
addition of iron to grown cultures of strain KC inhibits CCl4 transformation - possibly by
competing for a binding site (Figure 2.7), and (3) the second-order rate coefficients for
CCl4 transformation increase for cells grown in iron-limited media (Table 1).
Transformation of CCl4 apparently requires copper and probably involves a reducing
agent, as evidenced by the quenching action of oxidants, such as hydrogen peroxide and

oxygen (data not shown).

The present work disproves one proposed pathway of CCl4 transformation. Criddle et al.
(1990) hypothesized that C02 production from CCl4 might be explained by the oxidation
of the non-purgable non-filterable fraction: a two electron reduction of CCl4 could
produce a dichlorocarbene radical which could spontaneously undergo hydrolysis to form
formate; carbon dioxide could subsequently be produced by oxidation of the formate. In
the time series experiment with 14C (Figure 2.3), there was no evidence that the end
products were interconverted. This observation contradicts the original hypothesis and
suggests that transformation proceeds through a rapidly decomposing intermediate which
is subsequently transformed via parallel pathways to C02, the non-purgeable non-
filterable fraction, and a non-purgeable filterable fraction. A less likely explanation is

that more than one agent of transformation is operative.

47

48

Given the complex mechanism postulated for CCl4 uansformation, it is likely that a
complete kinetic description of the transformation will prove equally complex. The agent
of transformation must be identified and quantified. More information is needed to
understand changes in CCl4 transformation activity with cell growth stage and trace metal
speciation. As shown in Table 2.1, growing cells transform CCl4 faster than do
stationary phase cells. There is also evidence that the CCl4 transformation is affected by
other trace metals, notably cobalt and vanadium [3], and that these trace metals act
synergistically with iron to inhibit CCl4 transformation [Criddle, PhD. Thesis, 1989]. In
spite of these complexities, however, the present results do establish a simple first—order
dependence on CCl4 concentration over the CCl4 concentration range investigated. A
pseudo-first—order relationship with total protein concentration was also observed.
However, use of total protein in a pseudo-second-order kinetic expression must be
viewed as a temporary expediency. Total protein merely functions as a quantifiable
surrogate for the actual agent of transformation until such time as the agent itself can be
quantified. In the following chapter, I describe studies which further elucidate the

components of the CCl4 transformation system of P. stutzeri KC.

REFERENCES

1. Balch, W. E. and R. S. Wolfe. 1979. Transport of coenzyme M (2-
mercaptoethanesulfonic acid) in Methanabacterium ruminantium . J. Bact. 137. 264-273.

2. Criddle, C. S., J. T. DeWitt, and P. L. McCarty. 1990. Reductive dehalogenation of
carbon tetrachloride by Escherichia coli K-12. Appl. Environ. Microbiol . 56:3247—3254.

3. Criddle, C. S., J. T. DeWitt, D. Grbic-Galie, and P. L. McCarty. 1990.
Transformation of carbon tetrachloride by Pseudomanas sp. strain KC under
denitrification conditions. Appl. Environ. Microbiol. 56:3240-3246.

4. Egli, C., R. Scholtz, A. M. Cook, and T. Leisinger. 1987 . Anaerobic dechlorination
of tetrachloromethane and 1,2-dichloromethane to degradable products by pure cultures
of Desulfobacterium sp. Methanabacterium sp. FEMS Microbiology Letters 431257 -
261.

5. Egli, C., T. Tschan, R. Scholtz, A. M. Cook, and T. Leisinger. 1988.
Transformation of tetrachloromethane to dichloromethane and carbon dioxide by
Acetobacteriilm woodii. Appl. Environ. Microbial. 54:2819-2823.

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

7. Semprini, L., G. D. Hopkins, P. L. McCarty, and P. V. Roberts. 1992. In-situ
transformation of carbon tetrachloride and other halogenated compounds resulting from
biostimulation under anoxic conditions. Environ. Sci. Technol. 26: 2454-2461.

8. Sittig, M. (ed.) 1985. Handbook of Toxic and Hazardous Chemicals and Carcinogens,
Second Edition, Noyes Publications, New York.

9. Stumm and Morgan. 1981. p. 230-322. In Aquatic Chemistry, Second Edition, Wiley
and Sons, New York

49

CHAPTER 3

LOCALIZATION OF THE CARBON TETRACHLORIDE TRANSFORMATION
ACTIVITY OF PSEUDOMONAS STUTZERI KC

These studies were combined with end-product identification analyses and protonophore studies performed
by Michael J. Dybas and published in
Applied and Environmental Microbiology, 61: 758-762, 1995.

I gratefully acknowledge the co-development of the purification process for the secreted factor by Dr.
Michael J. Dybas

ABSTRACT

Previous research has established that rapid transformation of CCl4 by Pseudamonas
stutzeri KC requires the organism to be grown under denitrifying and iron-limited
conditions. The present study investigated the possible role of iron scavenging agents in
the transformation, and presents the finding that both extracellular and intracellular
factors are involved in the transformation. By themselves, washed cells of P. stutzeri KC
did not transform CCl4 to a significant degree. Occasionally, CCl4 transformation was
observed by cell-free culture supernatant, but this activity was not reliable. Rapid and
reliable CCl4 transformation was only obtained when washed whole cells were
reconstituted with culture supernatant, indicating that both extracellular and intracellular
factors are normally required for CCl4 transformation. Fractionation of culture
supernatant by ultrafiltration established that the extracellular f actor(s) is small, with an
apparent molecular weight of less than 500 daltons. Addition of micromolar levels of
iron inhibited CCl4 transformation in whole cultures, but the level of iron needed to
inhibit CCl4 transformation was over one hundred fold higher for washed cells
reconstituted with 10,000 dalton supernatant filtrate. Thus, the inhibitory effects of iron
are exacerbated by a supernatant factor(s) with a molecular weight greater than 10,000
daltons. The extracellular fraction was further purified following the discovery that it is
stable after lyophilization to powder and is extractable with acetone. A fraction
containing CCl4 transformation activity eluted at 27-28 minutes from a semi-preparative

reverse phase HPLC column at a flowrate of 7 mUmin with a methanol/water gradient.

51

INTRODUCTION

Pseudomonas stutzeri KC is a natural aquifer isolate that rapidly transforms CCl4 to C02
and non-volatile end product(s) without the production of chloroform under denitrifying
conditions. The ability of P. stutzeri KC to transform CCl4 is dependent on the presence
of iron-limiting conditions in the growth medium [1,5,12]. Additionally, ferric iron was
shown to inhibit CCl4 transformation by actively transforming cultures of P. stutzeri KC
[12]. Based on these observations, it has previously been hypothesized that an iron
scavenging system plays a key role in the transformation of CCl4 by P. stutzeri KC
[1,12]. In this chapter, I further evaluate the possibility of an iron scavenging system
fortuitously transforming CCl4 by focusing on the role of intracellular and extracellular
components involved in transformation activity. I describe studies which demonstrate
that an extracellular factor(s) is involved in the transformation and I describe progress
made toward partial purification of the extracellular factor(s). Additionally, I

characterize the extracellular f actor(s) by size and iron sensitivity.

52

MATERIALS AND METHODS

Organisms. P. stutzeri KC (DSM deposit no. 7136, ATTC deposit no. 55595), derived
originally from aquifer solids from Seal Beach, CA, [1] is routinely maintained in our
laboratories on nutrient agar plates. Pseudomonas fluorescens (ATTC deposit no. 13525)
was obtained from the culture collection of the Department of Microbiology at Michigan

State University.

Chemicals. CCl4 (99% purity) was obtained from Aldrich Chemical Co., Milwaukee,
Wis. All chemicals used were ACS reagent grade (Aldrich or Sigma Chemical Co.). All

water used in reagent preparation was 18 Mohm resistance or greater.

Glassware. All glassware used in the purification procedure was acid washed in 6M
HCl, rinsed with DI water, rinsed with HPLC grade acetone, and dried at 100°C prior to

use in any manipulation.

Media. Medium D was prepared and dispensed in 28 ml serum tubes or modified l-liter
Wheaton bottles as previously described in Chapter 2. Cultures of P. fluorescens were
grown in Medium D under aerobic conditions. Cultures of strain KC were also grown in
medium D at 20°C with 150 rpm shaking under aerobic or denitrifying (N2 headspace)
conditions. For purification of supernatant components, strain KC was grown in
simulated groundwater medium SGW (recipe provided by R. Skeen, Battelle Pacific
Northwest Laboratory). Medium SGW contained per liter of deionized water. 0.455 g of
Na2SiO3 - 9 H20, 0.16 g Na2CO3, 0.006 g of NaZSO4, 0.02 g of KOH, 0.118 g of MgC12
- 6H20, 0.0081 g of CaC12 - 2H20, 13.61 g of KH2PO4, 1.6 g of NaOH, 1.6 g of NaNOg,
1.6 g of acetate and lmL of trace element solution. The trace element solution contained

per liter of deionized water: 0.021 g of LiClz, 0.08 g of CuSO4 - 5H20, 0.106 g of ZnSO4

53

54

- 7H20, 0.6 g of H3803, 0.123 g of A12(SO4)3 - 18 H20, 0.11 g of NiC12 - 6 H20, 0.109
g of C0804 ~ 7H20, 0.06 g of TiCl4, 0.03 g of KBr, 0.03 g of K1, 0.629 g of MnClz .
41-120, 0.036 g of SnClz - 2H20, 0.3 g of FeSO4 - 7H20. The pH of SGW medium was
adjusted to 8.2 with 3 N KOH. The resulting medium was autoclaved at 121°C for 20

minutes then transferred to a Coy anaerobic glove box for degassing.

Analytical methods. CCl4 was assayed by removing samples of headspace gas above
liquid samples and injected the sample onto a gas chromatograph as described previously
in Chapter 1. Protein was assayed by the modified Lowry method, with bovine serum
albumin as the standard [7].

Bioassay for the secreted factor(s) using P. fluorescens. Tatara et al. [13] discovered
that rapid CCl4 degradation occurs when the secreted factor(s) generated by strain KC is
combined with diverse cell types, such as cells of P. fluorescens. This finding enabled
the development of a bioassay for the secreted factor(s). In the bioassay, P. fluorescens
cells were harvested by centrifugation (12,100 X g, 10 min, 4 °C) and resuspended to one
tenth the original culture volume in medium D, to a cell density of approximately 2 x 109
cf ulml. Five hundred microliters of the resulting 10 X concentrated cell suspension was
added to 4.5 ml samples generated during the fractionation procedure. The samples were
rendered anoxic by passage through the anaerobic interlock on the Coy anaerobic glove
box which pulled a vacuum of 20 in. of Hg and replaced the headspace with N2 nine
times. Samples were sealed under a N2 headspace in 28 ml Balch tubes and spiked with
CCl4. Levels of CCl4 were followed by headspace gas chromatography as previously
described.

Fractionation of CCl4 transformation activity. To identify the factors involved in

CCl4 transformation activity of strain KC, actively transforming cultures were

55

fractionated by cenuifugation and ultrafiltration. Cultures grown for 24 hours in SGW
medium under denitrifying conditions were first screened for CCl4 transformation
activity. Typically, about 300 nrl of actively uansforming culture was dispensed into
degassed Oak Ridge style centrifuge tubes in the anaerobic glove box. This type of
centrifuge tube has been shown to exclude oxygen and maintain the strict anaerobic
conditions required for methanogenesis during 10 minute centrifuge runs [3]. Cells were
harvested by centrifugation (10 minutes at 12,100 X g at 4°C), and the supernatant
filtered through a 0.2 pm filter. Occasionally, the cell-free supernatant was capable of
CCl4 transformation. In these instances, it was further fractionated by filtration through
Amicon 10,000 and 500 molecular weight cut -off filters in the anaerobic glove box
(95% N2 :1: 5% H2 atmosphere). Filtrate and retentate of each filter were assayed for
CCl4 transformation activity. The CCl4 transformation assay was performed on 4.5 ml
samples of the fractions with and without reconstitution with P. stutzeri KC cells or P.
fluorescens cells. Samples were dispensed under N2 and spiked with CCl4 to 10 jig/liter.
CCl4 levels were followed by gas chromatography as described in Chapter 2.

Effects of iron. To assess the effects of iron on CCl4 transformation, an actively
transforming culture was fractionated as described above, individual fractions were
spiked with 0-100 }4M iron (as ferric ammonium sulfate) and 10 yg/L CCl4, and each
fraction was assayed for CCl4 transformation. The 10,000 and 500 MW filtrate was
tested for activity by recombining (reconstitution) with cells of strain KC as described

above.

Siderophore assays. To determine the classes of siderophores produced by strain KC,
cultures were fractionated by cell removal and ultra filtration as described above.
General iron binding activity of the culture supernatant, 10,000 MW filtrate, 500 MW
retentate, and 500 MW filtrate was assayed by the method of Schwyn and Nielands [10].

56

This universal method to detect siderophores was developed by using their high affinity
for iron (III). The ternary complex Chrome Azurol S/iron (III)/hexa-
decyltrimethylammonium bromide, with an extinction coefficient of approximately
100,000 M"1 cm'1 at 630 nm, serves as an indicator. Because of the high sensitivity of
this assay, it is useful for the detection of siderophores in supematants of culture fluids.
The C'sasky assay [2] was utilized for the detection of hydroxamate class siderophores,
and the Rioux assay [9] was used for the detection of catechol class siderophores using

2,3-dihydroxy benzoic acid as a standard.

Partial acetone purification of transformation activity. Filtrate passed through an
Amicon 500 MW filter was lyophilized to dryness (yield = 12 mg dry weight per
milliliter filtrate). The lyophilized filtrate was suspended in approximately 5% of the
original filtrate volume (254 mg lyophilized filtrate/ ml) in ultra pure deionized water.
The sample was then transferred to a 28 ml test tube and 9 ml of HPLC grade acetone
were added to the sample. Samples were stored at 4 0C for 2 hours to allow precipitation.
A visible precipitate formed during the first minute after acetone addition. The
supernatant was decanted and filtered through a 0.45 pM pore size PTFE filter ( Gelman
Acrodisc®) to remove any particulate matter. The acetonezwater phase was then
evaporated under nitrogen at room temperature to a volume of approximately 4 ml. to
remove the majority of acetone. Approximately 80% of the bioassay activity was
recovered in the dried liquid fraction. The yield was approximately 7 pg partially
purified material per mg dry 500 MW filtrate precipitated.

Semi-preparative HPLC purification. Reverse phase semi-preparati ve HPLC
purification was performed on a Gilson HPLC equipped with a Whatrnan semi-
preparative HPLC column. The column had a length of 480 mm, an outer diameter of 16

min, and an inner diameter of 9.5 mm. The column was packed with Partisil® 10 ODS-3

57

with a 10 pm particle size. (Whatrnan), and a 23.62 mL void volume. The HPLC system
was also equipped with a UV detector, and absorbance was monitored at 260 and 340 nm.
These monitoring wavelengths were chosen based on maxima for absorbances observed
for the acetone purified preparation. Sample obtained following the partial acetone
purification procedure outlined above was loaded onto the column with a 1 mL sample
loop. The column temperature was maintained at 8°C using a continuous flow water
jacket around the column and a Fisher model 9001 isotemp digital circulator. Flowrate of
eluent was 7 mL/min, with 100% water for 5 min and a linear gradient to 100% methanol
at 35 min. Samples were collected at 1 minute intervals, pooled, stripped under nitrogen
to remove residual methanol, frozen under liquid nitrogen, and lyophilized to dryness.
Lyophilized samples were rehydrated in SGW medium or in deionized water. The
sample rehydrated with deionized water was saved for further analysis by storage at -20
°C and the portion rehydrated with SGW medium was combined with P. fluorescens and

assayed for CCl4 transformation.

Preparation of process blank. A process blank was prepared by filtering 1L of SGW
medium (pH 8.2) through 300 KDa and lKDa Filtron® ultracassettes, and lyophilizing
the filtrate to dryness, rehydration, acetone extraction, and separation and collection of
fractions by the same reverse phase HPLC method used in the purification procedure
developed for the secreted factor(s).

RESULTS

Reconstitution of fractionated CCl4 transformation activity. Figure 3.1 illustrates

CCl4 transformation by washed cells of strain KC, 500 MW filtrate, and reconstitution of

washed cells of strain KC and the filtrate fraction. By themselves, washed cells did not

58

carry out appreciable transformation. However, rapid and reliable transformation was
obtained when the washed cells were reconstituted with either of the filtrate fractions,
indicating that the secreted factor(s) required for transformation is small, with a nominal
molecular weight of less than 500 daltons. It should also be noted, however, that in some
cases (see, for example, Table 3.1), CCl4 transformation activity was observed with the
partially purified cell free supernatant. Tatara et al. [13] reported a pseudo-first-order rate
constant of 0.03 :1: 0.03 min'1 for the cell-free activity in 13 independently grown
cultures. The high standard deviation reflects the fact that for a large number of
experiments, there was minimal or no cell free transformation activity. An example of
the rates of CCl4 degradation that can be achieved by the cell-free activity are shown in
Table 3.1, however, it must be stressed that this example is not an average, but rather a
characterization of the activity on two of the relatively rare occasions in which significant

cell-free activity was observed.

Initially, strain KC cells were used in reconstitution assays. However, resuspended cells
of strain KC continued to produce additional units of supernatant transformation
component(s) during the time f rame of the assay. After finding that other cell types could
substitute for strain KC, a bioassay was developed using P. fluorescens to track
production of the secreted activity [12]. P. fluorescens cells were preferred for the
bioassay because, unlike strain KC, they are incapable of generating additional secreted

transformation activity during the time-frame of the assay.

