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Characterization of PCB (Polychlorobiphenyl)
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CHARACTERIZATION OF PCB (POLYCHLOROBIPHENYL) DECHLORINATION
BY ANAEROBIC MICROORGANISMS FROM HUDSON RIVER SEDIMENTS

by

Dingyi Ye

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1994

ABSTRACT

CHARACTERIZATION OF PCB (POLYCHLOROBIPHENYDDECHLORINATION
BY ANAEROBIC MICROORGANISMS FROM HUDSON RIVER SEDIMENTS

By

Dingyi Ye

Investigation on the anaerobic microbial dechlorination of polychlorobiphenyls
(PCBs) was conducted to identify the dechlorinating physiological groups and to
characterize their dechlorination processes. The investigation was performed with
microorganisms eluted from the Hudson River sediments. Addition of specific
inhibitors of sulfate-reducers (molybdate) and methanogens (2-bromoethanesulfonic
acid, BESA, henceforth), as well as sulfate, inhibited both PCB dechlorination and
methane formation, suggesting that either sulfate-reducing bacteria or methanogenic
bacteria, or both, were involved in the dechlorination. The observation that cultures
obtained under highly selective pressure for methanogens dechlorinated PCBs, mainly at
the para position, provided more evidence that methanogenic bacteria are probably among
the physiological groups capable of anaerobic PCB dechlorination. Ortho dechlorination
of 2-CB and 2-2-CB/2,6-CB by the transferred methanogenic cultures was observed,
indicating that the Hudson River contains
' microorganisms with the potential for complete dechlorination of PCBs to biphenyl.
Inocula that were pasteurized or treated with ethanol or both, retained a meta-preferential

dechlorination activity. This dechlorination activity was stable over time and through

and through serial transfers. Neither methanogens nor thermophiles were responsible for
this retained dechlorination activity. The results indicated that microorganism responsible
for the dechlorination by the pasteurized cultures were probably anaerobic sporeformers.
Dechlorination by the pasteurized cultures was also inhibited by BESA, sulfate, and
molybdate. It appeared that microorganisms capable of dechlorination in the pasteurized
cultures were probably sulfidogens. Only sporeforming bacteria were expected to survive
the harsh pasteurization, and among known sulfidogens the only genus forming spores is
Desulfotomaculum. Therefore, the experimental results suggest that the dechlorinating
microorganisms in our pasteurized enrichment are probably Desulfotomaculwn-like
sulfate-reducing bacteria. Incubation temperature was also used in combination with
pasteurization to more effectively differentiate three different activities Aroclor 1242, one

that removes meta chlorines and two that remove para chlorines.

To my wife

iv

ACKNOWLEDGEMENTS

I wish to express my deep thanks to Dr. Stephen A. Boyd and Dr. James M. Tiedje
for the Research Assistantship, for their valuable guidance and the continuous
encouragement throughout my years of graduate study.

Deep thanks to members of my thesis committee, Dr. Michael J. Klug and Dr. Boyd
G. Ellis for their precious time, their valuable advice, discussions, and suggestions.

special thanks to Dr. John A. Breznak for his helpful discussion and suggestions,
and for his generously permitting me to use the HPLC in his lab to analyze the fatty
acids.

I thank Dr. John F. Quensen, HI for PCB data summation and the helpful
discussions.

I also wish to thank Ms. Linda Schimmelpfenning for her assistance in extracting
some of my PCB samples, and thank Mr. Sven Bohm for sharing his excellent techniques
on ion chromatography.

Many thanks to all people in Dr. Boyd’s lab and Dr. Tiedje’s lab for discussions,

friendship, and encouragement during my graduate study.

TABLE OF CONTENTS

General Introduction

General information about PCBs
Microbial destruction of PCBs
The objectives of this Study
Overview of the thesis

References

Chapter 1: Effects of molybdate. 2-bromoethanesulfonic acid (BESA),

and sulfate on anaerobic microbial dechlorination of
polychlorobiphenyis (PCB’s)

Abstract

Introduction

Materials and methods
Results

Discussion

References

Chapter 2: Evidence for dechlorination of polychlorobiphenyis (PCB’s) by

methanogenic bacteria
Abstract
Introduction
Materials and methods
Results
Discussion

References

vi

10

12

13

14

15

16

32

33

35

38

55

Chapter 3: Anaerobic dechlorination of polychlorobiphenyis (Aroclor 1242) by
pasteurized and ethanol-treated microorganisms from sediments

Abstract

Introduction

Materials and methods

Results

Discussion

References

Chapter 4: Anaerobic dechlorination of polychlorobiphenyis (PCB’s) by

pasteurized microorganisms as affected by molybdate,
2-bromoethanesulfonic acid (BESA), and sulfate

Abstract

Introduction

Materials and methods

Results

Discussion

References

Chapter 5: Differentiation of Three Classes of Congener-specific
Dechlorination Activity on Aroclor 1242

Abstract

Introduction

Materials and methods
Results

Discussion

References

vii

58

59

65

72

76

78

79

81

83

100

101

103

112

119

Appendix
I. Effects of more selective pasteurization treatments
11. Attempts to stimulate PCB dechlorination by spore germinants
III. Isolation and testing sporeformers
IV. Evaluation of PCB bioavailability
V. Influences of some carbon and energy amendments
on PCB dechlorination by microorganisms from
Hudson River sediments
References

viii

121

126

126

136

137

139

143

157

LIST OF TABLES
Chapter 2

Table 1. Effects of BESA on PCB dechlorination and methane production 49

Chapter 4
Table l. The possible interpretation for the results 92
Summary
Table l. The observed dechlorination activities in this study 125
Appendix
Table 1. Results of the dilution experiment (8 weeks) 133
Table 2. Results of the dilution experiment (6 months) 133
Table 3. Results of the isolation experiment 137
Table 4. Differences among the four amendment groups 150

LIST OF FIGURES

General Introduction

Fig. 1.

Chapter 1
Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Polychlorobiphenyls (PCB’s). (A) The general structure of
PCBs. (B) The numbering and the chlorine substituted position
in the biphenyl ring system.

Effects of molybdate, BESA, and sulfate on the on-set
dechlorination of Aroclor 1242 by microorganisms eluted from
Hudson River sediments.

The initial dechlorination of Aroclor 1242 by microorganisms
eluted from Hudson River sediments incubated with either
molybdate, sulfate, or BESA.

Effects of molybdate (20 mM, added at 0 time only) and sulfate
on dechlorination when the concentration of sulfate was
maintained at 3 ~ 5 mM. (A) dechlorination of Aroclor 1242 by
microorganisms eluted from Hudson River sediments with
different amendments after 8 weeks of incubation; (B)
concentrations of sulfate in the cultures amended with sulfate
plus molybdate, or sulfate only; (C) methane produced by
different amended groups; (D) methane produced by different
amended groups as percentages of the non-amendment group.

Mole percentage of Aroclor 1242 peaks before and after 8 weeks
of incubation with the following amendments: sulfate only
(maintained at 3 ~5 mM); 20 mM molybdate (added at 0 time);
and sulfate (maintained at 3 ~5 mM) plus molybdate (20 mM,
added at 0 time).

Effects of BESA at 50 M, 5, and 50 mM on dechlorination:
(A) dechlorination of Aroclor 1242 by microorganisms eluted
from Hudson River sediments as effected by BESA at different
concentrations; (B) methane produced by different amendments;
(C) methane produced by different amendments as percentages
of the non-amendment group.

Mole percentage of Aroclor 1242 peaks before and after 8 weeks
of incubation with 5 mM BESA.

17

19

21

Chapter 2

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Chapter 3

Fig.