59

 

 

 

 

 

 

 

0.3 -
0.2
\ 1.-
CT *1 Alone
(”8) § 1* 50on
Filtrate Alone
0.] -
Whole Culture
.5me Filtrate+ 1.18
0.0 A I L l s I . l l - A -.. - l A j 1 . I
0 20 40 6O 80 100 120 140 160 180 200

Time (min)

Figure 3.1. P. stutzeri KC cells combined with 500 MW filtrate from strain KC. P.
stutzeri cells were grown for 24 hours in medium D (76 pg of protein per mL). Error
bars represent the standard deviation of triplicate samples.

Table 3.1. Transformation kinetics for fractionated cell-free activity.

60

 

 

 

 

 

 

 

 

Pseudo second order rate
coefficient ‘1
hacflon (L/nrg protein-d)
Example 1 Complete culture 054”
Supernatant 6.1
10,000 MW Filtrate 21.3
Example 2 Complete culture 1.3
500 mol weight filtrate 2.1

 

 

 

a Determined as per Tatara et al. (1993).
b Rate was calculated from the linear range of the transformation curve

Table 3.2. Iron binding properties of ultra-filtration fractions from culture supernatant
produced by P. stutzeri KC.

 

Measurements of iron binding activity

 

 

 

 

 

 

 

 

 

 

 

Rioux“ C'saskyb Neilandsc
Fraction (FM 23 DHBA) (A abs @ 520 nm) (A abs @ 630 nm)
Blank 0 0 0
Supernatant 3.08 0.04 0.38
10 kD filtrate 2.18 0.02 0.39
0.5 - 10 kD retentate 3.27 0.59 0.41
<0.5 kD filtrate 1.57 0.04 0.34

 

3 Presence of catechol quantified using 2,3-dihydroxybenzoic acid as standard

b Presence of hydroxymate was determined by measuring the absorbance at 520 nm. The
concentration of hydroxymate was unable to be quantified by use of a hydroxylamine

standard

6The level of iron binding activity was only tracked qualitatively.

61

Effects of iron and fractionation of iron binding activities. CCl4 transformation by
strain KC is inhibited by iron, and the possible role of a siderophore or siderophore-like
agent has been previously proposed [1,12]. We sought to characterize the fractions
generated during purification in terms of siderophore levels and sensitivity to iron
inhibition. The results are shown in Table 3.2. The 500 MW filtrate exhibited substantial
iron binding activity in the Neilands assay, accounting for approximately 50% of the total
supernatant catechol class activity. Retentate above the 500 molecular weight filter
exhibited iron binding activity in all three assays, indicating the production of multiple

iron-binding activities by strain KC.

In marked contrast to the complete culture of P. stutzeri KC, 10,000 MW filtrate and 500
MW filtrate did not exhibit high iron sensitivity . Whole culture activity is inhibited by
iron levels as low as l yM, but the combination of 10,000 MW filtrate and washed cells
was not inhibited by 100 fold higher iron levels as shown in figure 3.2. This suggests that
a high molecular weight secreted factor(s) (>10,000 MW) exacerbates or is responsible
for the inhibitory effects of iron.

62

 

 

 

0.20
0.18
0.16 ‘ I
I I E Control
0.14 -
0.12 -
I \
c. \
(1‘8) 0'10 _
0.08 r \\
0.06 - "“ E}
0.04 - \
.. \
(102 - ' 0.10 yMand
100 pMIron
0.“) A l n l A l A l a l s l 1 I n I
0 20 40 60 so 100 120 140 160
Time (min)

Figure 3.2. Effect on iron on transformation with 10,000 MW filtrate from strain KC.
Error bars represent the standard deviation of triplicate samples.

Partial purification of secreted factor(s). Anion exchange perfusion chromatography
revealed the presence of at least five constituents in the 500 MW filtrate. However,
recovery of strong activity from a salt gradient elution was hindered by inhibition of
transformation activity by ionic strength: 400 mM NaCl or KCI, as well as 200 mM
Na2P04 caused an approximate 50% inhibition of CCl4 transformation activity. This salt
concentration was in the center of the range in the salt gradient where elution of the major
protein peaks occurred. Ammonium sulfate precipitation of the activity was also

unsuccessful, possibly due to the observed effects of high ionic strength.

63

Acetone precipitation of concentrated lyophilized material was successful in removing
the great majority of contaminating material, which rapidly precipitated at 90%

acetone: 10% water (v/v). Approximately 80% of the supernatant f actor(s) (as measured
in the P. fiuorescens bioassay) remained soluble in the acetone: water phase, and was

rcadily recovered by drying under nitrogen (Table 3.3).

Table 3.3. Results of acetone precipitation. All samples were assayed by reconstitution
with P. fluorescens cells in SGW medium .

 

 

 

 

 

 

 

 

Fraction CCl4 removed Org)“
abiotic control 0.001 $0.001
positive control” 0.047 10.006
acetone supernatant 0.049 10.008
acetone pellet 0.013 $0.004

 

0- :1: one standard deviation
b. 10 mg of lyophilized 500 molecular weight filtrate

Semi Preparative HPLC Analysis. Semi-preparative HPLC analysis was performed to
purify the secreted factor produced by P. stutzeri KC. Figure 3.3 illustrates a typical
chromatogram for the initial HPLC separation of an acetone extract containing the
secreted factor. Figure 3.4 illustrates a CCl4 transformation activity plot of the fractions
collected. The majority of CCl4 transformation activity was recovered in fraction 28.
Adjacent fractions also contained some CCl4 transformation activity, but only fraction 28
was used for a second HPLC purification of the CCl4 transforming factor. Fraction 28
from the secondary HPLC run did not contain any CCl4 transformation activity, although
it did contain an absorbance peak at 28 minutes. Fraction 28 from the primary and

secondary rounds of HPLC purification were submitted for mass spectral analysis.

64

A process blank was prepared and analyzed by semi-preparative reverse phase HPLC to
determine if any contaminants introduced from the purification process co-elute with the
secreted factor. Figure 3.5 shows a chromatogram for initial HPLC separation of a
sample containing the secreted factor versus a chromatogram of the process blank. A
peak was present in the process blank at the same retention time as the CCl4 transforming
activity. Fraction 28 from the primary and secondary rounds of HPLC purification of the
process blank were also submitted for mass spectral analysis.

65

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aiiLIerr

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TLIJJ LLLII

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at

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—--——---.— —...___
~— -
-- -———_-.
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-— -._ _
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———-"'
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: II I III I I l I—I' II'I I II II" -. III 0 i. '.I III In" II'I II I' III I nIT' t.
I. .UI' I..|0||I0 Is a I rolIIII .III llII IIII "as IIIs'IIIs IsnIIIIII III: 0 III '00 Is. 0 -.III '0 I ooooooooo uIII, o.
II"'é'I'0'1"":‘10'2’0222'4'26 20'30'32

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Figure 3.3. Semi-preparative HPLC chromatogram of a primary run of a sample
containing the secreted factor involved in CCl4 transformation. Sample absorbance was
monitored at 260 nm.

0.10“

0.08 “

0.06 ‘

0.04 ‘

0.02 "

0.00- D

-0.02 ‘

1

Mass (pg) 01' Removed in Biousay

 

 

-0.“ 1' V U I I ' U l I I j 1 I ' V 1' I 1' 1 I I j I 1' 1 I T I I 1 1 I

0 2 4 6 81012141618202224262830323436

Fraction No.

Figure 3.4. Mass of CCl4 removed by fractions combined in a P. fluorescens bioassay
following HPLC purification of acetone extracted CCl4 transformation activity.

67

 

      

 

 

CI‘ transforming factor

 

 

 

 

 

n
"r till

.i ".1 -. I. . .. -. .. , ., 413.11.... 3114.34.11. 4.. .1114J...jj
Slow. _ m"..4.W1JW.31w.1:4m434w.j.%3445fi4_.W W M1 rm w .3qu m“ H mm

Figure 3.5. A semi-preparative HPLC chromatogram illustrating the initial separation of
CCl4 transforming activity. A chromatogram of the process blank is provided for

companson.

DISCUSSION

The underlying hypothesis evaluated in these studies was that an iron scavenging agent,
more precisely a siderophore, was responsible for the fortuitous transformation of CCl4
by P. stutzeri KC. The basis for this hypothesis was the inhibition of CCl4
transformation by trace-levels of iron. Initially, the results presented in this chapter
agreed with the iron scavenging hypothesis. The small size (< 500 daltons) of the
secreted factor(s) was in the range of known iron binding siderophores, with reported
molecular-masses between 360 to 1500 daltons [8]. Additionally, the occasionally
observed cell-independent transformation activity suggested that under certain conditions,
the transformation reaction is mediated by the secreted factor(s). However, the
production of cell free activity by P. stutzeri KC was rare and was not reproducible,
despite exhaustive efforts to understand its regulation. Consequently, further elucidation

of the mechanism of CCl4 transformation focused on cell-dependent transformation.

Evidence against the siderophore hypothesis was obtained from the experiments
investigating iron inhibition in culture supernatant subjected to ultrafiltration. The
finding that iron inhibition of CCl4 transformation was alleviated when the supernatant
was filtered through a 10,000 MW filter suggests that sensitivity to iron is conferred by a
high-molecular-mass factor(s). This hi gh-molecular-mass factor(s) may participate in
iron-scavenging activities. The insensitivity of the <10,000 MW filtrate to iron inhibition
suggests that the small secreted CCl4 transforming factor(s) either does not play a direct
role in iron binding or is still able to transform CCl4 after binding iron. A possible
explanation for these observations is that the normal physiological role of the low-
molecular-mass CCl4-transforming factor(s) is to shuttle electrons to a hi gh-molecular-
mass factor(s) that binds iron. When iron and the high-molecular-mass factor(s) are both

present, electrons are transferred preferentially to the iron-binding factor(s) rather than

69

CCl4. Such interpretation should be taken with caution, as further evidence is required to
confirm or refute this hypothesis. In future experiments, the role of iron might be

ascertained by monitoring reduction of Fe(III) to Fe(II).

Criddle et al. [1], Lewis and Crawford [5], and Tatara et al [12] have previously
determined that induction of CCl4 transformation occurs in iron-limited and copper-
containing medium. It has also been suggested that electron transfer from the secreted
factor to the CCl4 molecule occurs [4,5,6]. Recently, Lewis and Crawford [6] were able
to trap phos gene and thiophosgene as intermediates of CCl4 transformation. These
compounds are formed after an initial one-electron reduction of CCl4. Dybas et a1 [4],
recently showed that formate is an end-product of CCl4 transformation, which requires a
two net electron transfer onto the CCl4 molecule. In Chapter 2, it was demonstrated that
production of C02 and non-volatile products occurs simultaneously, with no
interconversion of the products. These observations taken together with the results
presented in this chapter suggests that a plausible model to explain the physiology of
CCl4 transformation may involve: (1) production and export of a CCl4-transforming
factor(s) from the cell in response to growth under iron-limitation and copper containing
media, (2) deactivation or loss of electrons from the factor(s) upon transformation of
CCl4, and (3) reactivation or reduction of the f actor(s) at the cell membrane. The
question of whether a one- or two-electron reduction is involved requires additional
investigation. The final elucidation of the CCl4 transformation mechanism may depend

on elucidation of the structure of the secreted factor involved in CCl4 transformation.

An advance in the purification of the secreted factor(s) was achieved with the finding that
CCl4 transformation activity partitioned into the liquid phase following acetone
precipitation. The partitioning of the secreted factor(s) into the acetone:water phase

indicated that it may be slightly hydrophobic in nature. This finding was used to develop

70

a technique using reverse-phase HPLC to further purify the secreted factor(s). As shown
in Figure 3.4, the majority of CCl4 transformation activity is found in fraction 28.
However, some activity was found in the adjacent fractions as well. This may be due in
part to the manner in which fractions were collected from the HPLC and then pooled
together for freeze drying. It is possible that the timing of collection for all fractions was
not precise, and therefore some crossing over of fractions could have occurred during the
collection process. Analysis of the process blank revealed that a contaminant peak also
eluted at a retention time of 27-28 minutes during HPLC analysis (Figure 3.5 ). In the
preliminary mass spectral analysis, common peaks were observed between both the

process blank and the active sample, with no significant difference immediately apparent.

The purification procedure cleariy needs additional development. Future HPLC runs
should be monitored at more than just the 260 and 340 nm wavelengths. For example,
absorbance maxima for polyenes are at 214 nm, aromatic rings at 253 nm and a, B
unsaturated ketones at 215 nm [11]. Additionally, substituent corrections exist for each
of these parent compounds [11]. Thus, knowing absorbance maxima may provided some
structural information as to nature of the secreted factor(s). Loss of activity following the
second-round of HPLC purification should also be examined. The nature of the secreted
factor(s) could have changed following HPLC analysis, and eluted at a different time.
Therefore, all fractions of the second purification should be combined with P.
fluorescens, rather than only examining the fractions containing activity in the first round
of HPLC purification. Finally, CCl4 transformation activity may require more than one
component. Thus, if a second HPLC purification run separates these components,
activity will not be observed. In future investigations, all fractions should be pooled

together and assayed for CCl4 transformation.

71

In the following chapter, I describe studies which utilize the bioassay with Pfluorescens

to examine properties of the CCl4 transforming factor(s) as well as the cell types that can

be combines with this factor(s) to result in CCl4 transformation.

 

REFERENCES

1. Criddle, C.S., J.T. DeWitt, D. Grbrié-Galié, and P.L. McCarty. 1990.
Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrifying
conditions. Appl. Environ. Microbiol. 56:3240-3246.

2. Csa'ky, T.Z. 1948. On the estimation of bound hydroxylamine in biological materials.
Acta Chem. Scand. 2: 450-454.

3. Dybas, MJ. and J. Konisky. 1989. Transport of coenzyme M (2-
mercaptoethanesulfonic acid) and methylcoenzyme M [(2-methylthio)ethanesulfonic

acid] in Methanacoccus voltae : Identification of specific and general uptake systems. J.
Bact. 171:5866-5871.

4. Dybas, M.J., G.T. Tatara, and C.S. Criddle. 1995. Localization and characterization
of the carbon tetrachloride transformation activity of Pseudomonas sp. strain KC. Appl.
Environ. Microbiol. 61:758-763.

5. Lewis, T. A., and R. L. Crawford. 1993. Physiological factors affecting carbon
tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC.
Appl. Environ. Microbiol. 59: 1635-1641.

6. Lewis, T. A., and R. L. Crawford. 1995. Transformation of carbon tetrachloride via
sulfur and oxygen substitution by Pseudomonas sp. strain KC. J. Bacterial. 177: 2204-
2208.

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

8. Neilands, J.B. 1981. Microbial iron compounds. Ann. Rev. Biochem. 50: 715-731.

9. Rioux, C., D.C. Jordan, and J.B.M. Rattray. 1983. Colorometric determination of
catechol siderophores in microbial cultures. Anal. Biochem. 133: 163-169.

10. Schwyn, B. and J.B. Neilands. 1987. Universal chemical assay for the detection and
determination of siderophores. Anal. Biochem. 160: 47-56.

11. Streitwieser, A. and G.H. Clayton. 1981. Introduction to organic chemistry. 2nd. ed.
MacMillan Publishing Co. New York

12. Tatara, G.M., M.J. Dybas, and C.S. Criddle. 1993. Effects of medium and trace
metals on kinetics of carbon tetrachloride transformation by Pseudomonas sp. strain KC.
Appl. Environ. Microbiol. 59:2126-2131.

13. Tatara, G.M., MJ. Dybas, and C.S. Criddle. 1995. Biofactor-mediated
transformation of carbon tetrachloride by diverse cell types, p. 69-77. In R Hinchee, A.
Leeson, and L. Semprini (ed.) The Bioremediaton Series, 3(3) , Bioremediation of
Chlorinated Solvents. Battelle Press, Richland, WA.

72

CHAPTER 4

BIOFACTOR-MEDIATED TRANSFORMATION OF CARBON
TETRACHLORIDE BY DIVERSE CELL TYPES

Portions of this chapter were published in: The Bioremediation Series 3(3), Bioremediation of Chlorinated
Solvents. ed. R. Hinchee. A. Ieeson. and L. Semprini. p. 69-77. Battelle Press, Richland WA. 1995.

I gratefuny acknowledge the intellectual contributions to experimental design by Dr. Michael J . Dybas

ABSTRACT

The transformation of CCl4 by Pseudomonas stutzeri KC requires a small (<500 dalton)
factor(s) secreted by strain KC and cells capable of activating the factor. Partially
purified supernatant from a culture of strain KC was combined with cells that do not
transform CCl4 or do so slowly. Rapid CCl4 transformation was obtained using related
Pseudomonads (P. stutzeri ; P. fluorescens); Escherichia coli K-12; a gram positive
bacterium (Bacillus subtilus); a bacterial consortium derived from groundwater at
Schoolcraft, MI (SC-1); a bacterial consortium derived from groundwater at Hanford,
WA (HC- 14); and yeast (Saccharamyces cerevisiae). Thus, specific cell types are not
required for activation of the factor. This finding was used to develop a bioassay for the
factor(s) in which samples to be assayed are inoculated with P. fiuarescens , spiked with
CCl4, and monitored for CCl4 degradation under anoxic incubation conditions. The
bioassay was used to establish that aerobically-grown strain KC cells secrete the factor,
oxygen reversibly inhibits CCl4 transformation by the factor, live cells are needed for
activation of the factor, the pH optimum for CCl4 transformation is 8.5, the factor is
readily transported through aquifer material, the factor is stable for up to six days in
synthetic groundwater at 16°C, and the factor is stable indefinitely after lyophilization to
powder and storage at -20°C. The possibility that organisms indigenous to aquifer solids
can recharge the factor was evaluated by addition of the factor to a slurry of Hanford
aquifer material. CCl4 transformation was only obtained when aquifer material was

inoculated with the secreted factor and biostimulated by addition of acetate and nitrate.

74

INTRODUCTION

The most significant strategies for in situ bioremediation are biostimulation, the addition
of nutrients and/or substrates to stimulate indigenous populations, and bioaugmentation,
the addition of non-indigenous organisms. Biostimulation avoids the problems associated
with transport and survival of introduced organisms because the indigenous organisms
are already present, and adapted to site conditions. A disadvantage of biostimulation is
that some or all of the stimulated organisms may be unable to degrade the target
contaminants, or, if they do degrade it, they may create undesirable products. The
principal advantage of bioaugmentation is that the introduced organism and the
transformation mediated by the organism, including the kinetics and pathway of
transformation, can be studied and optimized under laboratory conditions. However,
bioaugmentation also faces numerous challenges: the introduced organisms may fail to be
transported, they may fail to colonize, or they may be unable to compete with the
indigenous organisms. The ideal in situ remediation scheme would provide the benefits
of bioaugmentation (pathway control and kinetic optimization) with the advantages of

biostimulation (use of indigenous organisms).

Under denitrifying conditions, transformation of CCl4 typically results in the slow
accumulation of chloroform [4,7,13], a compound that is persistent and potentially
harmful to human health [14]. As a result, remediation strategies that avoid chloroform
production are desirable. P. stutzeri KC rapidly transforms CCl4 to carbon dioxide
[5,11,15], formate [6], and unidentified non-volatile product(s), without the production of
chloroform [5,15]. Thus, use of this transformation system offers pathway and kinetic
advantages. As shown by Dybas et al. [6], strain KC produces a small (< 500 dalton)
secreted factor that is required for CCl4 transformation. This secreted factor is

regenerated in the presence of viable whole cells of P. stutzeri KC.

75

76

In this chapter, evidence is provided that a diverse range of microorganisms regenerate
CCl4 transformation activity, including organisms that are indigenous to CCl4-
contaminated aquifers. Several characteristics of the factor activity are evaluated
including stability of the secreted factor, its production under aerobic conditions, oxygen
inhibition of CCl4 transformation, and transport of the factor through aquifer material.
While others have observed secreted xenobiotic degradative activities [2,8], this appears

to be the first report of a non-specific biological process for regeneration of extracellular

degradative activity.

MATERIALS AND METHODS

Chemicals. CCl4 (99% purity) was obtained from Aldrich Chemical Co., Milwaukee,
Wis. All chemicals used were ACS reagent grade (Aldrich or Sigma Chemical Co.). All

water used in reagent preparation was deionized 18 Mohm resistance or greater.

Media and growth conditions. Medium D prepared as described in Chapter 2.

Simulated groundwater medium (SGW) was prepared as described in Chapter 3.

Yeast medium contained per liter of deionized water: 75 g of glucose, 3 beef bouillon
cubes, 2 g of KHzPO4 and 3 g of Kzl-IPO4. The pH of the yeast medium was adjusted to
8.0 with 3 N KOH and autoclaved at 121°C for 20 minutes prior to use.

P. stutzeri KC (ATCC deposit no. 55595, DSM deposit no. 7136) was grown in medium
D or SGW medium under an atmosphere of nitrogen in modified 500 mL Wheaton
bottles (Wheaton cat. no. 219819). Openings to permit sampling were created in the
bottle caps by drilling 12 mm holes through the caps and inserting 30 mm teflon-faced
butyl rubber septa (Wheaton no. 224174). Sealed bottles were pressure tested for leakage

at 5 psi.

All cultures of strain KC were shaken at 150-200 rpm on a rotary shaker at a temperature
of 20 - 23°C. Culture manipulations were typically performed in the anaerobic glove box
under an atmosphere of 97:2% N2 and 312% H2. Oxygen and hydrogen levels were

monitored continuously with a Coy model 10 gas analyzer.

Analytical methods. CCl4 transformation assays were performed as described in
Chapter 2. The procedure described in chapter 2 was also used to evaluate the possibility

78

of chloroform production. In all assays, the method detection limit was 0.01 pg for
chloroform, corresponding to an aqueous phase concentration of 2 p g/L, as determined

by Standard Methods 103015 [1].

Preparation of partially purified culture supernatant. Cultures and supernatant were
generally prepared and manipulated in the anaerobic glove box. Cultures of strain KC
were screened for CCl4 transformation activity prior to the preparation of partially
purified culture supernatant. Actively transforming culture was transferred to 40-mL
Nalgene® centrifuge tubes and centrifuged at 4°C and 27,200 x g. Centrifuged
supernatant was decanted into a 115-mL sterile 0.2 pm filter unit, and a hand vacuum
pump was used to filter out any remaining cells from the culture supernatant The
resulting 0.2 pm filtrate was loaded into a model 8400 Amicon ultrafiltration stirred cell
and filtered at 30 psi through a preconditioned PM 10 (10,000 molecular weight cut-off)
ultrafiltration membrane (Amicon no. 13142). The 10,000 MW (molecular weight)
filtrate obtained in this manner was either used directly in CCl4 transformation assays or
further filtered at 55 psi through a preconditioned YC 05 (500 MW cut-oft) ultrafiltration
membrane (Amicon no. 13042). 500 MW filtrate was collected in a sterile serum bottle

on ice.

Experiments with diverse cell types. Pseudomonas fluorescens (ATCC deposit no.
13525), Escherichia coli K-12 (ATCC deposit no. 10798), and Bacillus subtilis (ATCC
deposit no. 6051) were obtained from the culture collection of the Microbiology
Department at Michigan State University. Pseudomonas stutzeri strain EPB-071388
110, an aquifer isolate from Seal Beach, Calif ., was provided by H. Ridgway (Orange
County Water District, CA). An aquifer consortium, designated SC-l, was obtained by
enrichment of organisms in a groundwater sample from a CCl4-contaminated aquifer at

Schoolcraft, MI. The enrichment was obtained from a 1% inoculum of Schoolcraft

79

groundwater in Nutrient Broth (Difco, Co.). A second aquifer consortium, designated
HC- 14, was obtained from R. Skeen of Battelle Pacific Northwest Laboratories
(Richland, WA). HC-14 was derived from a sample of aquifer material at the Hanford,
WA site. Saccharomyces cerevisiae (ATCC #58527) was obtained from Red Star Co. ,
Milwaukee, WI.

P. fluorescens , P. stutzeri , and SC-l were grown at 20-23°C under denitrifying
conditions in medium D (pH 8.2) or aerobically in medium D (pH 7.0) supplemented
with 10 pM FeSO4. B. subtilis and in some instances P. fluorescens were grown at 20-
23°C aerobically in nutrient broth (Difco Co.). E. coli was grown at 35°C aerobically in
medium D, but with glucose (3 g/L) instead of acetate as the electron donor. HC-l4 was
grown under denitrifying conditions in SGW medium (pH 7.5). S. cerevisiae was grown

aerobically at 35°C in yeast medium.

Cultures were transferred to an anaerobic glove box and dispensed into 40-mL centrifuge
tubes. Cells were collected by centrifuging at 12,100 x g for 5 minutes , wasting the
culture supernatant, and resuspending the pellet in 4 mL of anoxic medium D or anoxic
medium SGW at pH 8 or pH 7.5. Cultures of E. coli or B. subtilis were resuspended in
anoxic medium D with glucose as the carbon source, and S. cerevisiae cultures were
resuspended in anoxic yeast medium. A 0.5 mL sample of cell suspension (concentrated
ten fold by centrifugation) was added to 4.5 mL of filtered supernatant and assayed for

CCl4 transformation in 28-mL aluminum seal tubes as described previously.

Growth of P. straeri KC and secreted factor(s) production. Cultures of P. stutzeri KC
were grown under denitrifying conditions in SGW medium. Cultures were inoculated
with a 0.5% (v/v) inoculum taken from a 96 hour starter culture of strain KC grown in

SGW medium. Cultures were grown in 500 mL Wheaton bottles as previously described.

80

Initial and subsequent samples were taken by withdrawing 6mL of liquid, freezing 1 ml.
for protein analysis, and filtering 5 mL of sample through a 0.22 pm nylon filter. The 5
mL filtered sample was then frozen and lyophilized. Following completion of the growth
study, which was terminated at 100 hours following inoculation, the lyophilized samples
were rehydrated to 5 mL with deionized water and assayed for secreted factor using the

P. fluorescens bioassay.

Aerobic production of the supernatant factor. Cultures of strain KC were grown
aerobically in medium D. In the late exponential phase, cultures were filtered through a
0.2 pm filter. The resulting filtrate was made anoxic by transfer through the interlock of
an anaerobic glove box, filtered through a 10,000 MW filter, then filtered again through a
500 MW filter. A portion of the 500 MW filtrate was dispensed anaerobically as 4.5 mL
aliquots into 28 mL serum tubes, spiked with 0.5 mL of an anoxic cell suspension of P.
fluorescens, and assayed for CCl4 transformation. The remainder was removed from the
glove box, aerated, dispensed as 5 mL aliquots into 28 mL serum tubes (air headspace),

spiked with P. fluorescens cells, and assayed for CCl4 transformation.

Slurry experiments with aquifer material . Approximately 2 g (wet weight) of
Hanford aquifer solids (provided by R. Skeen, Battelle Pacific Northwest Laboratories,
Richland, WA) were placed into sterile 28-mL aluminum seal tubes. Each tube received
2 mL of SGW medium (pH 8.2) containing 800 mg/L of N03‘. One set of tubes was
autoclaved (sterile controls), one was amended with 800 mg/L acetate, and one received
no amendments. All tubes were spiked with 0.5 pg CCl4 and shaken at 20°C. After three
days, the acetate-fed tubes received an additional spike of 800 mg/L acetate and 800
mg/L nitrate. Two days later, half of the tubes from each set were supplemented with 2
mL of 10,000 MW filtrate from a culture of strain KC grown in SGW medium under

denitrifying conditions. Tubes that did not receive the filtrate were spiked with 2 mL of

81

anoxic SGW medium at pH 8.2. All tubes were incubated for 48 hours at 20°C and
heated at 70°C for two hours to release sorbed CCl4. Gas phase CCl4 mass was
determined using equivalent volume aqueous phase standards (prepared as previously
described) that were also heated to 70°C.

Stability of CCl4 transformation in the supernatant fractions. The stability of the
CCl4 transforming factor was evaluated under different storage conditions. In one test,
500 MW filtrate obtained from a culture of strain KC grown in medium D (pH 8.2) was
stored at 0°C under nitrogen in a 160-mL serum bottle sealed with teflon-lined butyl
rubber septum. Five milliliter factions were removed daily, combined with cells of P.
fiuorescens , and assayed for CCl4 transformation. In a second test, 10,000 MW filtrate
from a culture of strain KC grown in SGW medium at pH 8.2 (pH 8.3 after growth) was
divided into two fractions, and the pH of one fraction was adjusted to 7.5. Both fractions
were filter sterilized through a 0.2 pm filter, sealed under a headspace of nitrogen, and
stored at 16°C in a 160-mL serum bottle sealed with a teflon-lined butyl rubber septum.
Five milliliter fractions were removed daily, combined with cells of P. fluorescens, and

assayed for CCl4 transformation.

CCl4 transformation using lyophilized culture filtrate. 10,000 MW filtrate from strain
KC grown in SGW medium was frozen ovemight at -20°C and lyophilized. The resulting
powder was stored at - 0°C under aerobic conditions. Freeze-dried powder was
rehydrated in deionized water at one- or two-times its original concentration. Rehydrated
powder was then deoxygenated by uansfer through the interlock of an anaerobic glove
box, dispensed into 28 mL aluminum seal tubes, mixed with P. fluorescens , spiked with
CCl4, and assayed for CCl4 transformation. To obtain the transformation capacity of

f reeze—dried powder, samples were respiked with CCl4 until CCl4 transformation
stopped.

Requirement for live cells. Cultures of P. fluorescens were grown aerobically in
Nutrient Broth (Difco. Co.). The cultures were split into two equal fractions, one of
which was autoclaved at 121°C for 15 minutes. After autoclaving, the cultures were
confirmed killed by plating 100 pL of autoclaved culture onto nutrient agar (Difco Co.)
plates and incubating at 20°C under aerobic conditions for 5 days. Both the autoclaved
and live cultures of P. fluorescens cultures were transferred to an anaerobic glove box
and dispensed into sterile 40-mL centrifuge tubes. Cells were collected by centrifugation
at 12,100 x g, and pellets were resuspended in 4 mL of SGW medium at pH 8. A 0.5 mL
sample of cell suspension was added to 4.5 mL of rehydrated 10,000 MW filtrate and

assayed for CCl4 transformation in 28-mL tubes as described previously.

Transformation rate dependence on cell density. Freeze dried 10,000 MW filtrate
from P. stutzeri KC was prepared as previously described. P. fluorescens was grown 24
hours in SGW medium under aerobic conditions from a 1% nutrient broth inoculum.
Eighty milliliters of P. fluorescens was centrifuged at 10,000 rpm for 5 min. in a SS-34
rotor and concentrated 10X by resuspending the pellet in 8 mL of fresh SGW medium.
Various quantities of the resulting cell concentrate were added to tubes containing 5 mL
of 10,000 MW filtrate that had been rehydrated at its original concentration from freeze
dried powder. The cell concentration range was from zero to 1.4 x 108 CFU/mL Cell
concentration was determined by plating a dilution series of culture concentrate on

nutrient agar and counting colony forming units per milliliter.

pH optimum for CCl4 transformation. Freeze dried 10,000 MW filtrate from P.
stutzeri KC was prepared as previously described. Following rehydration, the filtrate was
dispensed into 20 mL aliquots, and the pH of individual aliquots was adjusted with 1 M
BC] or with 1 M NaOH to cover the pH range from 6 through 10 in pH increments of 0.5.

83

Three P. fluorescens cultures were grown 24 - 36 hours in SGW medium at pH 6.5, 7.5,
and 8.5 under aerobic conditions from a 1% nutrient broth inoculum. Eighty milliliters
from each culture were centrifuged and resuspended in 9 mL of phosphate buffer at the
desired pH level. The culture grown at pH 6.5 was used to test optimum at pH 6.0 and
6.5. The culture grown at pH 7.5 was used to test the pH optimum at pH 7.0, 7.5 and 8.0.
The culture grown at pH 8.5 was used to test the pH optimum at 8.5, 9.0 and 10.0. Cells
were combined with the pH adjusted filtrate, and CCl4 transformation activity was
measured as previously described. Final pH readings were recorded and graphed against

the first order rate coefficient for CCl4 transformation (k').

Transport of the secreted factor through aquifer material. Kontes® glass columns
(30 cm length, 2 cm i. d.) fitted with Teflon® luer lock stopcocks were sanitized by
soaking in a solution of 0.06% hypochlorite, rinsed with sterile deionized water, and
packed aseptically with Hanford or Schoolcraf t aquifer material under a laminar hood.
To create a slurry for packing, Hanford aquifer material (100 g wet weight) was
combined with SGW medium containing 400 mg/L nitrate. Fines were removed by
repeated elutriation with SGW medium, and large particles (>8 mm) were also removed.
These materials were removed to enable flow to pass through the solids at a reasonable
rate (2.5 mUmin) without creating a large back pressure. To create a slurry, Schoolcraft
aquifer material (courtesy of Brown & Root Environmental, Holt, MI) was combined
with SGW containing 400 mg/L NO3‘. In this case, adequate flow was achieved without
removing fines or large particles. Slurries of Hanford and Schoolcraf t aquifer material
were poured directly into columns containing SGW medium and periodically tapped
during filling to provide uniform packing and prevent the entrainment of air pockets.
After packing, each column received three exchanges of SGW medium to remove small
bubbles and to stabilize the packing. Each column was then connected to a Harvard®

syringe pump, and flow of SGW medium containing 10 pCi of 3H20 per liter was

84

initiated at 2.5 mL/min. Breakthrough was monitored by collecting 5 mL fractions of
column effluent and measuring the radioactivity in each fraction. Samples of each
fraction (300 pL) were injected into 10 mL of Safety Solve® (Research Products
International Corporation) liquid scintillation fiuor and counted on a Model 1500 Packard
Tri Carb liquid scintillation counter. The porosity, as estimated from the breakthrough
curves, was 0.48 for the Hanford packed column and 0.46 for the Schoolcraf t packed

column.

Mobility of the secreted factor(s) was evaluated by pumping 10,000 MW filtrate from
strain KC grown in SGW medium through both columns at a rate of 2.5 mL/min.
Effluent from each column was collected in 5 mL fractions and assayed for secreted

factor using the P. fluorescens bioassay.

Modeling. Tatara et al. [16] demonstrated that CCl4 transformation by strain KC is first
order with respect to CCl4 concentration over the CCl4 concentration range used in this
study. Assuming a first order kinetic expression and equilibrium between the gas and
liquid phase, the following equation can be written:

'd_M‘—l: _L_. V
dt V.,q-r-HCVg M“ “q

where MCCI4 is the micrograms of CCl4 in the bottle, t is time in minutes, k" is the
apparent first order rate coefficient (min '1 ), Vaq is the aqueous phase volume, V g is the
gas phase volume, and Hg is the dimensionless Henry's constant for CCl4 (1.0 at 200C).