Mole percentage of Aroclor 1242 represented by each chro-
matographic peak after 8 weeks incubation with the
methanogenic culture. (A) 0 time (B) 8 weeks (C) difference
between the 0 time and 8 weeks.

Molar ratios of several selected peaks to peak. 28 after 8 weeks
of incubation with different amendments.

Molar ratios of selected peaks to peak 28 (after 8 weeks
incubation with the methanogenic enrichment) as percentages of
those at 0 time (those at 0 time are defined as 100%).

Changes in Peak 15, 16, 36, and 37 after 8 weeks of incubation
with (A) the methanogenic culture, and (B) the negative control
group (with antibiotics, without the methanogenic substrates).
(C) the proposed dechlorination pathway for congener peak 36,
and 37.

Changes in mole percentage of Aroclor 1242 (represented by
each chromatographic peak) after 8 weeks incubation with the
heat- and ethanol-treated microorganisms from the Hudson River
sediments.

Effects of BESA on (A) dechlorination; and (B) methane
production; after 12 weeks of incubation.

Dechlorination by the initial cultures and the subsequent transfers
of both the methanogenic culture (with both methanogenic
substrates and antibiotics) and the group amended with
methanogenic substrates but without antibiotics. The
dechlorination activities are represented by accumulation of the
dechlorination products, peak 16 (normalized to peak 28), after
4 weeks incubation.

Mole percentage of Aroclor 1242 represented by each chro-
matographic peak after 4 weeks of incubation with the
transferred methanogenic cultures.

A brief flow diagram of the experiment procedure.

xi

39

41

42

47

Fig.

Fig.

Fig.

Fig.

Chapter 4

Fig.

Fig.

Chapter 5

Fig.

Fig.

Fig.

Dechlorination of Aroclor 1242 by heat and/ or ethanol treated
microorganisms eluted from the Hudson River sediments by
experiments performed in (A) September, and (B) May.

Mole percentage of Aroclor 1242 represented by each
chromatographic peak after 12 weeks incubation in the May
experiment with (A) Autoclaved (B) Treated (80°C, 15 min), and
(C) Untreated microorganisms eluted from the Hudson River
sediments. -

Homolog distribution of Aroclor 1242 after (A) 4 and (B) 12
weeks of incubation with treated (80°C, 15 min) and untreated
microorganisms (May experiment) eluted from the Hudson River
sediments.

Dechlorination of Aroclor 1242 by the cultures transferred from
the September experiment (1% transfer). (A) The second serial
culture (85°C, 15 min). (B) The third serial culture (90°C, 10
min).

Dechlorination of Aroclor 1242 by the pasteurized
microorganisms as affected by molybdate, sulfate, and BESA.
The microorganisms were eluted from Hudson River sediments
before the pasteurization.

Effects of molybdate, sulfate and BESA at different
concentrations on dechlorination by the pasteurized
microorganisms.

Dechlorination of Aroclor 1242 by (A) untreated, and (B)
pasteurized microorganisms eluted from Hudson River sediments
at different temperatures.

Methane production in the untreated cultures after 16 weeks of
incubation.

Temperature profiles of dechlorination of Aroclor 1242 by

microorganisms eluted from Hudson River sediments after 12
weeks incubation.

xii

67

70

71

104

105

106

Fig.

Fig.

Fig.

Fig.

Fig.

Appendix

Fig.

Fig.

Fig.

Fig.

Fig.

Mole percentage of Aroclor 1242 represented by each
chromatographic peak after 12 weeks incubation with the
untreated cultures at 30°C and 25°C.

Mole percentage of Aroclor 1242 represented by each
chromatographic peak after incubation at 30°C for 12 weeks with
untreated and pasteurized cultures.

Dechlorination of Aroclor 1242 by microorganisms from Hudson
River sediments.

Mole percentage of Aroclor 1242 represented by each
chromatographic peak after 12 weeks incubation at 25°C with
freshly prepared and aged Hudson River inoculum.

Mole percentage of Aroclor 1242 represented by each
chromatographic peak after 12 weeks incubation at 25°C with
original cultures and the transferred (25 %) cultures.

Dechlorination of Aroclor 1242 by microorganisms pasteurized
at 90°C for different time after (A) 4 and 8 weeks, and (B) 6
months of incubation.

Dechlorination of Aroclor 1242 by autoclaved microorganisms
eluted from the Hudson River sediments. The experimental
vessels were 160-ml serum bottles containing 25 g PCB-free
sediments and 75 ml RAMM with microorganisms. The serum
bottles were autoclaved at 121°C for 1 hour.

Dechlorination of Aroclor 1242 by microorganisms transferred
from the culture retaining the dechlorination activity after
autoclaving (see Fig. 2).

Results of dilution experiment after 4 weeks of incubation.
Error bars are the standard derivation (SD) of triplicate samples;
where not shown, the error bars are smaller than the symbols.

Dechlorination of aged (GFDS soil, experimental group B) and
freshly spiked (NBF soil) 2,3,4-3-CB and 2,3,4-4-CB by
microorganisms from Hudson River sediments after 16 weeks of
incubation. (A) normalized by 2,3,4-3,4-CB; (B) percentage
dechlorinated.

xiii

107

108

111

114

115

127

128

130

132

140

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Fig. 10.

Effects of different amendments on on-set dechlorination of
Aroclor 1242 by microorganisms from Hudson River sediments.

Influences of cellulose and glucose on dechlorination of Aroclor
1242 by the pasteurized microorganisms from Hudson River
sediments. The cellulose and glucose were added at 0 time and
4 weeks at a final concentration of 0.5 g/liter liquid.

Effects of methanol on dechlorination of Aroclor 1242 by (A)
non-preincubated and (B) preincubated cultures. The 0.5 %
amendments received methanol at 0 time and 4 weeks, while the
0.1% amendment only received methanol at 0 time.

Dechlorination of Aroclor 1242 by different experimental groups
of the preincubation experiment.

Mole percentage of Aroclor 1242 peaks after 12 weeks of
incubation with the preincubated and non-preincubated cultures.
The bottom panel shows differences in mole percentage between
preincubated and non-preincubated cultures after 12 weeks of
incubation. For peak assignments, please see reference 6.

xiv

143

145

151

152

153

GENERAL INTRODUCTION

Polychlorinated biphenyls (PCBs) were widely used in industry for almost 50 years
(1929-1978) (7), and during the 50-year period several hundred million pounds have been
released into the environment (10). Despite the fact that the production and application
of PCBs have been banned, their environmental fate is still a public concern because
PCBs are recalcitrant and ubiquitous environmental contaminants and have possible toxic
health effects (16).

In recent years microbially mediated anaerobic reductive dechlorination of PCBs
was observed in situ (5, 6, 7) and was later confirmed in the laboratory (14). This
process has also been reported by a number of laboratories with sediments from different
aquatic systems (for reviews see 1). However, despite the environmental significance
of anaerobic dechlorination of PCBs, research has, until now, failed to identify any
dechlorinating physiological group and to characterize their dechlorination processes.
Therefore, anaerobic dechlorination of PCBs by microorganisms from the Hudson River
sediments was investigated to tentatively identify the microbial physiological groups
capable of PCB dechlorination and to characterize the dechlorination processes performed

by the dechlorinating physiological groups.

General Information of PCBs
Structure and nomenclature. The polychlorobiphenyis (PCBs) are a family of

chlorine substituted biphenyls. The general structure, numbering in the biphenyl ring

Polychlorobiphenyls (PCB's)

Clx\/ \ / \/Cly (A)

209 congeners are theoretically possible
Half are actually produced

3 2 2' 3' in O o m
40- .4' “P a
5 6 .60 5' m 0 ° 0 m

p-para m-meta o-ortho

Numbering in the Biphenyl Ring System Substituted Position in the Biphenyl Ring System

Fig. 1. (A) The general structure of PCBs. (B) The numbering and the chlorine
substituted position in the biphenyl ring system.