Separation of variables and integration yields:

“(‘1 NHL]— v,,.n,v )‘

85

For a reaction that is first order with respect to CCl4 concentration, a plot of the logarithm
- 'v
of the mass of CCl4 in the bottle versus time yields a straight line with slope = —-k—°q—.
v,,q+H,,Vg
V
Rearrangement and solving for k" yields: k“ = -slope Vaq . The half-life (tl/z)

is given by: tm= 0:693

 

RESULTS

Transformation of CCl4 with diverse cell types. As illustrated in Figure 4.1, the
partially purified supernatant transformed CCl4 to a limited extent by itself, but its
activity was highly variable, as indicated by the large standard deviation for this sample
set in Table 4.1. In the presence of viable cells, the rate and reliability of transformation
increased dramatically. Figure 4.1 shows a typical transformation pattern. By
themselves, cells of P. fluorescens were unable to transform CCl4, but when combined
with 500 MW filtrate, rapid transformation resulted. Because P. fluorescens is unable to
transform CCl4 and does not produce any CCl4-transforming secreted factor, the relative
level of secreted factor in a sample can be determined by adding CCl4 to P. fluorescens
and monitoring the rate of CCl4 transformation. This simple procedure was used in

subsequent experiments as a bioassay for the secreted factor.

0.20

0.18

0.16

0.14

0.12

(1433.10 p

0.08

0.06

0.04

0.02

0.00

 

I P. fluorescens

500MW
filtrate

P. fluorescens

 

+
500 MW filtrate
l A l A l l l l J l I A I
15 30 45 60 75 90 115

Time (min)

Figure 4.1. Rapid CCl4 transformation results from the combination of P. fluorescens
with 500 MW filtrate. Error bars represent the standard deviation of triplicate samples.

As shown in Table 4.1, CCl4 transformation occurs when a wide range of cell types are
incubated in the presence of the secreted factor produced by strain KC. CCl4
transformation was obtained using cells from related Pseudomonads (P. fluorescens, P.
stutzeri ), another gram negative organism (E. coli ); a gram positive organism
(B.subtilus ); a consortium of organisms enriched from CCl4-contaminated groundwater
from Schoolcraf t, MI (SC-1);a consortium of organisms enriched from CCl4-
contaminated aquifer solids from Hanford, WA (HG-14); and yeast (S. cerevisiae ). By
itself, the Hanford enrichment was unable to transform appreciable CCl4 within the time
frame of this experiment, but when incubated in the presence of the secreted factor, a

CCl4 half -life of only 2.7 :t 0.2 minutes was obtained.

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The single gram-positive organism evaluated in this study (B. subtilus ) exhibited slower
CCl4 transformation upon combination with the supernatant factor than the gram negative
organisms (T able 4.1). This may be related to differences in membrane or peptidoglycan
structure, but additional confirmation is needed. The ability of a eukaryote (S. cerevisiae )
to transform CCl4 in the presence of the secreted factor produced by strain KC
demonstrates that the ability to transform CCl4 in the presence of the factor is very
general and may be universal. Also of interest is the finding that cultures that are
combined with the secreted factor do not need to be grown under iron-limiting
conditions. Cultures of P. fluorescens and B. subtilus grown in nutrient broth and
Schoolcraft consortium SC-l grown at pH 7 with 10 pM iron were all able to transform
CCl4 in the presence of the secreted factor (Table 4.1). Thus, while production of the
secreted factor by strain KC is dependent upon iron-limiting conditions [5,10,15],

transformation in the presence of the factor is not.

Chloroforrn was not detected during CCl4 transformation by any of the supernatant/cell
combinations tested (chloroform method detection limit = 2 pg/L). This is consistent

with previous reports of CCl4 transformation products for suain KC [10,15].

Growth of P. straeri KC and secreted factor(s) production. The ability of samples to
be stored and assayed for CCl4 transformation using the P. fluorescens bioassay was
used to determine the phase of growth during which P. stutzeri KC produces and secretes
the CCl4-transforming factor. Figure 4.2 illustrates the growth of P. stutzeri KC and
biomolecule production versus time as measured by the first-order-rate coefficient for
CCl4 transformation in the bioassay. A very rapid rate of growth is observed at 6 hours
following inoculation, during which time no biomolecule production is observed. After

approximately 12 hours of growth, rapid production of biomolecule is observed until the

90

culture reaches an age of 36 hours. After this point, no significant increase in

 

 

 

 

biomolecule production is observed.
100 r - 0.08
C mum - 0.07
I ’ ’
'- 0.06 p
' a!
A 10 .-
5 E - 0.05 I:
.5 : g
a - 0 04 In
8 .: ‘ I "
E Secreted Factor r 0.03 g
l r .
: 8
. - 0.02 a
L
- 0.01
. 1 k l v. 1 A l a L A l l l A l A l . l J l a l A 0.00
0 10 20 30 40 50 60 70 80 90 100 110 120
Time(Hours)

Figure 4.2. Secreted factor production during the growth of P. stutzeri KC. Error bars
represent the standard deviation of three independently grown cultures.

Production of the secreted factor under aerobic conditions. The bioassay with P.
fluorescens was used to determine whether secreted factor is produced by aerobically
grown cells of strain KC. Filtered supernatant from an aerobically grown culture of strain
KC was combined with cells of P. fluorescens. As shown in Figure 4.3, CCl4
transformation occurred when the filtered supernatant or filtered supematant/cell mixture

was incubated under anoxic conditions, indicating that strain KC can and does produce

91

the factor aerobically. Production of the factor appears to be growth-associated, as higher
biomass levels were obtained for aerobically grown cells. It is interesting to note,
however, that CCl4 transformation did not occur when the supernatant or supematant/cell
mixture was incubated aerobically, indicating that molecular oxygen or one of its
products inhibits the transformation. It should also be noted that production of the
secreted factor was inhibited by iron, regardless of the electron acceptor used for growth.
No CCl4-transforming secreted factor was produced when strain KC was grown

aerobically in medium containing more than 10 pM iron (data not shown).

 

 

 

 

025 _ 500 MW filtrate + P. fiuor. cells
aerobic incubation
\k A
0.20 d I
500 MW filtrate
aerobic incubation
0.15 -
CT
0th
0.10 -
500 MW filtrate
0.05 - anoxic incubation
‘ 500 MW filtrate + P. fiuor cells
1 .§ anoxic incubation
0.“) . i r r ' T f r r r ' r r m ' r r
0 20 40 60 80 100 120 140 160 180
Time (min)

Figure 4.3. Incubation under aerobic conditions inhibits CCl4 transformation. Error bars
represent the standard deviation of triplicate samples.

92

Stability of the secreted factor. To assess the potential for application of the secreted
factor, its stability was evaluated under various storage conditions. Initially, 500 MW
filuate from strain KC (grown in medium D) was stored under a headspace of N; at 0°C.
Under these conditions, CCl4 transformation activity was stable for 2 days but fell off on
day 3 (Table 4.2). No activity was observed on day 5. Subsequently, stability of 10,000
MW filtrate from strain KC (grown in SGW medium) was evaluated when stored at 16°C
under a headspace of nitrogen. Under these conditions, transformation activity persisted
for four days, began to decrease after six days, and was undetectable by the seventh day.
As shown in Table 4.2, initial transformation activity was significantly higher when CCl4
transformation assays were performed at pH 8.3 than at pH 7.5, indicating that

transformation kinetics are pH dependent.

Table 4.2. Stability of the secreted factor(s) as indicated by changes in the apparent first-
order rate constants (k‘) under varied storage conditions. a

   
   
 

 

Medium D, pH 8.2,

   

Storage Conditions

SGW medium, pH 8.3,

   

 
  
   

SGW medium, pH 7.5,

 

 

 

 

 

 

 

 

0°C, ndernitro _en 6°C,dr nitro -_ e 16°C, under nitro n

0 0.191 0.01b 0.251001 0.151001
1 0.25 1 0.01 0.19 1 0.02 0.06 1 0.01
2 0.211 0.01 0.201002 0.101004
3 0.02 0 0.151001 0.061001
4 ND d 0.15 1 0.01 0.05 1 0.00
5 0.001000 ND ND

6 ND 0.09 1 0.00 0.02 1 0.01
7 ND 00010.00 00010.00

 

 

 

 

 

a tabulated values represent the apparent first order rate constants [k" (min'1)], obtained
by combining the stored supernatant with P. fluorescens cells and measuring the initial
rate of CCl4 transformation.

b :1: values represent the standard deviation of triplicate samples.

C no :1: values are shown as this data point represents an average of duplicate samples
d ND = not determined.

To further assess the stability of the secreted factor and to provide a possible means of

concentrating it, 10,000 MW filtrate from strain KC grown in SGW was frozen at -20°C

and lyophilized to dryness. Lyophilized powder was stored at -20°C for six days prior to

rehydration. The lyophilized powder was rehydrated at one- and two-times its original

concentration, and then combined with cells of P. fluorescens . As shown in Table 4.3,

significant CCl4 transformation was observed. Combination of cells with powder

rehydrated to twice its original concentration resulted in more extensive transformation

than the combination of cells and powder rehydrated to its original concentration.

94

However, the increased mass of CCl4 transformed was not proportional to the increase in

powder concentration.

Table 4.3. Concentration dependence of the secreted factor on transformation of CCl4 by
P. fluorescens cells.

CCl4 removed after a 24

 

 

hour incubation (pg)
1) P. fluorescens alone 0.08:0.04
2) P. fluorescens + 1X 0.871003

rehydrated culture filtrate a

 

3) P. fluorescens + 2X 0.99:0.01
rehydrated culture filtrate b

 

 

 

 

a 1X represents a freeze-dried powder prepared from 10,000 MW filtrate that was
rehydrated to its original concentration.

b 2X represents a freeze-dried powder prepared from 10,000 MW filtrate that was
rehydrated to twice its original concentration.

Requirement for live cells. The bioassay with P. fluorescens was used to determine
whether live cells are required for CCl4 transformation. Rehydrated culture filtrate was
combined with live and dead (autoclaved) cells of P. fluorescens . As shown in Figure
4.4, CCl4 was rapidly transformed when the 10,000 MW filtrate was combined with live
cells, but transformation was not observed when filtrate was recombined with autoclaved

cells. Thus, live cells are required for the transformation.

95

3.0

  
     
 
  

 

 

 

 

 

E 11— —5\ T
2.5 I “I
10,000 MW filtrate +
autoclaved P. fluorescens cells
2.0 "
CT
(148) 1.5 -
1.0 "
10,000 MW filtrate +
live_P. fluorescens cells
0.5 "
0.0 ' I I I I I I I I I I I
0 15 30 45 60 75 90

Time (min)

Figure 4.4. Effect of cell viability on CCl4 transformation. Secreted factor was added as
freeze dried 10,000 MW filtrate from strain KC grown in SGW medium. Error bars
represent the standard deviation of triplicate samples.

Transformation rate dependence on cell density. The P. fluorescens bioassay was
used to establish that CCl4 transformation kinetics are cell density dependent.
Rehydrated culture filtrate was combined with various concentrations of P. fluorescens
and the first order degradation rate (k") was measured for each concentration. As shown
in Figure 4.5, the CCl4 transformation rate, as measured by the k" value, increased until
the cell density was 7.5 x 107 CFU per m1. Further increases in cell concentration did

not result in an increase in transformation rate. Thus, CCl4 transformation exhibited

saturation kinetics with respect to cell concentration.

0.08 '

 

0.06

k" 0.04

0.02

 

 

O 4 A l l l I I l l A 1 l l l J l L j l l L l I

0.0e+0 2.5e+7 5.0e+7 7.5e+7 1.0e+8 1.2e+8 1.5e+8
(CFU/mL)

Figure 4.5. The effect of cell density on transformation rates. Secreted factor was added
as freeze dried 10,000 MW filtrate from strain KC grown in SGW medium to various
concentrations of P. fluorescens cells. Error bars represent the standard deviation of
triplicate samples.

pH optimum for CCl4 transformation. The P. fluorescens bioassay was used to
establish the pH range for CCl4 transformation by the secreted factor. As shown in figure
4.6, the range of pH values over which CCl4 transformation can occur is broad. The pH
optimum for CCl4 transformation is approximately 8.5 as measured by the first order rate
coefficient for CCl4 transformation. Rates of CCl4 transformation were lowest near pH

6.5 and 10.

 

 

0.08 -
0.06 -
k" 0.04 -
0.02 -
0.00 . . . 1 . 1 .
6 7 9 10

pH

Figure 4.6. pH optimum of CCl4 transformation. Rates of CCl4 transformation are
presented as the first-order-rate coefficient in the P. fluorescens bioassay. Error bars
represent the standard deviation of triplicate samples.

Regeneration of activity by organisms indigenous to aquifer material. As shown in
Table 4.4., significant CCl4-removal was obtained when 10,000 MW filtrate was mixed
with a slurry of biostimulated Hanford aquifer material (test sample 6). Approximately
32% of the initial CCl4 mass (0.5 pg) was removed in two days. Addition of the
supernatant factor without biostimulation (acetate and nitrate addition) failed to promote
CCl4-removal beyond that of the sterile control. Biostimulation of the indigenous flora
without addition of supernatant factor also failed to bring about CCl4-removal beyond
that of the sterile control. No chloroform was detected in any of the samples with a

method detection limit of 2 pg/I..

Table 4.4.. Transformation of CCl4 in Hanford aquifer solid slunies.

 

 

 

 

 

 

 

 

 

 

 

 

CCl4 removed after 48 hr.
Test sample incubation (pg) a
1 Sterile Control 0.05 1 0.01b
2) Sterile Control + KC Supernatant Factor 0.05 :1: 0.05
3) Indigenous Flora 0.04 :1: 0.03
4) Indigenous Flora + KC Supernatant 0.06 :1: 0.02
Factor C
5) Stimulated Indigenous Flora d 0.01 :1: 0.05
6) Stimulated Indigenous Flora + KC Supernatant Factor 0.16 :1: 0.01

 

a An initial CCl4 mass of 0.5 pg was added to all samples. Final CCl4 mass was
determined by heating to 70 C to release solid bound CCl4.
b :1: values represent the standard deviation of three independent samples

C KC supernatant factor was added as 2 mL of 10,000 MW filtrate from an actively
transforming culture of P. stutzeri KC grown in SGW medium at pH 8.3

d Supplemented with two pulse additions of 800 mg/L acetate and 800 mg/L nitrate.

Transport of the secreted factor through aquifer material. Transport of secreted
factor through aquifer material was evaluated by pumping 10,000 MW filtrate from
SGW-grown strain KC through columns packed with aquifer solids from Hanford, WA.
and Schoolcraft, MI. Figure 4.7a illustrates the breakthrough profile of 31120 and
secreted factor for Schoolcraft aquifer material as quantified by the CPMICPM° ratio and
the ratio of influent to effluent first-order bioassay rates using P. fluorescens . Figure

4.7b demonstrates the breakthrough profile of 3H20 and the secreted factor through

Hanford aquifer material. The breakthrough profile for the tritiated water was similar to

99

that of secreted factor in both of the aquifer solid systems tested, indicating that the

secreted factor was not retarded in either of these aquifer materials.

 
   
   

 

 

 

1 z_ [— 1.2
Tritiated water

1 .0- l . - 1.0
0.01- - 0.8

i secreted factor

91

Q 00 - 0.6

I:

hi

0
0.41- - 0.4
0.2 r 0.2
0.0 ' ‘ ‘ ‘ ‘ ' 0.0

0 20 40 60 80 100 120

Volume (ml)

Figure 4.7a. Breakthrough profile for tritiated water and the secreted factor in a column
packed with Schoolcraft, MI, aquifer material. Water was pumped through the column at
a flow rate of 2.5 mL/min.

Bioaasay ratio {1' of effluent! k' of influent}

100

        

 

 

 

1 .2 I- . . - 1
Tritiated water
1.0 l- *
L1
0 0.8 _
E secreted factor
0 - O.
i 0.6
8 - o.
0.4 ‘
0.2 _ o.
0.0 L‘ ' J k 0.
0 20 40 60 80 100 120

Volume (ml)

Figure 4.7b. Breakthrough profile for tritiated water and the secreted factor in a column
packed with Hanford, WA, aquifer material. Water was pumped through the column at a
flow rate of 2.5 mL/min.

DISCUSSION

My results indicate that rapid CCl4 transformation is obtained when diverse microbial
cell types are combined with the secreted factor produced by P. stutzeri KC. Previous
research has established that the transformation of CCl4 by strain KC is linked to the
trace metal scavenging activities of the cell, and the role of iron was especially important
[5,10,15]. In fact, the hypothesis that iron-scavenging agents, such as siderophores,
might be involved in the transformation led me to speculate that organisms other than

strain KC might be able to transform CCl4 in the presence of the secreted factor. Cross

iv '4:-

b
Biousay ratio {1' of effluent! k' of influent}

j
.0
A m

N

O

101

reactivity of siderophores between different bacterial species is well known [3, 9, 11].
The finding that mixtures did result in CCl4 transformation indicates that cross reactivity
does occur. However, the role of trace metal scavenging systems is unclear - although
organisms grown in iron-rich medium exhibited no evidence of extracellular iron-
scavenging activity, as measured with a universal chemical assay for siderophores [12],
they were still able to transform CCl4 in the presence of the secreted factor produced by
strain KC. Nevertheless, the patterns of growth of P. stutzeri KC and production of the
secreted factor [Figure 4.2], suggest that the factor does participate in trace-nutrient
delivery. A very rapid initial phase of growth was observed during which no secreted
factor production occurred. However, at approximately 12-hours of growth, the rate of
protein increase slowed and secreted factor production accelerated. This pattern suggests
that the factor is produced due to the limitation of a required nutrient or metal. To further
test this concept, iron concentrations and siderophore activity should be quantified
throughout the growth cycle. Understanding of iron uptake patterns could shed some

light on questions regarding the natural role of this factor.

The transformation of CCl4 by P. stutzeri KC appears somewhat analogous to reduction
of nitroaromau'c compounds mediated by Streptomyces sp. strain Tu 2484 exudates.
Secondary metabolites cinnaquinone and dicinnaquinone, secreted during the growth
phase of strain Tu 2484, have been shown to mediate electron transfer between hydrogen
sulfide and various nitrobenzenes [8]. The secreted factor produced by strain KC may
play a similar role, possibly functioning as a mediator of electron transfer between the
cell and the CCl4 molecule. Evidence supportive of such a hypothesis includes the
following: (i) formate is one of the nonvolatile products of CCl4 transformation by strain
KC [6], and this product can only be generated by a two-electron reduction of CCl4; (ii)
actively metabolizing cells are required - no CCl4 transformation is observed for late

stationary phase cells [6,16], cells that have not been stimulated by growth substrate

102

addition [Table 4.2], and autoclaved cells with or without secreted factor(s) (Figure 4.4);
(iii) certain oxidizing agents such as hydrogen peroxide [15] quench the reaction
(although oxygen appears only to inhibit it - Figure 4.3); and (iv) rates of transformation
by the secreted factor alone are significantly slower than in the presence of cells (Table
4.1). This hypothesis needs further support as proof of electron flux between the secreted
factor and CCl4 has not been demonstrated. Additionally, it would be interesting to
determine if combination of the secreted factor with an abiotic electron donor such as
titanium citrate would result in CCl4 transformation. This type of experiment could help
solidify the hypothesis that electron flux is involved and also provide evidence as to the

role of the cell in this transformation process.

The inhibition of the CCl4 transformation reaction by oxygen presents an interesting
conflict in data between the work presented in this chapter and work reported by Lewis
and Crawford. Lewis and Crawford [10] reported limited transformation of CCl4 by
cultures of strain KC grown under an oxygen headspace. However, the differences in the
reported results may be attributable to differences in experimental procedures. In Figure
43, the time-frame of the CCl4 transformation experiment was only a few hours, while
Lewis and Crawford monitored CCl4 transformation for 72 hours during the growth of P.
stutzeri KC. Additionally, CCl4 transformation was observed in their cultures after
dissolved 02 levels began to drop. The limited extent of CCl4 transformation observed in
the growing cultures may have occurred in areas of the culture medium where the
metabolic rate of the growing cells was sufficient to drop localized dissolved 02 levels to
the point where CCl4 transformation would occur. Despite the differences observed in
the experimental systems, it is impossible to discount Lewis and Crawford's finding that
product distribution in the aerobically grown cultures differed significantly from that in
the anaerobic cultures. Under aerobic conditions, a larger proportion of the transformed

CCl4 was found converted to C02 than in the anaerobically grown cultures. This may

103

result from competition for the tri-cloromethyl radical between 02 and other reacting
species. Clearly, it would have been interesting to determine if a product distribution
change existed between the biassay and whole cultures of P. stutzeri KC. The removal of
a large portion of supernatant constituents by ultrafiltration may have resulted in a
"cleaner” reaction, possibly resulting in a larger portion of the CCl4 being converted to
C02. This data may have helped to refute or support the hypothesis of a trichloromethyl
radical intermediate.

The present work presents a generally favorable outlook for use of the secreted factor in
field applications. Use of the secreted factor in combination with biostimulated
indigenous populations may provide the benefits of both bioremediation and
biostimulation. As with bioaugmentation, kinetic and pathway control are possible
because the performance of the secreted factor can be studied and optimized in the
laboratory. The pH range for CCl4 transformation was broad (Figure 4.6), but it was
optimal at a moderately alkaline pH values that are close to the values needed for iron
limitation and efficient production of the factor. I detected no chloroform in any of the
supematant/cell combinations studied. In all likelihood, CCl4 is transformed by the
secreted factor faster than chloroform can be produced by a parallel pathway. As shown
in Table 4.4, the amount of CCl4 removed from slurries of Hanford aquifer material that
were both biostimulated and inoculated with secreted factor was significantly more than
the CCl4 removed by biostimulation alone. Because the cells required to regenerate the
secreted factor can be native to the contaminated site, many of the ecological and transport
issues raised by the introduction of non-native organisms are avoided. Transport of the
secreted factor is still necessary, but we observed no difficulties in moving it through
aquifer material (Fig. 4.7a and 4.7b). Other data supporting possible application of the
secreted factor are its ability to be concentrated by freeze drying (Table 4.3), and its

favorable storage properties after f reeze-drying. Its stability in water is a possible

104

drawback to application and will need further evaluation, as only limited stability was

obtained in medium D and SGW medium (Table 4.2).

In the following chapter, these studies are further expanded upon by the identification of a
cell type that is incapable of regenerating the secreted factor. Additionally, evidence is
provided which supports the finding that the cell may function to transfer electrons to the

CCl4 molecule in a reaction mediated by the secreted factor.

REFERENCES

l. APHA/AWWA/WEF. 1992. Standard methods for the examination of water and
wastewater. American Public Health Assoc. Washington, DC.

2. Armenante, P.M., N. Pal, and G. Lewandowski. 1994. Role of mycelium and
extracellular protein in the biode gradation of 2,4,6-trichlorophenol by Phanerochaete
chrysosporium. Appl. Environ. Microbial. 60: 1711-1718.

3. Buyer, J.S. and J. Leong. 1986. Iron transport-mediated antagonism between plant
growth promoting and plant-deleterious Pseudomonas strains. J. Biol. Chem. 261:
791-794.

4. Criddle, C.S., J.T. DeWitt, and P. L. McCarty. 1990. Reductive dehalogenation of
carbon tetrachloride by Escherichia coli K-12. Appl. Environ. Microbiol. 56: 3247-
3254.

5. Criddle, C.S., J.T. DeWitt, D. Grbie-Galie and P. L. McCarty. 1990.
Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under
denitrifrcation conditions. Appl. Environ. Microbiol. 56: 3240-3246.

6. Dybas, M.J., G. M. Tatara, and C.S. Criddle. 1994. Localization and
characterization of the carbon tetrachloride transforming activity of Pseudomonas sp.
strain KC. Appl. Environ. Microbiol. 61: 758-762.

7. Egli, C., T. Tschan, R. Scholtz, A.M. Cook, and T. Lekinger. 1988. Transformation
of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium
woodii. Appl. Environ. Microbiol. 54: 2819-2823.

8. Glaus, M.A., C.G. Heijman, R.P. Schwarzenbach, and J. Zeyer. 1992. Reduction of

nitroaromatic compounds mediated by Streptomyces sp. exudates. Appl. Environ.
Microbial. 58: 1945: 1951.

9. Hohnadel D. and J..M. Meyer. 1988. Specificity of pyoverdine-mediated iron
uptake among fluorescent Pseudomonas strains. J. Bacteriol. 170: 4865-4873.

10. Lewis, T.A. and R.L. Crawford. 1993. Physiological factors affecting carbon
tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain
KC. Appl. Environ. Microbial. 59: 1635-1641.

11. Morris, J., D.J. O'Sullivan, M. Koster, J. Leong, P. J. Weisbeek, and F. O'Gara.
1992. Characterization of fluorescent siderophore-mediated iron uptake in
Pseudomonas sp. strain M114: evidence for the existence of an additional ferric
siderophore receptor. Appl. Environ. Microbiol. 58: 630-635.

12. Schwyn, B. andJ.B. Neilads. 1987. Universal chemical assay for the detection and
determination of siderophores. Anal. Biochem. 160: 47-56.

13. Semprini, L., G. D. Hopkins, P.L. McCarty, and P.V. Roberts. 1992. ln-situ

transformation of carbon tetrachloride and other halogenated compounds resulting
from biostimulation under anoxic conditions. Environ. Sci. Technol. 26: 2454-2461.

105

106

14. Slttig, M. (ed.). 1985. Handbook of toxic and hazardous chemicals and carcinogens,
2nd. Noyes Publications, New York.

15. Tatara, G.M., M.J. Dybas, and C.S. Criddle. 1993. Effects of medium and trace
metals on the kinetics of carbon tetrachloride transformation by Pseudomonas sp.
strain KC. Appl. Environ. Microbiol. 59: 2126-2131.

CHAPTER 5

ROLE OF CELL MEMBRANES IN THE TRANSFORMATION OF CARBON
TETRACHLORIDE BY PSEUDOMONAS STUTZERI KC

107

ABSTRACT

Previous research has established that the transformation of CCl4 by Pseudomonas
stutzeri KC proceeds via a complex mechanism involving both a secreted (cell-free)
factor and cell-associated factors. The combination of the secreted factor and a diverse
range of cell types results in rapid CCl4 transformation. The present study provides
further insight into the cell associated factor(s) required for CCl4 transformation. The
combination of the secreted factor with fermenting cells of Escherichia coli resulted in
rapid CCl4 transformation, but a strictly fermenting bacterium, Lactabacillus acidophilus
, did not mediate rapid CCl4 transformation. L. acidophilus lacks a membrane bound
electron transport chain and cytochromes, suggesting participation of these constituents in
regeneration of the secreted activity. Crude cell membranes supplemented with secreted
factor and NADH rapidly transformed CCl4, demonstrating the need for a membrane-
associated redox component(s). The metalo-center inhibitors cyanide and pyridine both
inhibited CCl4 transformation. Chloramphenicol also inhibited CCl4 transformation.
These data led to the development a new model which posits that CCl4 transformation
activity requires both a secreted factor and a non-respiratory membrane-associated

electron transport agent.

108

INTRODUCTION

The transformation of carbon tetrachloride (CCl 4) by Pseudomonas stutzeri KC involves
both a cell-associated and a small (< 500d) cell-free component [4]. Previous studies
demonstrated that rapid CCl4 transformation is obtained when diverse microbial cell
types are combined with the secreted factor produced by P. stutzeri KC [9]. Forrnate, a
two electron reduction product of CCl4, is an end product of the transformation,
suggesting that the secreted factor is a reductant capable of a two-electron transfer [4].
One possible hypothesis is that regeneration of the CCl4 transforming factor secreted by

strain KC is reduced by one or more of the respiratory chain components.

In this chapter, I describe experiments designed to assess the role of electron transport in
regeneration of the secreted factor. Inhibitor studies were performed with Pseudomonas
fluorescens , an organism capable of rapid CCl4 transformation in the presence of the
secreted factor produced by strain KC [4,9]. Additionally, a simplified electron transport
system consisting of NADH and crude cell membranes of P. stutzeri KC is shown to
reconstitute CCl4 transformation activity. Finally, a revised model is postulated which

more accurately describes CCl4 transformation.

109

MATERIALS AND METHODS

Chemicals. CCl4 (99% purity) , sodium cyanide, and sodium azide, were obtained from
Aldrich Chemical Co., Milwaukee, Wis. INT (2-(p-iodo-phenyl)-3-(p-nitrophenyl)-5-
phenyl tetrazolium chloride), rotenone (1,2,12,12a-tetrahydro-8,9-dimethoxy-2-(1 -
methylethenyl-[l]benzo-pyrano[3,4—b]furo[2,3h]-[1]-benzopyran-6(6l-I)-one), quinicrine
dihydrochloride, dicumarol (3,3'-methylenebis[4—hydroxy-2H-1-benzypyran-2-one]),
HOQNO (2-heptyl-4-hydroxyquinoline N-oxide), DCCD (N ,N '-dicyclohexyl-
carbodiimide), DNP (2,4-dinitrophenol), and Chloramphenicol were obtained from Sigma
Chemical Co. All chemicals used in media preparation were ACS reagent grade (Aldrich
or Sigma Chemical Co.). All water used in reagent and inhibitor preparation was

deionized 18 Mohm resistance or greater.

Media. Hanford simulated groundwater (SGW) was prepared as described in Chapter 3.
Lactobacilli broth AOAC (Dif co) was prepared according to the manufacturer's
instructions. The resulting medium was autoclaved at 121°C for 20 minutes and

transferred to an anaerobic glove box (Coy Laboratories, Ann Arbor, M1) for degassing.

Preparation of partially purified culture supernatant. Preparation of filtered and
lyophilized culture supernatant was prepared as described in Chapter 4.

Preparation of crude cell membranes. Two liters of P. stutzeri KC (ATCC deposit no.
55595, DSM deposit no. 7136) culture was grown from a 1% inoculum in SGW medium
under aerobic conditions in 4L erlenmeyer flasks. 2 L of culture was transferred to 250
mL Nalgene® centrifuge tubes and centrifuged at 8,200 x g at 4 0C. Pellets were
resuspened in 50 mL of 50 mM potassium phosphate buffer (pH 8.0) and were pooled

together into 2 centrifuge tubes containing 200 mL of cell resuspension each. These

110

111

resuspended pellets were cenuifuged again at 8,200 x g for 15 minutes at 4 0C. The
resulting cell pellets had a total mass of 5 g and were resuspended in 12 mL of 50 mM
phosphate buffer (pH 8.0). The washed and resuspended cell pellet was sonicated for 10
min. at 1 sec. bursts with 50% time on and 50 % time off. DNAse I was added at final
concentration of 250 ng/mL, and the lysate was incubated for 30 minutes at 20°C. The
lysate was centrifuged at 8,200 x g, for 10 minutes at 4 °C to remove unbroken cells. The
cell-pellet was discarded and the lysate centrifugation process was repeated. The
resulting lysate was ultracentrifuged at 150,000 x g at 4 °C for 120 min. Following
ultracentrifugation, the cytoplasmic fraction was decanted and the membrane fraction was
gently resuspended in 12 mL of 50 mM potassium phosphate buffer pH 8.0. The crude
cell membrane and cytoplasmic fractions were used immediately in a CCl4

transformation assay with partially purified secreted factor.

CCl4 transformation assays with crude cell membrane preparations. Lyophilized
500 MW filtrate was rehydrated to its original volume (5 mL) as follows. 4.5 mL of 500
MW filtrate was transferred to 28 mL aluminum seal tubes (Bellco Glass ), then
supplemented with 500 yL of fresh cytoplasmic fraction, 500 yL of fresh membrane
preparation, or 500 FL of 50 mM potassium phosphate buffer. Test samples were made
anoxic by passage through the interlock of an anaerobic glove box. B-NADH (Sigma)
was added to select samples to give a final concentration of 20 yM. The serum tubes
were sealed under anoxic conditions with Teflon-lined butyl rubber septa (West) and
aluminum crimp seals (West). CCl4 transformation was monitored as described in

Chapter 2.

Measurement of electron transport system activity. Electron transport activity
measurements were performed by the method of Zimmerman et al. [10] and Trevors et al.

[14]. A 10 mL quantity of sample was poured into sterilized serum tubes, and 1 mL of a

112

0.2% (w/v) tetrazolium dye was added to each tube. At various time intervals, 1 ml
aliquots were removed from the sample tubes. The 1 mL aliquots were extracted with 10
mL of HPLC grade methanol, filtered through 0.22 pm nylon filters, and measured
spectrophotometrically at 480 nm. CCl4 transformation was monitored as described

previously.

CCl4 transformation with Lactobaciau: acidophilus. L. acidophilus (ATCC no. 4356)
was obtained from the American Type Culture Collection. L. acidophilus was grown
anaerobically in Lactobacilli broth AOAC in 120 mL serum bottles at 35 0C. Cultures of
L. acidophilus were transferred to an anaerobic glove box and dispensed into 40 mL
centrifuge tubes. Cells were collected by centrifuging at 12,100 x g for 5 minutes,
wasting the culture supernatant, and resuspending the pellet in 4 mL of anoxic
Lactobacilli broth AOAC. A 0.5 mL sample of cell suspension (concentrated ten fold by
centrifugation) was added to 4.5 mL of filtered supernatant and assayed for CCl4

transformation in 28 mL aluminum seal tubes as previously described.

Lactate production was measured using the lactate reagent kit from Sigma Chemical Co.
A standard curve was prepared using lactate over the concentration range 0-10 mg/mL,

and measuring the absorbance at 540 nm.

CCl4 transformation with fermenting E. coli. E. coli K-12 (ATCC no. 10798) was
obtained from the culture collection of the Microbiology Department at Michigan State
University. E. coli was grown at 35°C under strictly fermenting conditions with 3 g/L of
glucose as the carbon and energy source in SGW medium. SGW medium for the growth
of E. coli under fermenting conditions was prepared nitrate free to avoid the production
of anaerobic respiratory chains. Cultures were grown to an OD660 of approximately 0. 18,

transferred to an anaerobic glove box, dispensed into 40-mL Nal gene centrifuge tubes,

113

f itted with butyl rubber septa, and centrifuged at 12,100 x g for 5 min. The culture
supernatant was decanted, and the pellet was resuspended in 4 mL of nitrate free anoxic
SGW medium with glucose as the electron donor. A 0.5 mL sample of cell suspension
(concentrated ten fold by centrifugation) was added to 4.5 mL of filtered supernatant and
assayed for CCl4 transformation as described previously.

\
Inhibitor Studies. Relevant information pertaining to the concentration and proper
solvent for each inhibitor is summarized in Table 1. All inhibitor stock solutions were
prepared fresh daily at concentrations sufficiently high so that a minimal volume of
solvent was added in the inhibitor studies. This was to minimize the chances of
secondary effects attributable to solvent addition. Appropriate controls were also

prepared to ensure that solvents themselves were not responsible for inhibition.