3

system, and substituted position in the biphenyl ring system are shown in Fig. 1.

Chlorines can be attached to one ring or both, the number of the substituted
chlorines may vary from 1 to 10. There are 209 theoretically possible congeners,
however, only about half are actually produced (9).

Commercial PCBs were usually complex mixtures differing in the average
chlorination levels under different trade names such as ”Aroclor" .

Physical and chemical properties of PCB. The major physical properties of PCBs
are low water solubility, low vapor pressures, and excellent dielectric properties (9).
The major chemical properties of PCBs are flame resistant and low reactivity, such as
stable to oxidation and hydrolysis, and resistant to bases and acids (9).

Use of PCBs and the resulted environmental problems. Due to the relative
inertness, flame resistant, and the excellent dielectric properties, PCBs were widely used
in industry for almost 50 years. PCBs were mainly used as dielectric fluids (capacitors
and transformer), industrial fluids (hydraulic system, gas turbines, and vacuum pumps),
fire retardant, heat transfer applications, and plasticizers (adhesives, textiles, surface
coatings, sealants, printing, copy paper) et a1. (9).

it is estimated that during the 50—year period about several million pounds have
been released into the environment, and this has caused environmental problems because
PCBs have possible health effects, they are thermally and chemically stable, and the
lipophilic nature of these compounds enables them to accumulate in food chain, to

undergo bio-magnification, and to distribute worldwide.

4
Microbial Destruction of PCBs

Aerobic microbial degradation. Since PCBs are thermally and chemically stable,
therefore, their environmental fate mainly depends on microbial destruction. Intensive
studies on aerobic microbial degradation of PCBs have been reported (for review please
see reference 1). These studies include soil communities, pure strains, and recombinant
microorganisms. Although most soils contaminated with PCBs contain organisms with
some level of PCB-degrading ability (1), the aerobic degradation is usually limited to the
less chlorinated congeners, possibly because that higher chlorination levels may have
steric hindrance effect on 2,3-dioxygenation which is a general pathway in aerobic
degradation of PCBs (1).

Anaerobic reductive dechlorination. The anaerobic reductive dechlorination is a
process in which chlorine is replaced by hydrogen and the aromatic rings remain intact.
Unlike the aerobic degradation of PCBs which is generally limited to the less chlorinated
congeners, the anaerobic dechlorination of PCBs converts a large array of highly
chlorinated PCBs to lesser chlorinated congeners and the dechlorination products are
known substrates for aerobic microorganisms (1). Anaerobic reductive dechlorination
of PCBs removes chlorines from the biphenyl rings, hence reduces the toxicity of PCBs.
Moreover, the reductive dechlorination of PCBs is especially noteworthy for the ability
to transform highly chlorinated PCBs which are stable in the environment. The
anaerobic reductive dechlorination is actually a major initial step to destruct most highly
chlorinated PCBs. Without this initial destruction step, Those highly chlorinated
congeners would, otherwise, likely persist in the environment.

However, despite the environmental significance of the reductive dechlorination of

5
PCBs, until now, no physiological group of the dechlorinating microorganisms has been

identified.

The Objectives of This Study
The objectives of this study are: (1) to identify the physiological group(s) of the
dechlorinating microorganisms from the Hudson River sediments, and (2) to characterize

the dechlorination processes correlated to the defined physiological groups.

Overview of The Thesis

Effects of Molybdate, BESA, and Sulfate on Dechlorination of PCBs (Chapter
1). Both sulfate-reducing bacteria and methanogenic bacteria are major terminal electron
sinks in the anaerobic food chain, and play important roles in the degradation of organic
pollutants in anaerobic environments (for reviews, see references 3, 8, 9, 13). To clarify
the roles these two physiological groups play in the anaerobic dechlorination of PCBs,
specific inhibitors of sulfate reducers (molybdate) and methanogens (2-
bromoethanesulfonic acid, BESA henceforth) (2, 17) were used to probe the functions
of methanogens and sulfate-reducers in anaerobic dechlorination. Effects of sulfate on
the dechlorination were also investigated. Sulfate is not a specific inhibitor of any
microbial physiological group, however, addition of sulfate to sulfate-limited cultures
usually stimulates growth of sulfate-reducing bacteria and concomitantly inhibits
methanogenic bacteria (11, 12, 15). Therefore, sulfate may be used as both an inhibitor
of methanogens and a stimulator of sulfidogens. The experimental results showed that

addition of molybdate, BESA, and sulfate inhibited concomitantly PCB dechlorination

6

and methane formation, suggesting that either sulfate-reducing bacteria or methanogenic
bacteria, or both, were involved in PCB dechlorination.

Methanogen experiment (Chapter 2). Results presented in Chapter 1 suggested
that either sulfatereducing bacteria or methanogenic bacteria, or both, were involved in
PCB dechlorination. To further examine whether methanogens are capable of PCB
dechlorination and to obtain an in—depth of physiological and ecological understanding
about the microbial dechlorinating community, methanogen experiment was conducted.
The experimental results showed that methanogenic bacteria are probably among the
physiological groups capable of anaerobic dechlorination of PCB’s. Furthermore, ortho
dechlorination of 2-CB and 2-2-CB/2,6-CB by the transferred methanogenic cultures was
observed, indicating that the Hudson River contains microorganisms with the potential
for complete dechlorination of PCBs to biphenyl.

The heat- and ethanol-treatment experiments (Chapter 3). In one batch of the
inhibition experiments described in Chapter 1, one of the triplicated autoclaved (121°C,
1 h) cultures retained the dechlorination activity (Appendix Section 1). After the
contamination was carefully examined and ruled out, this unexpected result suggested that
the responsible microorganism(s) was(were) particularly heat resistant. Therefore, a
PCB-dechlorinating inoculum was treated with either heat or ethanol, or both, to
determine whether anaerobic sporeformers are capable of the PCB dechlorination. All
treated cultures retained a meta-preferential dechlorination activity. Methanogens were
not responsible for the retained dechlorination activity since no methane production was
detected in any treated culture. Thermophiles were also not responsible for the

dechlorination activity as evidenced by the fact that no dechlorination was observed at

7

37°C and above. These results indicate that anaerobic sporeformers are at least one of
the physiological groups responsible for the reductive dechlorination of PCBs.

Effects of molybdate, BESA, and sulfate on the dechlorination by the
pasteurized cultures (Chapter 4). Pasteurization eliminates methanogens which play an
important role in anaerobic microbial processes (4, 20), but a partial dechlorination
activity was still retained (19). Pasteurization also minimized the microbial diversity in
the system since only sporeforming bacteria maybe expected to survive the treatment.
Therefore, the effects of molybdate, BESA, and sulfate on PCB dechlorination by the
pasteurized cultures were investigated to examine the relationship between the sulfate-
reducer surviving the pasteurization and the retained dechlorination activity. BESA and
sulfate completely inhibited dechlorination at their lowest screened concentrations, 1 mM
and 2 mM, respectively. Molybdate partially inhibited the dechlorination at 2, 4, and
8 mM, and completely inhibited the dechlorination at 16 mM. It appeared that
microorganisms capable of anaerobic dechlorination of PCBs in the pasteurized cultures
were probably sulfidogens. Molybdate probably directly inhibited sulfidogens, while
both sulfate and BESA probably inhibited the dechlorination by competing with PCB as
electron acceptors. Only sporeformers were expected to survive the harsh pasteurization,
and among sulfidogens the only known genus forming spores is Desulfotomaculwn.
Therefore, the experimental results suggest that microorganisms responsible for the
dechlorination of PCBs in our pasteurized enrichment are probably Desulfotomacuium-
like sulfate-reducing bacteria.