Cultures of P. fluorescens were grown aerobically in SGW medium to a desired optical
density of OD660 = 0.15, a value chosen to ensure that the cultures were in the
exponential stage of growth. Cultures were dispensed into 40-mL Nalgene® centrifuge
tubes. Cells were collected by centrifuging at 12,100 x g for 5 minutes , wasting the
culture supernatant, and resuspending the pellet in 4 mL of SGW medium at pH 8.2. A
0.5 mL sample of cell suspension (concentrated ten fold by centrifugation) was added to
4.5 mL of partially purified culture filtrate in a 28 mL serum tube and the desired
inhibitor or solvent was added. The resulting test samples were made anoxic by transfer
through the interlock of a Coy anaerobic glove box. Tubes were sealed under anoxic

conditions and CCl4 transformation was monitored as previously described.

Modeling. The first-order-rate coefficient or k" value was calculated as described

previously in Chapter 4.

114

Table 5.1. Summary of inhibitors used

 

 

 

 

 

 

 

 

 

 

Chemical Inhibitor (31:23:23,333? Solvent InhrercqzrmSlrte or
Rotenone 10 - 1,000 Acetone NADH Dehydgggnase
Quinacrine

dihydrochloride l - 1,000 Water Flavins

Dicumarol 1 - 50 Pyridine Quinones

HOQNO 0.7 - 50 Ethanol Cytochrome b

NaCN (cyanide) 10 - 3,000 Water Cytochrome c oxidase
NaNJjazide) 20 - 20,000 Water Cytochrome c oxidase
DCCD l - 100 Acetone ATPase (Fo subunit)
DNP l - 500 Acetone Uncoupler
Chloramphenicol 100 - 200 Ethanol Bacteriostatic Agent

 

 

 

 

 

a. The concentrations of inhibitors used in this study were chosen based on
concentrations referenced in a similar study performed by Arnold et al [2].

RESULTS

Transformation of CCl4 by crude cell membrane preparations. Crude cell membrane
and cytoplasmic fractions were prepared to determine if membrane bound or cytoplasmic
proteins provide reducing equivalents to the secreted factor produced by P. stutzeri KC.
NADH was added to determine if it enhances transformation or if it can provide reducing
equivalents to the secreted factor with an additional mediator protein. Freshly prepared
membrane and cytoplasmic fractions were combined with the secreted factor with and
without NADH. As shown in Figure 5.1, rapid CCl4 transformation occurred when the

secreted factor was combined with crude membrane preparation from P. stutzeri KC in

115

the presence of 20 pM NADH. Transformation of CCl4 also occurred when the secreted
factor was combined with crude membranes without NADH present. However, the rates
were not as fast as with NADH. No CCl4 transformation was observed when the secreted
factor was combined with the cytoplasm of P. stutzeri KC cells with or without NADH
present. The secreted factor did not transform CCl4 when combined with NADH, thus

suggesting that a membrane-bound component mediates transfer of reducing equivalents

 

 

 

 

from NADH to the secreted factor.
0-20 _ q .. . sr: + KC
"‘ " o Cytoplasm
_ ' SF + KC Cyto. +
' 20pM NADH
O. 15 ' ' .
CT '
(F8) . SF + Crude KC
L Membranes
0.10 E
r SF + _Pfluor.
r . cells
0-05 ’ ‘ sr= + Crude KC
- Membranes
+ ZOyM NADH
0.00 a 1 n l 4 A 4 l A . n l . n . l . A . l A
0 3O 6O 90 120 150

Time (min)

Figure 5.1. Transformation of CCl4 with crude cell membrane preparations from P.
stutzeri KC. Error bars represent the standard deviation of triplicate samples.

INT-Formazan inhibits CCl4 Transformation. An active electron transport system is
an almost universal component of respiring organisms, which reduces INT (2-(p—iodo-

phenyl)-3-(p-nitrophenyl)-5-phenyl tetrozolium chloride) to INT-formazan [12]. The

116

conversion of INT to INT -formazan was measured as a means of determining the
effectiveness of inhibition of electron transport system activity. As shown in Figure 5.2,
the production of INT-formazan inhibits CCl4 transformation by cultures of P. stutzeri
KC. As a result, INT-formazan production could not be utilized in the inhibitor studies a

means of determining the effectiveness of a given inhibitor.

 

 

 

0.30 '
0.25 -
y. . A)
O. 20 Abiotic CODIIOI
‘~
L \? ‘
CT 0.15 -
(1‘8)
INT-Formazan ‘
0.10 -
0.05 _ Positive Control
0.00 A l A l A l A I A l A l A l A 1 A I
0 30 60 90 120 150 180 210 240 270

Time (min)

Figure 5.2. Effect of INT -Formazan on CCl4 transformation by P. stutzeri KC. Error
bars represent the standard deviation of triplicate samples.

Comparison of CCl4 transformation with B. subtilu: and L. acidophilus . L
acidophilus was combined with the secreted factor from P. stutzeri KC to determine if a
strictly fermenting bacterium could regenerate the secreted factor to result in CCl4
transformation. As shown in Figure 5.3, L. acidophilus does not rapidly transform CCl4
in the presence of the secreted factor. In fact, rates for the secreted factor alone were very

similar to those observed for the secreted factor in combination with L. acidophilus.

117

Since L. acidophilus is a gram positive organism, B. subtilus was used a positive control.
Cultures of L. acidophilus were actively fermenting glucose to lactate (data not shown)
during the transformation assay. Therefore, the alkaline pH necessary for CCl4

transformation was not inhibitory to the metabolism of L. acidophilus.

0.3 1
4:!“ o B. subti____l_us alone
V‘V V L. acidophilus alone

0.2 ‘ i 1 ’ MR 500 Alone

 

 

 

L.acidophlius
CT ‘ ' +Factor
(F8)
01‘
Bsubtilus
+Factor
0.0 AAAlAAAIAAAlAAAI4AL1AAAIAAAIAAAIAAAI
O 20 40 60 80 100 120 140 160 180

Time (min)

Figure 5.3. Comparison of CCl4 transformation with B. subtilus and L. acidophilus when
combined with the secreted factor from P. stutzeri KC. Error bars represent the standard
deviation of triplicate samples.

Combination of the secreted factor with fermenting E. coli. E. coli was grown under
strictly fermenting conditions to determine if recharge of the secreted factor required
respiring cells (either aerobically or anaerobically). The combination of E. coli under
fermenting conditions with the secreted factor from strain KC resulted in CCl4

transformation (Figure 5.4).

118

Abiotic CC

Fermenti ng
_1_':‘._. coli.

 

 

 

0.0 I I r I ' I ' I fl I ' I ‘ I ' I ' I ' I
0 20 40 60 80 100 120 140 160 180 200
Time (min)

Figure 5.4. Transformation of CCl4 by strictly fermenting E. coli combined with the
secreted factor from P. stutzeri KC. Error bars represent the standard deviation of
triplicate samples.

119

Table 5.2. Effect of electron transport inhibitors as measured by first-order rate

coefficients.

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chemical ngztzg): k " (min' 1)‘1 Inhibition
Inhibitor
Rotenone o - 1,000 (0.08 :1: 0.01) - (0.06:1: 0.01) Slight__
Quinacrine
dihydrochloride o - 1,000 (0.06:1: 0.01) - (0.05: 0.1) No
Dicumarol o - so (o.os¢o.or) - (0.07: 0.01) No
limo o - so (0.07:0.02) - ( 0.06:0.01) No
NaCN (cyanide) 0 -3,000 (0.07:0.01) - (0.00) Yes
NaN3 (azide) o - 20,000 (00710.01) - (0.05:0.01) Sngh_t_
DCCD o - 100 (00610.01) - (0.08:0.01, No
DNP o - soo (0.0&0.0l) - (00910.01) No
Chloramphenicol o-zoo (0.19:0.02) - (0051001)!) Yes
Pyridine o- 25 pL (0.08 :l: 0.01) - (00210.01, Yes

 

 

a first-order-rate coefficient (k") values represent the average of triplicate samples,
standard deviations are not shown.

b rate calculated on a whole culture of P. stutzeri KC

Inhibitors of electron transport. Specific respiratory chain inhibitors were evaluated in

order to determine if components of the respiratory electron transport system are involved

in transformation of CCl4 with the secreted factor produced by P. stutzeri KC. Table 5.2

shows that 1 mM rotenone slightly inhibits the CCl4 transformation reaction. A

concentration of 5 mM rotenone (data not shown) had a greater inhibitory effect on CCl4

transformation, but the rotenone was added in 500 FL of acetone, which also significantly

inhibited CCl4 transformation. Lower concentrations of rotenone did not inhibit the

CCl4 transformation reaction. Quinicrine dihydrochloride, an inhibitor of flavins, does

 

120

not inhibit the CCl4 transformation reaction of P. fluorescens combined with the secreted
factor from P. stutzeri KC. Dicumarol also does not affect the CCl4 transformation
reaction. However, pyridine which was the solvent used for the addition of dicumarol
significantly inhibited CCl4 transformation. HOQNO, a cytochrome b inhibitor, does
not inhibit CCl4 transformation, and either does DCCD, an inhibitor of the F0 subunit of
ATPase. DNP, which is an uncoupler and renders the cytoplasmic membrane permeable

to H+, also does not inhibit transformation.

Cyanide, a cytochrome oxidase inhibitor significantly inhibited the transformation of
CCl4 as shown in Table 5.2. Concentrations of cyanide as low as 10 14M completely
inhibited CCl4 transformation. Another cytochrome oxidase inhibitor, azide, only
slightly inhibited CCl4 transformation at a concentration of 2 mM. Both cyanide and
azide were added in water, so solvent effects were not a concern in this experiment
Chloramphenicol, a bacteriostatic agent which blocks protein synthesis and porins

significantly inhibits CCl4 transformation at a concentration of 170 FM.

DISCUSSION

P. stutzeri KC transforms CCl4 by a complex mechanism involving both cell-free and
cell-associated factors. [4,9]. I had previously hypothesized that mediation of the CCl4
transformation may be linked to an interaction between the secreted factor and a cell-
associated factor in the electron transport system of bacteria. Three lines of evidence
support the hypothesis that a membrane-associated redox component(s) is required to
mediate CCl4 transformation activity: (1) experiments with crude cell extracts, (2)
inhibition of activity by INT-formazan, and (3) lack of transformation by the L
acidophillus system. Crude cell extract experiments established that CCl4 transformation

required a source of reducing equivalents (NADH), a cell membrane, and the secreted

121

factor (Figure 5.1). In addition, several previous hypotheses were refuted by this
experiment. A cytoplasmic component is not required for transformation, and NADH
does not reduce the secreted factor directly . Clearly, a membrane bound component is
required for transfer of reducing equivalents from NADH to the secreted factor.
Inhibition of CCl4 transformation by INT-formazan suggests that an electron transport
chain is required for regeneration of the secreted factor (Figure 5.2). Dehydrogenases
reduce INT to INT -formazan, which is deposited as optically dense purple crystals inside
the bacteria. INT-forrnazan may compete with the secreted factor for reducing
equivalents entering an electron transport chain. L. acidophilus, an organism lacking any
membrane bound electron transport system [5], facilitated only a very limited level of

CCl4 transformation when combined with the secreted factor (Figure 5.3).

The nature of the membrane-associated redox component was further explored in
experiments with fermenting E. coli , which does not utilize respiratog electron transport
chains for growth. Ferrnenting E. coli was capable of regenerating activity (Figure 5.4).
This suggests that the membrane-associated redox component(s) involved in CCl4
transformation may not be a respiratory chain. Not all of the electron transport chains
present in E. coli are required respiratory electron transport-dependent ATP synthesis

[61-

Additional support for the hypothesis that a non-respiratory redox component(s) is
involved in the reaction was obtained from inhibitor studies. Reducing equivalents enter
the respiratory chain via one of several dehydrogenases (often flavin containing enzymes,
e.g. NADH and succinate dehydrogenases) and are passed sequentially down a chain of
carriers which includes iron-sulfur proteins, coenzyme Q, and a series of cytochromes
[2]. Table 5.2 shows that rotenone, an inhibitor of NADH dehydrogenase, only slightly

inhibited CCl4 transformation at a concentration of 1 mM. Quinicrine dihydrochloride,

122

an inhibitor of the flavin groups in dehydrogenases, did not inhibit CCl4 transformation.
These results argue against the idea that the secreted factor accepts reducing equivalents
from a respiratory chain enzyme. If an enzyme in the respiratory chain were involved in
CCl4 transformation, then these dehydrogenase inhibitors should impact CCl4
transformation. However, dicumarol (a quinone inhibitor) and HOQNO (a cytochrome b
inhibitor) did not inhibit CCl4 transformation . Therefore, a model in which the secreted
factor accepts reducing equivalents from a component of the respiratory chain does not

adequately describe the mechanism of CCl4 transformation.

It could be argued that inhibition by cyanide implicates a respiratory chain cytochrome as
the step where secreted factor interacts to result in CCl4 transformation. Both bacteria and
eukaryotic organisms contain cytochrome oxidase [l3], and both organism types
regenerated activity. Cyanide is known to inhibit cytochrome oxidase, and it strongly
inhibited CCl4 transformation. However, if cytochrome oxidase were capable of
regenerating activity, it should have been blocked by inhibitors acting higher up on the
electron transport chain. In addition, azide, which is a known cytochrome oxidase

inhibitor, only slightly inhibited CCl4 transformation at a concentration of 20 mM.

Pyridine also inhibits CCl4 transformation when added in sufficient quantity. Both
pyridine and cyanide bind tightly to the metal centers of many enzymes. Cyanide is a
nucleophile and can attack electrophilic sites. In addition, cyanide complexes with the
Fez+ form of iron bound to the heme cofactor [12]. Therefore, even though cyanide is

listed as an inhibitor of cytochrome oxidase, it is not a specific inhibitor of this enzyme.

DCCD, a specific inhibitor of the F0 subunit of ATPase did not inhibit CCl4
transformation, indicating that in the short term, ATP is not involved in the

transformation reaction. Additionally, a proton gradient is not required for CCl4

123

transformation as DNP, an uncoupler which renders the cytoplasmic membrane
permeable to 11+ ions, did not inhibit CCl4 transformation. This is not inconsistent with a
previous observation by Dybas et al. [4], who observed that CCCP, which uncouples
respiration from ATP synthesis, stimulated resting cells to transform CCl4. The presence
of either DNP or CCCP collapses the proton gradient and stimulates rapid electron
transfer. Therefore, over the short-term, maintenance of a proton gradient and production

of ATP are not necessary for regeneration of the secreted factor.

A few other clues for the mechanism of transformation were obtained. Chloramphenicol
inhibited CCl4 transformation. One possible mechanism of Chloramphenicol inhibition is
related to its capacity to stop translation at the ribosome level. This mechanism can be
discounted because: (1) other experiments conducted with Chloramphenicol indicated
inhibition in a time frame too short for new protein synthesis (<10 minutes) and (2)
Chloramphenicol inhibited CCl4 transformation in a bioassay with P. fluorescens, an
organism that does not produce the secreted factor. A second inhibitory mechanism is
blockage of bacterial outer membrane porin channels [3]. For the secreted factor to
obtain reducing equivalents at the inner membrane and transform CCl4 outside the
bacterial cell, it must be able to freely diffuse in and out of the outer membrane porin
channels which typically allow free diffusion of molecules smaller than 600MW. The
small size of the secreted factor (< 500 daltons) would make this diffusion possible,

unless the porin channels were blocked by Chloramphenicol.

Tatara et al. [10] showed that copper is required for CCl4 transformation by P. stutzeri
KC. Both cyanide[1] and pyridine [8] are strong ligands to the metal centers of enzymes.
Thus, one possible mode of inhibition might involve blockage of a copper containing

enzyme needed for regeneration of the secreted factor. It is also possible that cells grown

124

in the absence or at very low copper concentrations could lack copper containing

enzymes needed to regenerate or produce the secreted factor.

As a result of the data presented above, a new model is proposed for recharge of the
secreted factor. The new model posits that a non-respiratory membrane-associated
electron transport chain is required for regeneration of CCl4 transformation activity.
Additional research is needed to understand the role of trace metals in the regeneration of
CCl4 transformation activity. Additional work is also needed to identify the specific

f actor(s) that transfers reducing equivalents to the secreted factor.

 

REFERENCES

1. Alexander K. and SJ. Baskin. 1987. The inhibiton of cytochrome oxidase by
diaminomaleonitrile. Biochemica et Biophysica Acta. 912: 41 -47.

2. Arnold, R. G., T.,]. DiChristina, and M. R. Hoffman. 1986. Inhibitor studies of
dissimilative Fe(III) reduction by Pseudomonas sp. strain 200 (”Pseudomonas
ferrireductans"). Appl. Environ. Microbiol. 52: 281-289.

3. Chopra, 1. 1990. Penetration of antibiotics to their target sites. Journal of
Antimicrobial Chemotherapy. 26: 607-609.

4. Dybas, M.J., G. M. Tatara, and C.S. Criddle. 1995. Localization and
characterization of the carbon tetrachloride transformation activity of Pseudomonas sp.
strain KC. Appl. Environ. Microbiol. 61: 758-762.

5. Gottshalk, G. 1986. Bacterial Metabolism, Second Edition. Springer-Veflag,
New York.

6.Haddock, B.A. and C.W. Jones. 1977. Bacterial respiration. Bacteriological Reviews.
41: 47 - 99

7. Shienke, A.K. W.A, Kaplan, C.L. Hamilton, J. A. Shelnutt, and R.A. Scott. 1989.
Structural and spectroscopic characterization of exogenous ligand binding to isolated
factor F430 and its configuration isomers. J. Biol. Chem. 246: 7267-7284.

8. Peterson, J. N., R.S. Skeen, K. M. Amos, and B.S. Hooker. 1994. Biological
destruction of CCl4: I. experimetnal design and data. Biotech. Bioeng. 43: 521-528.

9. Tatara, G.M., M.J. Dybas, and C.S. Criddle. 1995. Biofactor-mediated
transformation of carbon tetrachloride by diverse cell types, p. 69-77. In R. Hinchee, A.
Leeson, and L. Semprini (ed.) The Bioremediaton Series, 3(3) , Bioremediation of
Chlorinated Solvents. Battelle Press, Richland, WA.

10. Tatara, G.M., M.J. Dybas, and C.S. Criddle. 1993. Effects of medium and trace
metals on kinetics of carbon tetrachloride transformation by Pseudomonas sp. strain KC.
Appl. Environ. Microbiol. 59:2126-2131.

11. Trevors, J .T., C.I. Mayfleld, and W.B. Inniss. 1982. Measurement of electron
transport activity in soil .Microb. Ecol. 8: 163 -168.

12. Walsh , C. Enzymatic reaction mechanisms. W.H. Freeman and Company, New
York. 1979

l3.Yamanaka, T., Y. Fukumori, M. Numata, and T. Yamazaki. 1988. The variety of
molecular properties of bacterial cytochromes containing Heme a, p.39-46. In M. Brunori
and B. Cance (ed), Cytochrome oxidase: Structure, function, and physiopathology. New
York Academy of Sciences, New York.

14. Zimmerman R., R. Iturriaga, and J. Becker-Birick. 1978. Simultaneous

determination fo the total number of aquatic bacteria and the number thereof involved in
respiration. Appl. Env. Micro., 36: 926-935.

125

CHAPTER 6

THE USE OF VEGETABLE OILS FOR THE ENGINEERED APPLICATION OF
PSEUDOMONAS STUTZERI KC

The C02 trapping technique described in these studies was performed with the assistance
of Dr. Michael Dybas

126

ABSTRACT

Vegetable oil may serve as a low cost non-toxic electron donor that could provide a
controlled release of growth substrate to microorganisms for extended time periods.
Pseudomonas stutzeri KC was tested for its ability to grow and transform carbon
tetrachloride(CCl4) when vegetable oils are used as the carbon and energy source. P.
stutzeri KC achieved growth yields on vegetable oils under denitrifying conditions that
were similar to those observed when acetate was the carbon and energy source. 14 C-
CCl4 studies revealed that 40 - 50% of the originally added CCl4 was converted to C02.
Corn oil was used to evaluate the long term growth characteristics and CCl4
transformation capacity of P. stutzeri KC and of an enrichment derived from CCl4
contaminated aquifer solids from Schoolcraft, MI. CCl4 masses of 18.3 :1: 0.8 and 17.2 :r:
1.3 pg were removed by P. stutzeri KC and P. stutzeri KC combined with the Schoolcraft
flora respectively. No significant CCl4 transformation was observed for the Schoolcraft
flora alone. Approximately 5% of the initially added CCl4 was converted to chloroform
by P. stutzeri KC. Growth curves, pH, and nitrate analysis revealed that the yield and

rates of growth were limited by the addition of nitrate.

127

INTRODUCTION

Three hundred to four hundred thousand subsurface hazardous waste sites have been
identified which impact groundwater [7]. A majority of these sites contain various levels
of toxic organics, and many current remediation technologies are designed to treat these
wastes [3]. In addition, many groundwaters are contaminated with high levels of nitrate
from agricultural operations. Nitrate contamination can render groundwater unusable as
drinking water. Thus, remediation technologies that remove both nitrate and organic

contamination are desirable.

Bioremediation, the use of microorganisms (either native or introduced) to degrade
contaminants to harmless endproducts and biomass, is a promising approach for
environmental cleanup. Many highly chlorinated contaminants are only biodegraded by
cometabolism, which is defined as the fortuitous transformation that depends on the
previous or concurrent utilization of a growth substrate[1]. By virtue of their oxidized
state, these highly chlorinated organic solvents are poor energy sources. Growth
substrates (electron donors) must be provided to allow for the growth of cometabolic
populations and to sustain the cometabolic reaction. A major factor limiting
cometabolism is delivery of the growth substrate needed to sustain a biomass capable of
degrading the target contaminant. Of primary concern is the cost associated with
repeated delivery and mixing of nutrients in subsurface environments. Use of a low cost

electron donor that does not need to be delivered repeatedly would be highly desired.

In this chapter, I report on the growth of P. stutzeri KC on vegetable oils and its
subsequent cometabolism of CCl4 with nitrate as the electron acceptor. Additionally, I
report on the transformation capacity of P. stutzeri KC and the organisms long term

growth characteristics using vegetable oil as a growth substrate.

128

MATERIALS AND METHODS

Organisms. P. stutzeri KC (DSM deposit no. 7136, A'I'I‘C deposit no. 55595), derived
originally from aquifer solids from Seal Beach, CA, [2] is routinely maintained in our
laboratories on nutrient agar plates. An enrichment of groundwater microorganisms was
obtained from CCl4-contaminated aquifer solids from Schoolcraf t, MI. The enrichment
was obtained by incubating 1 g of aquifer solids in 100 mL of SGW media at pH 8.2
under denitrifying conditions at 20-23 0C. The enrichment was used as an inoculum in

CCl4 transformation experiments after protein levels reached 89 :1: 12 mg/L.

Chemicals and radioisotopes. All chemicals used were ACS reagent grade (Aldrich or
Sigma Chemical Co.). All water used in reagent preparation was 18 Mohm resistance or
greater. CCl4 (99% -purity) was obtained from Aldrich Chemical Co., Milwaukee, Wis.

l4C-labeled CC14(3.4 mCi/mMol) was obtained from NEN DuPont (Boston, MA).

Media. Hanford simulated groundwater (SGW) was identically prepared as described in
Chapter 3, with the exception that acetate was not added to the growth medium. After pH
adjustment, media was transferred to growth vials and supplemented with vegetable oils

as the electron donor source. Medium D was prepared as described in Chapter 2.

Initial screening of CCl4 transformation activity for P. W KC cultures grown
on vegetable oils. P. stutzeri KC was screened for growth and transformation of CCl4 on
corn oil, soybean oil, and canola oil. All three types of oil were obtained from a local
food market in Lansing, MI. One liter of SGW medium (acetate free) was prepared at pH
8.2. Fifteen milliliters of medium were dispensed into 30 mL serum bottles (Wheaton),
and 50 pL of oil was dispensed into test vials receiving oil. Serum bottles were degassed

through the interlock of an anaerobic glove box, (Coy laboratories, Ann Arbor, MI) and

129

130

sealed under anoxic conditions with 20 min Teflon-lined butyl rubber septa (West) and
aluminum crimp seals (West no. 5120-1180). Samples were then autoclaved at 121°C for
20 minutes. Following autoclaving, Tween 80 (polyethylenesorbital monooleate) was
added as an emulsifier to half of the samples at a final concentration of 0.01% (v/v). A
mass of 2.1 :r; 0.7 yg of 14C-CC14 was added to all vials prior to inoculation. P. stutzeri
KC was added as a 1% inoculum (v/v) from a 48 hour medium D grown culture that was
washed and resuspended to its original volume (100 mL) in 50 mM phosphate buffer at
pH 8.2. Abiotic controls received CCl4 but no organisms. Cultures were grown for 72

hours before being assayed for CCl4, l4C-C02, and protein .

Due to the partitioning of CCl4 into the oil phase, the 30 mL serum bottles from the initial
screening experiment were heated at 90 0C for 2 hours prior to GC analysis. Bottles
removed from the water bath were immediately assayed by removing 100 pL of
headspace gas with a 500 pL Pressure-Lok® gas syringe (Supelco) and injecting the
sample into a gas chromatograph as described in Chapter 2. External calibration curves
were prepared by addition of a primary standard (8.37 ng of CCl4 per pL of methanol) to
secondary aqueous solutions having the same gas/water ratio, water/oil ratio, ionic
strength, incubation temperature, and speed of shaking as the assay samples. A five point
calibration curve was prepared over a concentration range bracketing that of the assay

samples.

Protein was assayed by the modified Lowry method, with bovine serum albumin as the
standard [6].

l“C-CO; determination. Following GC analysis, both P. stutzeri KC cultures and
controls were acidified with 300 pL of 6 N HCl (final pH of 2). N2 gas (100 mL) was
flushed through the headspace of the vials and transferred via teflon lines to 1 mL of 3N

131

KOH in 2 mL serum vials sealed with 11 mm teflon lined aluminum seals. N 2 gas (100
mL) was then flushed through the base trap to remove any residual CCl4. The ratio of
transfer gas to vial headspace was 10:1, and the ratio of stripping gas to headspace was
100:1 in the base trap. Base (100 pL) was added to 10 mL of scintillation cocktail
(Beckrnan). All samples were counted for 5 minutes on a Packard tri-carb liquid

scintillation counter (Model 1500).

Transformation capacity and long term growth characteristics. Corn oil was chosen
as the growth substrate in an experiment to determine the long term growth
characteristics and transformation capacity of oil grown cultures. One liter of SGW
medium (acetate free) was prepared at pH 8.2 with 2X phosphate buffer (27.22 g/L
1(1-12P04). The 2X phosphate concentration increased the buffering capacity in order to
prevent the strong alkaline conditions that frequently arise during growth under
denitrifying conditions. Ten milliliters of medium were dispensed into 20 mL automated
headspace sampler vials (Alltech), and 50 FL of corn oil was added to each vial. Vials
were degassed through the interlock of an anaerobic glove box and sealed under anoxic
conditions using teflon lined butyl rubber septa and aluminum crimp seals. Vials were
autoclaved at 121°C for 20 min. CCl4 was added from a sterile aqueous phase stock at a
final mass of 9.5 :1: 0.6 pg. P. stutzeri KC was added as a 1% inoculum (v/v) from a 72
hour culture grown in SGW medium with corn oil as the electron donor under
denitrifying conditions. The initial protein level for samples receiving P. stutzeri KC was
approximately 12 mg/L (6.9 x 107 cells/mL). The Schoolcraft samples received a 1%
inoculum (v/v) from an enrichment of aquifer solids as described in the organisms section
of materials and methods. Schoolcraft samples had an initial protein concentration of
approximately 0.9 mg/L. Samples labeled P. KC + Schoolcraf t received a 1% inoculum
(v/v) of each culture. The abiotic controls received CCl4 but no organisms. On days

indicated in the figures, 3 vials from each set were assayed for CCl4, protein, and nitrate.

132

CCl4 and chloroform were analyzed on a Perkin Elmer Auto System equipped with a PE
624 - 0.533 diameter - 50 meter column (PE No. N9312844) and an electron capture
detector with nitrogen carrier (30 psi) and helium make-up gas. The GC had a cycle time
of 5.0 minutes, an oven temperature of 80°C, and detector and injector temperatures of
250°C. The GC was connected to a Perkin Elmer Model HS-40 automated headspace
sampler. The autosampler had a thermostating temperature of 90 0C, a needle
temperature of 120 0C, a thermostating time of 12 minutes, a pressurization time of 2.0
seconds, and an injection time of 0.15 minutes. The headspace sampler had nitrogen
carrier gas with a pressure of 30 psi. External calibration curves were prepared by
addition of a primary standard (0.44 pg of CCl4 per pL of methanol and 0.2 pg of
chloroform per pL of methanol) to secondary aqueous solutions having the same
gas/water ratio, water/oil ratio, ionic strength, and incubation temperature as that of the
assay samples. An eight point calibration curve was prepared over a concentration range

bracketing that of the assay samples.

Aqueous phase samples were filtered through 0.22 pm nylon Titon® syringe filters and
diluted 1:10 prior to analysis for nitrate. Nitrate concentrations were determined by
capillary electrophoresis with indirect photometric detection (CE-IPD) using a model 270
capillary electrophoresis system (Applied Biosystems Inc.) equipped with a 72 cm x 50
pm capillary column micro-coated for anion analysis with a 2% ethylene glycol solution.
The detection electrode was anodic using 30 kV of applied voltage. The capillary
electrophoresis system was interfaced to a PC via a 900 Interface with data acquired
within Turbochrome (PE-Nelson) chromatography processing software. A five point
calibration curve was prepared over a concentration range bracketing that of the assay

samples and was analyzed by the same method as that of the test samples.

133

RESULTS

Initial screening of CCl4 transformation activity for P. stutzeri KC cultures grown
on vegetable oils. Transformation of CCl4 by P. stutzeri KC generates C02, a cell
associated product(s), and a non-volatile product(s) [2]. Table 6.1 illustrates that com,
soybean, and canola oil were all suitable growth substrates for P. stutzeri KC, with
growth yields similar to those observed in Medium D with acetate as the growth
substrate. An emulsifier, Tween 80, was not necessary to facilitate the growth of the
organism. All of the originally added CCl4 was transformed by the organism during the
72 hour growth period. l4C-C02 measurements showed that approximately 40 - 50% of
the original CCl4 was converted to C02. The C02 values for the samples containing
Tween 80 were approximately five times lower, suggesting that Tween 80 affects the

pathway of transformation.

134

Table 6.1. Growth of P. stutzeri KC on various vegetable oils and subsequent CCl4
transformation.

I T 4 ’ ProtemYleld ‘Mass CCl4 ‘
Sam le
_-

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Abiotic Control, Soybean 141123 2 1:0 7 0
Oil
Abiotic Control, Soybean 51:6 1 8:1:0.1 0
Oil and 0.01% Tween 80b
Abiotic Control, No Oil 11:5 1 7:03 0
Added
Abiotic Control, No Oil and 51:7 1 810.2 0
0.01% Tween 80
P. KC and Com 0“ 2644:17 0.01:0.0 l4,681:1:3,878
P. KC, Corn Oil and 0.01% 306:1:32 (11:00 39413559
Tween 80
P. KC and Soybean Oil 237119 0.0100 21,404¢4,751
P. KC, Soybean Oil and 260:1:1 0.21:0; 4977:1922
0.01% Tween 80
P. KC and Canola Oil 270115 0.0100 15,727:1:3,070
P. KC, caHOIa OI], and 228m 0.0i0.0 7 036%
0.01% Tween 80
a. Mass of CCl4 added was 2.0 pg

b. Tween 80 (Poloxethylenesorbitan Monooleate) was added as an emulsifier

c. CCl4 transformation was not monitored for the P. stutzeri KC that was grown in
Medium D.

d. 14c - ccra had a specific activity of 3.4 mCi/mmole

135

Transformation capacity and long term growth characteristics. Corn oil was
evaluated as a long term growth and energy substrate using both P. stutzeri KC and a
consortium of groundwater microorganisms obtained from an enrichment of aquifer
solids from a CCl4-contaminated aquifer in Schoolcraft, MI. Figure 6.1 illustrates the
cumulative mass of CCl4 transformed by cultures of P. stutzeri KC over 13 days. The
mass of CCl4 removed was equivalent to a concentration of 1.83 :t 0.08 mg/L. Fr gure 6.1
also indicated chloroform was produced by cultures of P. stutzeri KC grown on corn oil.
The final mass of chloroform produced was approximately 5% of the originally added
CCl4 mass. Figure 6.2 illustrates the mass of CCl4 removed by the Schoolcraf t
consortium. When compared to the abiotic loss of CCl4 (Figure 6.3), it cannot be
concluded that the Schoolcraft consortium achieved significant removal of CCl4. The
Schoolcraf t consortium removed 4.59 :t 0.75 pg of CCl4 while the abiotic losses were
4.13 :1: 0.61 pg of CCl4. Although difficult to observe on Figure 6.2, chloroform (0.08 :1:
0.04 pg) was observed in the Schoolcraf t consortium samples. Figure 6.4 shows the mass
of CCl4 removed and chloroform produced by a mixture of P. stutzeri KC and
Schoolcraft consortium. P. stutzeri KC was added to Schoolcraf t consortium at a ratio of
12:1 based on initial protein levels. The similarity of Figure 6.4 to Figure 6.1 indicates

that CCl4 removal and chloroform production were attributable to P. stutzeri KC.

 

136

Cummulative Mass CCl4 Added

J:

20w

 

 
    

15"

 

 

 

Cummulative Mass
of CCl4 Removed

 
  
  

 

Cummulati ve Mass
CHC13 Produced
as I

r

 

 

1 U I V T T T I I T I H V I I I I V I

0 3 6 9 12 15
Time (Days)

Figure 6.1. The cumulative mass of CCl4 removed and chloroform produced by P.
stutzeri KC. CCl4 was added initially and on day 3. Error bars for the mass of CCl4
added represent the cumulative error of addition. Error bars for experimental samples
represent a standard deviation of three independently grown cultures.

 

137

Cummulative Mass CCl4 Added

ir—H P i

201

 

15‘

 

ES
5.

   
  
    

Cummulati ve Mass
of CCl4 Removed

Ii
0 U V T Y I I "

 

‘ CHC13 Produced

6 9 12 15
Time (Days)

Figure 6.2. The cumulative mass of CCl4 removed and chloroform produced by an
enrichment of organisms from Schoolcraft aquifer solids. CCl4 was added initially and
on day 3. Error bars for the mass of CCl4 added represent the cumulative error of
addition. Error bars for experimental samples represent a standard deviation of three
independently grown cultures.

 

138

 

 

 

20 .. Cummulative Mass CCl4 Added
15 '

CT v .

(11910 g 4g

5 ‘ Cummulative Mass
of CCl4 Lost

 

 

 

Time(Days)

Figure 6.3. Abiotic loss of CCl4. Error bars represent the standard deviation of 3
independent control samples. CCl4 was added initially and on day 3. Error bars for the
mass of CCl4 added represent the cumulative error of addition. Error bars for
experimental samples represent a standard deviation of three independently grown
cultures.