Differentiation of congener-specific dechlorination activity on Aroclor 1242

(Chapter 5). In the pasteurization experiment (Chapter 3), it was necessary to identify

8

if thermophiles were responsible for the dechlorination activity retained in the pasteurized
culture. Therefore, effects of the incubation temperature were screened. Since
temperature is also a pressure to select the dominant population, combination of both
pasteurization and the incubation temperature will more effectively differentiate the
microbial dechlorinating community. The experimental results confirmed that the
dechlorination activity in the pasteurized cultures was not due to thermophiles. In
addition to this conclusion, since both pasteurization and incubation temperature are
selective pressures, combination of both pressures effectively differentiated the para and
meta dechlorination activities of the system and hence demonsn’ated and partially
characterized three classes of congener-specific meta and para dechlorination activity
present in the Hudson River sediments.

The relationships among the Chapters. Despite that five chapters report different
investigations on the anaerobic dechlorination of PCBs, these chapters are related each
other.

Chapter 1 reports the preliminary investigations, and chapter 2 is a further
investigation based on the conclusion of Chapter 1.

In one batch of the experiments described in Chapter 1, unexpected dechlorination
by an pasteurized culture was observed, and it was this unexpected result that prompted
the author to initiated the heat- and ethanol- treatment experiments summarized in
Chapter 3.

Results of Chapter 3 indicate that anaerobic sporeformers are probably among the
microorganisms capable of PCB dechlorination. However, it remained unclear which

microorganism(s) the dechlorinating sporefomer(s) might be. Chapter 4 reports the effort

9

to tentatively identify and characterize the dechlorinating microorganisms in the
pasteurized cultures.

As mentioned above, the original purpose of the experiment reported in Chapter
5 was to determine if thermophiles were responsible for the dechlorination by the
pasteurized cultures. The results did clearly answer this question. In addition, this
experiment successfully differentiated the para and meta dechlorination activities in the
system. As shown in Chapter 5, three classes of dechlorination activity were
demonstrated and partially characterized.

Besides the above relationships, the five chapters share common objectives: to
identify the physiological groups of the dechlorinating microorganisms, and to
characterize their dechlorination processes.

The appendix. The appendix consists of some experiments which are not included

in the five chapters.

10

References

1. Abramowicz, D. A. 1990. Aerobic and anaerobic biodegradation of PCBs: a
review. p.241-251. In G. G. Steward and 1. Russell (ed.), CRC Critical reviews in
biotechnology. Vol. 10(3), CRC Press. Inc., Boca Raton. Fla.

2. Balch, W. E., and R. S. Wolfe. 1979. Specificity and biological distribution of
coenzyme M (2-mercaptoethanesulfonic acid). J. Bacteriol. 137:256-263.

3. Berry, D. F., A. J. Francis, and J. -M. Bollag. 1987. Microbial metabolism of
homocyclic and heterocyclic aromatic compounds under anaerobic conditions.
Microbiol. Rev. 51: 43-59.

4. Bhatnagar, L., M. K. Jain, and J. G. Zeikus. 1991. p. 251-270. Methanogenic
bacteria. In Variations in autotrophic life. Academic Press, Orlando, FL.

5. Brown, J. F., R. E. Wagner, D. L. Bedard, M. J. Brennan, J. C. Carnahan,
R. J. May, and T. J. Toffiemire. 1984. PCB transformations in upper Hudson
sediments. Northeast. Environ. Sci. 3:167-179.

6. Brown, J. F., D. L. Bedard, M. J. Brennan, J. C. Carnahah, H. Feng, and R.
E. Wagner. 1987 . Polychlorinated biphenyl dechlorination in aquatic sediments.
Science 236:709-712.

7. Brown, J. F., R. E. Wagner, H. Feng, D. L. Bedard, M. J. Brennan, J. C.
Carnahah, and R. J. May. 1987. Environmental dechlorination of PCBs. Environ.
Toxicol. Chem. 6:579-593.

8. Evans, W. C., and G. Fuchs. 1988. Anaerobic degradation of aromatic
compounds. Ann. Rev. Microbiol. 42:289-371.

9.Hutzinger, 0., S. Safe, and V. Zitko. 1979. p. 1-8. In the Chemistry of PCB’s.
CRC Press Inc. Boca Raton, Fla.

10. Hutzinger, O. and W. Veerkamp. In T. Leisinger (ed.), Microbial degradation
of xenobiotics and recalcitrant compounds, Academic Press, New York.

11. Lovley, D. R., D. F. Dwyer, and M. J. Klug. 1982. Kinetic analysis
competition between sulfate reducers and methanogens for hydrogen in sediments.
Appl. Eviron. Microbiol. 43:1373-1379.

12. Lovley, D. R., and M. J. Klug. 1983. Sulfate reducers outcompete methanogens
at fresh water sulfate concentrations. Appl. Environ. Microbiol. 45: 187-192.

11

13. Mohn, W. W., and J. M. Tiedje. 1992. Microbial reductive dechlorination.
Microbial. Rev. 56:482-507.

14. Quensen, J. E, III, J. M. Tiedje, and S. A. Boyd. 1988. Reductive
dechlorination of polychlorinated biphenyls by anaerobic microorganisms from
sediments. Science 242:752-754.

15. Robinson, J. A. and J. M. Tiedje. 1984. Competition between sulfate-reducing
and methanogenic bacteria for H2 under resting and growing conditions. Arch.
Microbial. 137 :26-32

16. Safe, S. 1989. Polychlorinated biphynels (PCBs): mutagenicity and
carcinogenicity. Mutat. Res. 220:31-47.

17. Taylor, B. F., and R. S. Oremland. 1979. Depletion of adenosine triphosphate
in Desulfovibia by oxyanions of group VI elements. Curr.Microbial. 3:101-103

18. Widdel, F. 1988. Microbiology and Ecology of sulfate- and sulfur-reducing
bacteria. p. 469-585. In A. J. B. Zehnder (ed.), Biology of anaerobic
microorganisms. John Wiley & Sons, Inc. , New York.

19. Ye, D., J. F. Quensen III, J. M. Tiedje, and S. A. Boyd. 1992. Anaerobic
dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-
treated microorganisms from sediments. Appl. Environ. Microbiol. 58:1110-1114.

20. Zeikus, J. G.. 1977. The biology of methanogenic bacteria. Bacterial. Rev.
41:514-541.

Chapter 1

Effects of Molybdate, 2-Bromoethanesulfonic Acid (BESA),

and Sulfate on Anaerobic Microbial Dechlorination of Plychlorobiphenyls (PCB’s)

12

13

ABSTRACT

Addition of molybdate, 2-bromoethanesulfonic acid (BESA), and sulfate inhibited
concomitantly PCB dechlorination and methane formation by microorganisms from the
Hudson River sediments, suggesting that either sulfate-reducing bacteria or methanogenic
bacteria, or both, were involved in the dechlorination. The inhibitory effects depended
on experimental concentrations. BESA (50 mM) completely inhibited the on-set of
dechlorination after 2 weeks of incubation, while 50 pM had no effect. When
methanogensis was completely inhibited by 5 mM BESA, the cultures retained a partial
dechlorination activity different from that of pasteurized cultures, indicating that the
responsible microorganism(s) is(are) non-methanogenic and cannot survive pasteurization.
In the presence of 3 ~ 5 mM sulfate, partial dechlorination was observed. This activity
is noteworthy for its ability to attack 2,4-4-chlorobiphenyl (2,4—4—CB) and 2,4,6-2-CB.
This specificity of dechlorination, together with different dechlorination specificities
expressed by the BESA- and molybdate-amended cultures, as well as those observed with
our different enrichments and other reported dechlorination patterns, indicate that diverse
types of microorganisms in Hudson River sediments have the capacity to remove
chlorines from certain positions of specific PCB congeners. The presence or absence of
these active microorganisms may determine the actual PCB-dechlorination pattern

observed in-situ.

14
INTRODUCTION

Both sulfate-reducing bacteria and methanogenic bacteria are major terminal
electron sinks in the anaerobic food chain, and play important roles in the degradation
of organic pollutants in anaerobic environments such as ground water and sediments, etc.
(for reviews, see references 2, 5, 12, and 22). To investigate their activities in mixed
communities, specific inhibitors of these two physiological groups are widely used. These
inhibitors include molybdate which specifically inhibits sulfate-reducing bacteria, and 2-
bromoethanesulfonic acid (BESA) which specifically inhibits methanogenic bacteria. The
inhibitory properties of these reagents are based on unique physiological characteristics
of the bacteria (1, 21), and are purported to have little effect on other microorganisms
(14, 20). Thus, they may serve as powerful tools to probe the function of different
physiological groups in the microbial community (14).

Unlike molybdate and BESA, sulfate is not a specific inhibitor of any microbial
physiological group. However, addition of sulfate to sulfate-limited cultures usually
stimulates growth of sulfate-reducing bacteria and concomitantly inhibits methanogenic
bacteria, mainly because sulfate-reducing bacteria out compete methanogenic bacteria for
hydrogen or acetate, and also due to bioenergetics reasons (7, 8, 17). Therefore, sulfate
may be used as both an inhibitor of methanogens and a stimulator of sulfidogens.

In order to examine the role that methanogenic bacteria and sulfate-reducing
bacteria play in PCB dechlorination, effects of molybdate, BESA, and sulfate on
dechlorination were investigated. These studies are part of the efforts to characterize
anaerobic microbial PCB dechlorination activity observed in mixed communities present

in PCB contaminated sediments of the Hudson River (3, 15, 15a)

15

MATERIALS AND METHODS

Inoculum. The sediments for preparation of the inoculum were collected from the
upper Hudson River near Hudson River Falls, NY. (site H7 in reference 3).
Microorganisms were eluted from the sediments with the reduced anaerobic mineral
medium (RAMM) (18) as described elsewhere (23). Sediment collection, shipment, and
inoculum preparation were under strictly anaerobic conditions.

Preparation of the experimental batch. Both loo-ml or 60-ml serum bottles were
used for different experiments. The 60-ml serum bottles received 10 g of PCB-free
Hudson River sediments (collected from the Hudson River at River Miles 205 , refer to
reference 15) and 10 ml of RAMM containing 0.1% methanol and 10% inoculum, and
was then incubated at 37°C for 1 week. After methane was detected, the bottles were
autoclaved for 1 h on 3 consecutive days with incubation at 37°C between each
autoclaving. After the third autoclaving, 10 ml of inoculum, 10 ml of RAMM, and 80
pl of 10% PCBs (Aroclor 1242, Monsanto Co., St. Louis, Mo.) in acetone were added.
To evaluate the effects of inhibitors after on—set of dechlorination, similar experiments
were set up using 50 pl of 10% PCB in acetone and 25 g dry sediment per 160-ml serum
bottle. Stock solutions of molybdate, BESA, and sulfate were adjusted to pH 7 and
autoclaved. The controls were autoclaved twice, 1 h each time with a 5 h incubation at
37°C before PCBs were added. The samples were shaken for 1 h and then incubated at
25°C in dark. Preparation of the experimental batches with 160-ml serum bottles
followed the same procedures; the amounts of sediments, RAMM, PCB, and inoculum

were proportionally increased.

16
Analysis of sulfate, molybdate, and determination of methane production.

Sulfate and molybdate were analyzed with a high pressure ion chromatograph (Dionex)
equipped with a HPIC AS4A column (20 cm) and a conductivity detector. The mobile
phase was 1.7 mM NaHCO3/1.8 mM Na2CO3, and the flow rate was 2.3 ml/min.
Before being sampled, the culture bottles were shaken for 30 min, the headspace gas was
analyzed for methane production, and 2 ml of the liquid portion of the samples were
taken while being flushed with N2, acidified with 1 N HCl, and bubbled with N2 for 5
min to drive out H28. The samples were then centrifuged, filtered (0.22 pm, Millipore
Co. , Bedford, Mass), and analyzed for sulfate and molybdate.

Methane production was measured by gas chromatography using a flame ionization
detector.

PCB analysis and data summation. These procedures were as previously

described (15).

RESULTS

Effects of molybdate, BESA, and sulfate after the on-set of dechlorination.
After the on-set of dechlorination of Aroclor 1242 (200 pg/ g dry sediment) was
confirmed by GC analysis at 4 weeks, molybdate, BESA, and sulfate were introduced
at a final concentration of 5, 25, and 20 mM, respectively, and then added again at these
same concentrations 1, 2, and 3 weeks henceforth. PCB analysis showed that during 4
weeks of incubation no further dechlorination was observed in any culture including the
non-amended cultures, suggesting that those congeners susceptible to microbial

dechlorination probably had been dechlorinated to below a threshold concentration.

17

Aroclor 1242 Dechlorination
Inhibitor Effects

 

 

 

 

 

 

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Fig. 1. Effects of molybdate, BESA, and sulfate on the on-set dechlorination of Aroclor
1242 by microorganisms eluted from Hudson River sediments. Error bars are the

standard deviation (SD) of triplicate samples; where not shown, the error bars are smaller
than the symbols.

18
Therefore, 400 pg/ g sediment (dry weight) of Aroclor 1242 along with 5, 25, and 20

mM molybdate, BESA and sulfate were added, and that time was re-designated as the
0 time. The cultures were continuously incubated, and sampled at 1, 2, 3, 5, and 8
weeks. The cultures were re-amended with 5 mM of molybdate and 25 mM of BESA
at 1 and 2 weeks; and 20 mM of sulfate at 1, 2, and 3 weeks. As shown by Fig. 1,
dechlorination resumed in the non-amended cultures, whereas no dechlorination was
observed in those cultures amended with either molybdate, BESA, or sulfate. The non-
amended cultures produced methane, whereas no methane was detected in any amended
culture.

Effects of BESA, molybdate, and sulfate on initial dechlorination. In this
experiment BESA, molybdate and sulfate were added at the initiation of incubation. The
sulfate experiment was performed using a different batch of inoculum from that in the
molybdate and BESA experiments. The initial concentrations of molybdate and BESA
were 5 and 50 mM, respectively. The same amounts of molybdate and half the amount
of BESA added initially were re-fed at 2, 4, 6, and 8 weeks. The initial concentration
of sulfate was 20 mM, and the same amount was added again at 4 and 8 weeks. The
non-amended cultures produced methane, however, no methane production was detected
in any amended cultures, except one of the triplicated molybdate-amended cultures which
produced methane between 4 and 6 weeks (13 .6 % of that produced by the non-
amendment group, data not shown). After supplement of molybdate at 6 weeks, no
additional methane was detected in this culture.

BESA and sulfate completely inhibited dechlorination at the experimental

concentration used. Molybdate delayed and partially inhibited the dechlorination.