 

139

 

 

 

 

 

20 _ Cummulative Mass CCl4 Added
15 ‘
« Cummulative Mass
CCl4 Removed
CI‘
(#8) 10 j '
5 -
Cummulative Mass
‘ CHC13 Produced
.. {r i i
()4 ..,...,.......,....
3 6 9 12 15
Time(Days)

Figure 6.4. The cumulative mass of CCl4 removed and chloroform produced by a mixture
of P. stutzeri KC and an enrichment of organisms from Schoolcraft aquifer solids. CCl4
was added initially and on day 3. Error bars for the mass of CCl4 added represent the
cumulative error of addition. Error bars for experimental samples represent a standard
deviation of three independently grown cultures.

Figure 6.5 shows the growth of P. stutzeri KC, Schoolcraf t consortium, and P. stutzeri
KC + Schoolcraft consortium on corn oil under denitrifying conditions. Figure 6.6
illustrates the concentrations of nitrate remaining in the three test conditions over the time
of the study. In viewing the two figures together, it becomes apparent that growth
stopped after the nitrate was consumed. On day 12, a final nitrate concentration of
approximately 62 mg/L was added to the samples, which may account for the late

increase in growth observed for the P. stutzeri KC + Schoolcraft samples. The final pH

 

140

of the samples did not exceed 8.42 :1: 0.11, and it should be noted that visible oil still

remained in the sample vials. Therefore, I concluded that nitrate was the growth limiting

 

 

factor in the experiment.
1000
3. KC + Schoolcraf t flora
g.”
100 ri KC
A
1:1
2) Schoolcraftfiora
V ;
.5 10
8
E
91
1
.1....,...,...,...,-...
0 3 6 . 9 12 15
Time(Days)

Figure 6.5. Growth of P. stutzeri KC, Schoolcraf t consortium, and P. stutzeri KC «1-
Schoolcraft consortium. Error bars represent the standard deviation of 3 independently
grown cultures.

 

141

15-

‘ Cummulative Mass of N03 Added ¥ }

10"

 

 

 

5 " Mass of N03
Remaining for
Schoolcraf t Consorti

 
  

Mass of N03 (mg)

 
 

5-4

   
  

Mass of N03
Remaining for
2. KC and_I_’. KC + Schoolcraft

0 r V I I V T I K V 1 I ‘I' V ? V V l I fii I

0 2 4 6 8 10 12 14
Time (Days)

 

 

 

 

Figure 6.6. Mass of N03 remaining in cultures of P. stutzeri KC, Schoolcraft consortia,
and P. stutzeri KC + Schoolcraf t consortia Error bars represent the standard deviation of
3 independent cultures.

DISCUSSION

The present work provides a favorable outlook for the application of vegetable oils as
growth and carbon sources for in situ bioremediation. Table 6.1 illustrates that P. stutzeri
KC is capable of growth on various vegetable oils and subsequent transformation of
CCl4. The addition of an emulsifier, Tween 80, was not required to aid the organisms is
breaking down the long chain fatty acids that comprise vegetable oils. A conversion of

40 -50% of the originally added CCl4 to l4C-C02 indicates that the transformation

142

pathway for oil grown cells is very similar to that seen for cells grown on acetate. The
lower values for C02 observed for cells grown in the presence of Tween 80 suggests that
the emulsifier alters the pathway of transformation. Lewis and Crawford [4] reported that
amount of organics present in the culture medium can greatly influence product
distribution, with high levels of organics lowering the percentage of CCl4 that is
converted to C02.

The cumulative mass of CCl4 removed by P. stutzeri KC (Figure 6.1) in the long term
growth experiments is very similar to the 24 hour transformation capacity reported by
Tatara et al [8] for P. stutzeri KC grown in medium D. As Figures 6.1 and 6.5 illustrate,
the cultures continued to transform CCl4 and grow over the 13 day time period, with rates
influenced by NO3' concentrations (Fr gure 6.6). These results indicate the advantage that
oil presents over acetate as a growth substrate for in situ bioremediation. Higher growth
rates are typically observed with acetate grown cultures. P. stutzeri KC will typically
reach stationary phase within 72 hours following inoculation in Medium D [8]. Using oil
as a growth substrate, organisms are limited by the solubility of oil in water, and by the

supply of electron acceptors (N03') and other essential nutrients.

To the best of my knowledge, this is only the second report of P. stutzeri KC producing
appreciable chloroform as a product of CCl4 transformation (Figures 6.1 and 6.4) Lewis
and Crawford [5], reported that approximately 5% of the originally added CCl4 was
converted to chloroform in a HEPES buffered medium under denitrifying growth
conditions. They also [4] reported that medium composition and levels of organics can
greatly influence end-products of CCl4 transformation and also influence the distribution
of these products. In this study, the mass of chloroform produced was also approximately
5% of the mass of CCl4 added, and the presence of oil may also have influenced this

product formation. Figure 6.7 illustrates the pathway for chloroform production in the

143

presence of lipids [Criddle, Ph. D. Thesis]. The trichloromethyl radical is capable of
abstracting hydrogen from lipids to form chloroform. Therefore, if a tri-chloromethyl
radical is produced during the transformation of CCl4 by P. stutzeri KC, then the
presence of oil may explain the production of low levels of chloroform. Alternatively,
since this is the first observed production of chloroform seen in our laboratory, method
changes must also be considered as well. It is possible that chloroform production was
not detected previously because I typiwa performed assays with much lower CCl4
concentrations. At CCl4 concentrations of 20 pg/L, the conversion of 5% of the initially
added CCl4 to chloroform would result in chloroform levels below detection limits
(method detection limit of 2 pg/L). The new GC-protocol developed for this experiment
has increased sensitivity to chloroform (method detection limit of 0.5 pg/L). In addition,
approximately 50-fold higher concentrations of chloroform were utilized in this set of
experiments, therefore chloroform was easily detected at the concentrations produced.
Despite the varying explanations for the observation of chloroform as an end-product of
CCl4 transformation, this work warrants further pursuit as the growth of P. stutzeri KC on

oil may provide insight on the pathway of CCl4 transformation.

 

 

 

 

 

 

 

cr\ 0 e- 0 3 — RHLipidR° or H
\
a a a / a o/ \a
carbon tetrachloride trichl-(Tomethyl—ra-dical

 

 

 

chloroform

Figure 6.7. Production of chloroform from the reaction of a trichloromethyl radical with
a lipid molecule. Adapted from Criddle, Ph.D. Thesis [1a].

The growth of P. stutzeri KC and transformation of CCl4 in the presence of
microorganisms enriched from aquifer material is also promising for field application
(Figure 6.4). Little or no transformation was observed for Schoolcraf t consortium (Figure

6.2 and 6.3) despite the fact that the consortium grew very well with oil as a growth

144

substrate (Figure 6.5). This indicates that P. stutzeri KC is able to successfully compete
with groundwater bacteria when KC is provided with an inoculum advantage and the pH

is optimum.

The use of vegetable oil as a growth substrate could enable P. stutzeri KC to transform
higher levels of CCl4 than previously reported. Concentrations of CCl4 higher than 5
mg/L can be toxic to P. stutzeri KC (data not shown). In an oil/water system, CCl4 will
tend to partition into the oil phase. This would tend to reduce aqueous phase
concentrations of CCl4. For a given total mass of CCl4 in the system, organisms
experience lower aqueous phase concentrations of CCl4. Rates of transformation would
therefore be dependent upon equilibrium chemistry and diffusion of secreted factor
and/or CCl4 out of the oil phase. These physical and chemical properties may enable
biological removal of CCl4 in subsurface environments where CCl4 concentrations were
previously considered excessive. However, delivery of vegetable oils to the subsurface
environment poses a major engineering challenge. The hydrophobic nature of oil and its
viscosity make it difficult to evenly disperse in the subsurface. Future studies need to be
conducted on methods of oil introduction. Column and model aquifer studies should be

performed to further asses the feasibility of oil as a long term growth substrate for in situ

bioremediation.

 

REFERENCES

1.Crlddle, C.S. 1993. The kinetics of cometabolism. Biotech. Bioeng. 41: 1048 - 1056.
1a. .Criddle, C.S. 1989. Ph.D. disseratation. Stanford Universtiy, Stanford, Calif.

2.. Criddle, C. S., J. T. DeWitt, D. Grbic-Galic, and P. L. McCarty. 1990.
Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under
denitrifieation conditions. Appl. Environ. Microbial. 56:3240-3246.

3. Fredrickson, et. al. 1993. Enhancement of in-situ microbial remediation of aquifers.
United States Patent. Patent Number: 5,265,674.

4. Lewis, T.A. and R.L. Craword. 1995. Transformation of Carbon tetrachloride via
sulfur and oxygen substitution by Pseudomonas sp. strain KC. J. Bact. 177: 2204-2208.

5. Lewis, T. A., and R. L. Crawford. 1993. Physiological factors affecting carbon
tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC.
Appl. Environ. Microbiol. 59:1635-1641.

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

7. Office of Emergency and Remedial Response. 1993. Evaluation of the likelihood of
DNAPL presence at NPL sites. EPA Report 540-R-93-073 PB93-963343)

8. Tatara, G.M., M.J. Dybas, and C.S. Criddle. 1993. Effects of medium and trace

metals on kinetics of carbon tetrachloride transformation by Pseudomonas sp. strain KC.
Appl. Environ. Microbial. 59:2126-2131.

145

 

CHAPTER 7

CONCLUSIONS AND FUTURE INVESTIGATIONS

146

147

The main goal at the outset of this study was to elucidate the biochemical components
and processes responsible for CCl4 degradation by P. stutzeri KC. Although several
questions remain unanswered at the conclusion of my work, I have made significant

progress in understanding many aspects of this interesting and complex system.

Preliminary studies performed on P. stutzeri KC focused on understanding how trace
metals affected the kinetics of CCl4 transformation. These studies were designed to
examine an initial hypothesis proposed by Criddle et. al [1991], which stated that an iron
scavenging system cometabolically transformed CCl4. My initial work provided support
for this hypothesis by determining that the cometabolic transformation of CCl4 was
dependent on the growth of P. stutzeri KC in iron limited and copper-containing
medium. These preliminary investigations also established that addition of ferric iron to

an actively-transfonning-culture inhibited CCl4 transformation.

Based on the hypothesis that the secreted factor was a siderophore, I hypothesized that
other cell-types will be able to transform CCl4 in the presence of the secreted factor.
This hypothesis was based on previously published iron uptake studies which
demonstrated that many species of microorganisms are able to use exogenously supplied
siderophores from other cell-types to obtain iron. The finding that P. fluorescens was
able to transform CCl4 in the presence of the secreted factor from P. stutzeri led to the
discovery that many diverse cell types, including gram positive and even eukaryotic
organisms are capable of rapid CCl4 transformation in the presence of the secreted factor.
The ability of other cells to transform CCl4 when combined with the secreted factor led to
the development of a bioassay which was critical in the purification processes, and also
provided a tool to perform many interesting physiological studies. Using the bioassay for
the secreted factor, I was able to determine that aerobically-grown P. stutzeri KC secretes

the factor, oxygen reversibly inhibits CCl4 transformation, live cells are required for

 

148

activation of the factor, the factor is readily transported through aquifer material, the
factor is stable indefinitely after lyophilization to powder, and the pH optimum for CCl4

transformation is approximately 8.5.

To further test the iron scavenging hypothesis, 1 subjected culture supernatant to
ultrafiltration, and combined the size fractionated supernatant with washed cells of P.
stutzeri KC. This process established that both extracellular and intracellular factors were
involved in the transformation. By themselves, washed cells of P. stutzeri KC did not
transform CCl4 to a significant degree. Occasionally, CCl4 transformation observed in
cell-free culture supernatant, but this activity was not reliable. Rapid and reliable CCl4
transformation was only obtained when washed whole cells were combined with culture
supernatant. Fractionation of culture supernatant established that the extracellular factor
is small with an apparent molecular weight of 500 daltons. I also established that the
inhibitory effects of iron are largely due to a supernatant factor with a molecular weight
greater than 10,000 daltons. This data conflicted with the siderophore hypothesis,
suggesting that elucidation of the normal physiological role of the CCl4 transformation
system might best be achieved by purification and identification of the secreted factor.
Although this goal was not fully achieved, significant progress was made toward
purification of the secreted factor. A procedure was developed following the discovery
that the secreted transforming factor is stable after lyophilization to dryness, and is
extractable with acetone. Additionally, a fraction containing transformation activity
eluted at 27 - 28 minutes from a semi-preparative reverse phase HPLC column at a
flowrate of 7 ml/min with a methanol/water gradient However, this peak of activity
contained several constituents as evaluated by mass spectrometry, thus further

purification is required.

 

149

To localize the cell dependent transformation activity, I performed crude cell extract
experiments with P. stutzeri. These studies established that CCl4 transformation required
a source of reducing equivalents (NADH), a cell membrane, and the secreted factor. This
finding led me to hypothesize that the secreted factor accepted reducing equivalents for
CCl4 transformation from a respiratory chain enzyme. To test this hypothesis, 1
performed respiratory chain inhibitor studies. The results of these studies did not support
my hypothesis, but did support the hypothesis that non-respiratory electron transport
proteins might be important This hypothesis was supported by an experiment in which
L. acidophilus, an organism lacking any membrane bound cytochromes or other electron
transfer enzymes did not significantly transform CCl4 when combined with the secreted

factor.

The above studies have aided in the development of engineering applications of P.
stutzeri KC. Studies on the effects of trace metals, pH, and kinetics of CCl4
transformation by P. stutzeri KC provided critical baseline information for a
bioaugmentation experiment conducted in a CCl4-contaminated aquifer in Schoolcraft,
MI. Additionally, studies using vegetable oil as a slow release growth substrate may led
to the development of novel technologies to more effectively remediate CCl4 and other

hazardous waste sites.

The attempt to determine the mechanism and pathway of CCl4 transformation through
elucidation of the biochemical components responsible for the transformation was not
successful. However, 1 established that the production of C02 and a non-volatile fraction
by P. stutzeri KC did not involve any interconversion of these products. This data refuted
a previous hypothesis which stated that the production of C02 resulted from the oxidation
of non-volatile product(s), and suggested instead that production of C02 and non-volatile
products occurs via parallel pathways. This data also supported the conclusion that

150

formate was not further oxidized to C02. Therefore, the most acceptable current model
would involve the production of phos gene and/or thiophos gene from a dichlorocarbene
radical as proposed in Chapter 1. However, at this point it is not yet possible to

discriminate between one- and two-electron reduction pathways to exist

Below, I provide a speculative model for CCl4 transformation by P. stutzeri KC, which is
consistent with the known features of this transformation. The model posits that CCl4
accepts electrons from a reduced form of the secreted factor produced during growth in
medium that contains copper and is iron limited. The oxidized form of the secreted factor
is capable of further transformations only after reduction at the cell membrane by a non-
respiratory enzyme. Additional data in support of such a model are as follows: 1) cell-
free transformation activity is occasionally observed, 2) changes in product distribution
based on changes in the buffer composition of the medium were observed by Lewis and
Crawford [1995], 3) low levels of cell-associated products are produced even though very
reactive intermediates such as phosgene and thiophosgene are produced [Lewis and
Crawford, 1995], 4) very low chloroform production is observed indicating that
intermediates are not reacting with membrane associated lipids, and 5) iron inhibition is

linked to a large molecular weight extracellular component

As an alternative model to that of Figure 7.1, it might be argued that the secreted factor
binds CCl4 for subsequent transformation at the cell membrane, possibly by a

f errisiderophore reductase. Such a model deserves consideration, but does not appear
consistent with the observation that cells of P. stutzeri strain EP3-071388 transform CCl4
when combined with the secreted factor following growth in iron excess conditions, nor
with the observation of occasional cell-free transformation activity. Furthermore, in

studies performed by Lewis and Crawford, it was demonstrated that medium composition

 

151

greatly affects product distribution, thus providing further evidence that the

transformation occurs extracellularly.

 

 

 

NADH NAD+
k Dehydrogenase?
5:323:39:-:3:-:-:-:.:.:-:-:-:-:3:1:1 ...... 444723233335:-:-:-:-:-:-:-:-:-?-I
13:3:3'3:3:3:3:3:3:323:3:3:3:323:3:3:32354085158815:Cémbbhshfifiii23:3
-I-IM9'PPF9P‘FZ-I-I~2-2-2-(0siidizeid).-I-I-I-' 2(Re'd'ucéd)I-I-I-I-I-I-I

 

 

 

Secreted Factor
0 Secreted Factor Reduced

/ Oxidized

 

[amine

 

Native.
Secreted factor production Physfifi'fig‘w
Iron limited ?
Cu- containing media
Dentrifying or Xenobiotic
Aerobic Growth Transformation Role
CO2 + Formate CCl
& 4
Non-Volatiles

Figure 7.1 Model of proposed CCl4 transformation process by P. stutzeri KC.

 

152

Future studies should focus on refining or refuting the model presented in Figure 7.1.

The stoichiomeuy of the CCl4 transformation reaction is needed to determine electron
flux through the reaction(s). Using the crude membrane system, the electron balance
should be determined for the oxidation of NADH, along with a chloride and carbon
balance. These studies would determine the electron flux in the reaction so that
conclusions can be drawn as to the flow of electrons in the reaction, as well as providing
information to aid in elucidating the mechanism(s) for the transformation. Additionally,
improvements in the purification protocol should be made by recombining fractions,
altering wavelengths of absorbance for HPLC, changing the buffer in the eluent to
possibly achieve better separation of the peaks, and possibly removing the filters from the
purification procedure. Genetic analysis should focus on testing the secreted factor with
f enisiderophore reductase deficient mutants to determine if this enzyme is the membrane
associated membrane responsible for transformation. These studies, may answer many of
the questions that remain unanswered and will further clarify the molecular basis of this

transformation.

 

 

 

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