19

 

 

 

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Total Chlorines Per Biphenyl
i5

 

 

 

0 2 4 6 8 10 12

' ’ Incubation Time (Weeks)

Fig. 2. The initial dechlorination of Aroclor 1242 by microorganisms eluted from
Hudson River sediments incubated with either molybdate, sulfate, or BESA. Error bars
are the standard deviation (SD) of triplicate samples; where not shown, the error bars are
smaller than the symbols.

20
The color of the cultures amended with sulfate and with BESA turned dark black

after 2 weeks of incubation, and these cultures gave off a strong sulfide-smell. In
contrast to the dark black color of the sulfate- and BESA-amended cultures, the
molybdate-amended cultures turned somewhat yellowish after 2 weeks of incubation and
there was no sulfide-like odor.

Inhibition of dechlorination at .low sulfate concentrations. In the experiments
shown in both Fig. 1 and Fig. 2, dechlorination was completely inhibited by the 20 mM
sulfate amendments. Therefore, an experiment with a lower concentration of sulfate was
conducted. In this experiment, the initial concentration of sulfate was 5 mM, and we
attempted to maintain this concentration during the entire incubation period. In another
amendment group, 20 mM molybdate was added (at 0 time only) with 5 mM sulfate to
ensure the presence of sulfate in the absence of sulfate reduction. As a control 20 mM
molybdate was also added alone at 0 time. Because of the instrumental problems, both
3 and 4 week sulfate samples were not assayed until 5 weeks, and consequently, the
concentration of sulfate was maintained between 3 ~ 5 mM.

As shown by Fig. 3A, dechlorination was significantly inhibited by all three
amendments. After 8 weeks of incubation, the non-amended culture removed 0.96
chlorines from each biphenyl molecule; in contrast, only 0.31, 0.25, and 0.24 chlorines
were removed from each biphenyl molecule by the cultures amended with molybdate,
sulfate, and molybdate plus sulfate, respectively.

Inhibition by 5 mM sulfate was not reversed in the presence of molybdate since
molybdate at the experimental concentration also inhibited the dechlorination. However,

molybdate did inhibit sulfate consumption (Fig. 3B). After 2 weeks of incubation, the

21

 

 

 
  
   
   

     

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

     
   

 

 

 

 

 

 

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Fig. 3. Effects of molybdate (20 mM, added at 0 time only) and sulfate on dechlorination
when the concentration of sulfate was maintained at 3 ~ 5 mM: (A) dechlorination of
Aroclor 1242 by microorganisms eluted from Hudson River sediments with different
amendments after 8 weeks of incubation; (B) concentrations of sulfate in the cultures
amended with sulfate plus molybdate, or sulfate only; (C) methane produced by different
amended groups; (D) methane produced by different amended groups as percentages of
the non-amendment group. The hollow arrows indicate supplement of sulfate. Error
bars are the standard deviation (SD) of triplicate samples; where not shown, the error
bars are smaller than the symbols, except for Fig. (D).

22

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Fig. 4. Male percentage of Aroclor 1242 peeks before and after 8 weeks of incubation
with the following amendments: sulfate only (maintained at 3 ~5 mM); 20 mM
molybdate (added at 0 time); and sulfate (maintained at 3 ~ 5 mM) plus molybdate (20
mM, added at 0 time). For peek assignments, see reference (15).

23

concentration of sulfate decreased 0.9 mM in the cultures amended only with sulfate,
while in presence of 20 mM molybdate the concentration of sulfate only decreased 0.06
mM. HPIC analysis showed that after 2 weeks of incubation the concentration of
molybdate decreased 1.0 and 1.8 mM in the with- and without- sulfate cultures,
respectively.

Both sulfate and molybdate inhibited methane production. The inhibitory effect of
20 mM molybdate on methanogensis was stronger than that of 5 mM sulfate (Fig. 3C,
3D). As shown in Fig. 3D, sulfate resulted in suppression of methane production, except
that supplement of sulfate to the cultures amended with sulfate plus molybdate at 5 weeks
(3B) failed to inhibit methane production (3D). Methane production in this amendment
group increased between 5 and 6 weeks until sulfate was added at 6 weeks (3D).
Hydrogen in the headspace was also monitored with gas chromatography; no hydrogen
cumulation was found in any culture (data not shown). Fig. 4 shows the dechlorination
patterns by the three amended cultures. The sulfate-amended culture is noteworthy for
its ability to attack peak 18, which represents 2,4—4-chlorobiphenyl (2,4-4-CB) and 2,4,6-
2-CB (mainly 2,4—4—CB).

Effects of BESA at different concentrations. Cultures were amended with 50 pM,
5 mM, and 50 mM (25 mM at 0 time and 2 weeks) BESA added at 0 time only. A 2
week lag time was observed in the 5 and 50 mM amendments (Fig. 5A). The 50 p.M
amendment had no effect. The 50 mM BESA treatment continued to inhibit
dechlorination during the 12 week incubation, and the 5 mM BESA delayed and partially
inhibited the dechlorination (Fig. 5A). Methane formation was partially inhibited by 50

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of Aroclor 1242 by microorganisms eluted from Hudson River sediments as effected by
BESA at different concentrations; (B) methane produced by different amendments; (C)
methane produced by different amendments as percentages of the non-amendment group.
Error bars are the standard deviation (SD) of triplicate samples; where not shown, the
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25

 

 

 

 

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26
after 3 weeks. Methane formation was also partially inhibited by 50 [TM BESA between

2 and 8 weeks (Fig. SB, 5C). The dechlorination pattern of the cultures amended with
5 mM BESA (Fig. 6) showed that this activity includes some dechlorination activity
eliminated by pasteurization (23). The dechlorination pattern after 8 weeks, instead of
12 weeks, was plotted to facilitate comparison of this activity with those of sulfate and

molybdate amended cultures (F ig. 4).

DISCUSSION

BESA, and sulfate completely inhibited PCB dechlorination in two separate
experiments where Aroclor 1242 and the inhibitors were added together with the
inoculum and where they were added to a culture which had already dechlorinated added
PCBs. In the former case, molybdate delayed and reduced the extent of initial
dechlorination, and in the latter case completely inhibited dechlorination. The different
effects of molybdate on these two dechlorination experiments may be due to
concentration effects. When molybdate was added along with Aroclor 1242 (400 ppm)
to cultures that had already dechlorinated 200 ppm Aroclor 1242 (Fig. l), a total amount
of 25 mM of molybdate had been added. The cultures were continuously incubated with
further supplements of molybdate (5 mM) at l and 2 weeks. Therefore, after 8 weeks,
35 mM of molybdate had been added. However, in the other dechlorination experiment
(Fig. 2), at 8 weeks only 20 mM of molybdate had been added (5 mM added at 0 time
and 2, 4, 6 weeks). '

The results suggest that either sulfate-reducers or methanogens, or both, were

involved in dechlorination. In these experiments, methanogensis and dechlorination were

27
simultaneously inhibited. Inhibition of methanogensis by BESA and sulfate was expected

since BESA is a specific inhibitor of methanogens, and sulfate should stimulate sulfate-
reducers and hence inhibit methanogens. Molybdate is generally regarded as a specific
inhibitor of sulfidogens (21 , 14) and thus is widely used in microbiology studies due to
its specificity (14). However, inhibition of methanogensis by higher concentration of
molybdate has been reported (9, l9) and also was observed in our experiments. All three
anions also affected sulfate-reducers. As mentioned above, molybdate inhibited sulfate-
reducers while sulfate stimulated sulfate reduction. The sulfonic acid moiety of BESA
is a potential electron acceptor for sulfate-reducers (14). In our experiments the dark-
black color, indicating the formation of metal sulfide, and the strong sulfide-smell of the
BESA-amended cultures support this suggestion. Since molybdate, BESA, and sulfate
affected both sulfate reducers and methanogens, and concomitantly inhibited
dechlorination, the results suggest that either sulfate-reducers or methanogens, or both
were involved in the dechlorination.

Inhibition of sulfate reducers by molybdate was evidenced by the following
observations: (i) in presence of molybdate inhibition of sulfate consumption was observed
(Fig. 3B). (ii) Compared to the non-amended cultures, the black color of sulfate-
amended cultures indicated formation of metal sulfide, while the yellowish color of the
molybdate-amended cultures probably suggested that reduction of sulfate was more or
less inhibited. (iii) As shown in Fig. 3D, supplement of sulfate at 5 weeks suppressed
methane production in the cultures amended only with sulfate, but not the cultures
amended with sulfate plus molybdate, indicating that sulfate-reducers were less active in

presence of molybdate.

28

Previously we have demonstrated that methanogens or methanogensis are not
required for PCB dechlorination (23). Results reported here present another example that
some non-methanogenic microorganisms which cannot survive the pasteurization are also
capable of PCB dechlorination. When methanogensis was completely inhibited by 5 mM
BESA after 3 weeks of incubation, partial dechlorination occurred in these cultures (Fig.
5A). The dechlorination activity (Fig 6) was different from that of pasteurized cultures,
indicating that the responsible microorganism(s) is(are) non-methanogenic and cannot
survive pasteurization. May et al. (11) also reported that dechlorination by a non-
methanogenic culture produced a dechlorination pattern different from that produced by I
the pasteurized cultures. Comparison between the dechlorination patterns reported by
May et a1. and that shown here for the 5 mM BESA-amended cultures suggests that these
two dechlorination activities are different. For example, the culture reported by May et
al. only removed para chlorines from congeners containing adjacent para and meta
chlorines (l 1). In contrast, the 5 mM BESA-amended cultures were able to dechlorinate
25-2-CB and 4-4—CB (peak 9). These results demonstrated that among the
microorganisms eliminated by pasteurization, there are at least two different non-
methanogenic populations capable of PCB dechlorination.

Our results demonstrate that PCB dechlorination can occur in the presence of 3 ~ 5
mM sulfate. Inhibition of dehalogenation of many organic compounds (12) including
PCBs (10, 13, 16) by sulfate have been reported in many investigations. Morris et al.
(13) found that addition of 10 mM sulfate reduced the dechlorination activity by 50 %
compared with C02 treatment. Rhee et al. (16) reported that dechlorination of PCB was

inhibited partly by 10 mM sulfate, and May et al. (10) observed that a culture enriched

29
with 20 mM sulfate expressed no dechlorination activity until the sulfate was depleted

(9 weeks). In contrast, Hiiggblom and Young (6) reported chlorophenol degradation
coupled to sulfate reduction, with reductive dechlorination seeming to be the initial step.
May et al. (1 1) also reported transfers from a sulfate-amended enrichment dechlorinated
2,3,4—CB after the addition of sulfate, and the activity was maintained for 15 weeks in
presence of > 20 mM sulfate. Our observation that partial dechlorination occurred in
the presence of sulfate is in agreement with previous reports (11) that sulfate inhibits
PCB dechlorination by microorganisms from Hudson River sediments, and also in
agreement with the conclusion by May et al. (10) that despite the inhibitory effects of
sulfate on dechlorination, different populations with ability to dechlorinate PCBs in
presence of sulfate can be obtained.

The dechlorination activity of sulfate-amended cultures observed here is different
from that observed by May et al.. This, together with different dechlorination activities
expressed by the BESA- and molybdate-amended cultures, as well as those observed with
our pasteurized cultures (23), methanogenic enrichment (Chapter 2), and other reported
dechlorination patterns (4), indicate that diverse microorganisms in Hudson River
sediments have the capacity to remove chlorines from certain positions of specific PCB
congeners. The presence or absence of these active microorganisms may determine the

actual PCB-dechlorination pattern observed in-situ.

30

REFERENCES

l. Balch, W. E., and R. S. Wolfe. 1979. Specificity and biological distribution of
coenzyme M (2-mercaptoethanesulfonic acid). J. Bacteriol. 137:256-263.

2. Berry, D. F., A. J. fiancis, and J. -M. Bollag. 1987. Microbial metabolism of
homocyclic and heterocyclic aromatic compounds under anaerobic conditions. Microbiol.
Rev. 51: 43-59.

3. Brown, J. F., D. L. Bedard, M. J. Brennan, J. C. Camahan, H. Feng, and R.
E. Wagner. 1987. Polychlorinated biphenyl dechlorination in aquatic sediments. Science
236:709—712.

4. Brown, J. F., R. E. Wagner, D. L. Bedard, M. J. Brennan, J. C. Carnahan, R.
J. May, and T. J. Tofflemire. 1984. PCB transformations in upper Hudson sediments.
Northeast. Environ. Sci. 3:167~179.

5. Evans, W. C., and G. Fuchs. 1988. Anaerobic degradation of aromatic compounds.
Ann. Rev. Microbiol. 42:289-371.

6. Hfiggblom, M. and L.Y. Young. 1990. Chlorophenol degradation coupled to sulfate
reduction. Appl. Env. Microbiol. 56:3255-3260.

7. Lovley, D. R., D. F. Dwyer, and M. J. Klug. 1982. Kinetic analysis competition
between sulfate reducers and methanogens for hydrogen in sediments. Appl. Eviron.
Microbiol. 43:1373-1379.

8. Lovley, D. R., and M. J. Klug. 1983. Sulfate reducers outcompete methanogens at
fresh water sulfate concentrations. Appl. Environ. Microbiol. 45: 187-192.

9. Madsen, T., and J. Aamand. 1991. Effects of sulfuroxy anions on degradation of
pentachlorophenol by methanogenic enrichment culture. Appl. Env. Microbiol. 57:
2453-2458.

10. May, H. D., A. W. Boyle, W. A. Price, 11, and C. K. Blake. 1992.
Dechlorination of PCBs with Hudson River bacterial cultures under anaerobic conditions:
effect of sulfate, sulfite and nitrate. 1992. Annu. Meet. Am. Soc. Microbiol.

11. May, H. D., A. W. Boyle, W. A. Price 11, and C. K. Blake. 1992. Subculturing
of a polychlorinated biphenyl-dechlorinating anaerobic enrichment on solid media. Appl.
Environ. Microbiol. 58:4051-4054.

12. Mohn, W. W., and J. M. Tiedje. 1992. Microbial reductive dechlorination.
Microbiol. Rev. 56:482-507.

31

13. Morris, P. J., W. W. Mohn, J. F. Quensen III, J. M. Tiedje, and S. A. Boyd.
1992. Establishment of a polychlorinated biphenyl-degrading enrichment culture with
predominantly meta dechlorination. Appl. Environ. Microbiol. 58:3088-3094.

l4. Oremland, R. S., and D. G. Capone. 1988. Use of "specific" inhibitor in
biogeochemistry and microbial ecology. In K. C. Marshall (ed.), Advances in microbial
ecology. vol. 10. Plenum Publishing Co.

15. Quensen, J. F., III, S. A. Boyd, and J. M. Tiedje. 1990. Dechlorination of four
commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic microorganisms
from sediments. Appl. Environ. Microbiol. 56:2360-2369.

15a. Quensen, J. F., 111, J. M. Tiedje, and S. A. Boyd. 1988. Reductive
dechlorination of polychlorinated biphenyls by anaerobic microorganisms from sediments.
Science 242:752-754.

16. Rhw, Go'Yo, Bo BUSh, Co Mo BCthony, A. DeNllCCi, Ho'Me Oh, and Re C0 SOROI.
1993. Anaerobic dechlorination of Aroclor 1242 as affected by some environmental
conditions. Environ. Toxicol. Chem. 12:1033-1039.

17. Robinson, J. A. and J. M. Tiedje. 1984. Competition between sulfate-reducing and
methanogenic bacteria for H2 under resting and growing conditions. Arch. Microbiol.
137:26-32

18. Shelton, D. R., and J. M. Tiedje. 1984. General method for determining anaerobic
biodegradation potential. Appl. Environ. Microbiol. 47:850-857.

19. Smith, L. R. and M. J. Klug. 1981. Electron domors utilized by sulfate-reducing
bacteria in eutrophic lake sediments. Appl. Environ. Microbiol. 42:116-121.

20. Sparling, R. and Daniels L. 1987. The specificity of growth inhibition of
methanogenic bacteria by bromoethanesulfonate. Can. J. Microbiol. 33:1132-1136.

21. Taylor, B. F., and R. S. Oremland. 1979. Depletion of adenosine triphosphate in
Desulfovibio by oxyanions of group VI elements. Curr.Microbiol. 3: 101-103

22. Widdel, F. 1988. Micribiology and Ecology of sulfate- and sulfur-reducing bacteria.
p. 469-585. In A. J . B. Zehnder (ed.), Biology of anaerobic microorganisms. John Wiley
& Sons, Inc., New York.

23. Ye, D., J. F. Quensen 111, J. M. Tiedje, and S. A. Boyd. 1992. Anaerobic
dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-treated
microorganisms from sediments. Appl. Environ. Microbiol. 58:1110-1114.

Chapter 2

Evidence for Dechlorination of Polychlorobiphenyls (PCB’s)

by Methanogenic Bacteria

32

33

ABSTRACT

When microorganisms eluted from the upper Hudson River (HR) sediment were
cultured without any substrate except PCB-free HR sediment, methane formation was the
terminal step of the anaerobic food chain. In this food chain, fermentative bacteria
converted organic matter into substrates for methanogenic bacteria. In sediments
containing Aroclor 1242, addition of eubacteria—inhibiting antibiotics, which should
directly inhibit fermentative bacteria and thereby indirectly inhibit methanogens, resulted
in no dechlorination activity nor methane production. However, when substrates for
methanogenic bacteria were provided along with the antibiotics (to free methanogens
from dependence on eubacteria), concomitant methane production and dechlorination of
PCB’s were observed. The dechlorination of Aroclor 1242 was distinctly different from,
and more limited than, that observed with untreated or pasteurized inocula. Both
methane production and dechlorination in the culture amended with antibiotics plus
methanogenic substrates were inhibited by BESA, the specific inhibitor for methanogenic
bacteria. These results suggest that methanogenic bacteria are probably among the
physiological groups capable of anaerobic dechlorination of PCB’s. Ortha dechlorination
of 2-CB and 2-2-CB/2,6-CB by the transferred methanogenic cultures was observed,
indicating that the Hudson River contains microorganisms with the potential for complete

dechlorination of PCBs to biphenyl.

34
INTRODUCTION

Methanogenic bacteria play an important role in anaerobic destruction of organic
compounds (4, 13). There are many observations of reductive dechlorination of
chlorinated compounds either under methanogenic conditions (15 , 16, 25), or by pure
methanogenic strains (14, 18, 20).

In PCB dechlorination experiments when dechlorination of PCB occurred, methane
production was always concomitantly observed (21, 22, 27). However, these studies
could not distinguish whether methanogenic bacteria were among the microorganisms
capable of PCB dechlorination or they functioned as the terminal step to maintain a
continuing energy flow through the food chain, so that the dechlorination could proceed.
Heat- and ethanol-treatments of mixed PCB dechlorinating populations eliminated non-
sporeforming bacteria including methanogens, but a meta-preferential dechlorination
activity was maintained (27), suggesting that involvement of methanogenic bacteria was
not necessary for dechlorination. However, heat- and ethanol-treatments resulted in loss
of para dechlorination activity perhaps due to elimination of some microorganisms.
Methanogenic bacteria were among the microorganisms eliminated by the heat- and
ethanol- treatments, however, the relationship between methanogens and the activities lost
from the heat- and ethanol-treatments was unknown. It is important to identify the role
methanogenic bacteria play in anaerobic reductive dechlorination of PCBs, so that an in-
depth physiological and ecological understanding of the microbial community involved
in anaerobic PCB dechlorination can be obtained. This understanding will improve PCB
bioremediation strategies, and facilitate isolation of microorganisms capable of PCB

dechlorination.

35
MATERIALS AND METHODS

Approaches. Methanogenic bacteria depend on fermentative bacteria for
substrates. Addition of eubacteria-inhibiting antibiotics (penicillin-G plus D-cycloserine)
will directly inhibit eubacteria and thus, indirectly inhibit methanogens. However, when
substrates for methanogenic bacteria are provided along with the antibiotics to free
methanogens from dependence on eubacteria, methanogens should be enriched. There
are three kinds of archaebacteria: extreme halophiles, extreme thermophiles, and
methanogens. Only methanogens can be enriched if the cultural conditions are not
favorable for the other two groups. Therefore, if such a culture has any PCB
dechlorination capacity, the responsible microorganisms are most likely methanogenic
bacteria. Insight into the role methanogens play in the dechlorination can also be
examined determining the effects of 2-bromoethanesulfonic acid (BESA), the specific
inhibitor for methanogens (2), on PCB dechlorination by the methanogenic culture. The
observation that BESA inhibits both dechlorination activity and methane production in
the methanogenic culture, but does not inhibit dechlorination activity in the untreated
culture would support the dechlorination capacity of methanogens.

Inoculum preparation. Inoculum was prepared with sediments collected from the
upper Hudson River near Hudson River Falls, N. Y. , and the inoculum preparation
procedure was as previously described (23).

Preparation of experimental vessels. The medium for this experiment was
Barker’s medium (19) which is generally used for enrichment of methanogenic bacteria.

The medium was modified with addition of N aHCO3 (2.4 g/liter) and Na2S.9H20 (0.28

g/liter)

36
For the initial methanogenic enrichments and subsequent transfers, l60-ml serum

bottles were used. A preincubation procedure (23) was followed to ensure that the
experimental vessels strictly anaerobic. The preincubated l60-ml serum bottles contained
25 g PCB-free air-dried Hudson River sediments, 15 ml of Barker’s medium (19)
containing 5 % of inoculum and 0.1% of ethanol. After formation of methane was
detected, all vessels were then autoclaved at 121°C for l h on 3 consecutive days with
incubation at 37°C between each autoclaving.

After the third autoclaving, the following were added to each bottle while the bottle
was being flushed with filter-sterilized 02-free N2/C02 (80:20, vol/vol) with a Hungate
apparatus: 25 ml of Barker’s medium, 25 ml of inoculum, 25 ml of sediment extract, 100
[1.1 of 10% (wt/wt) Aroclor 1242 in acetone. Sediment extract was prepared by
autoclaving PCB-free Hudson River sediment with deionized water (1:1, wtzvol) for l
h, the mixture was then shaken for another hour, centrifuged and filtered. The final
concentration of PCB’s was 400 ug/ g of dry sediment. Besides the reductant (sodium
sulfide) in the Barker’s medium titanium citrate (28) was also added at a final
concentration of 0.2 mM. Penicillin-G (Sigma Co., St. Louis, Mo.) and D-cycloserine
(Sigma Co. , St. Louis, M0.) were made as stock solutions, bubbled with 02-free N2/C02
(80:20, vol/vol) to drive out the dissolved oxygen, and then filter-sterilized (0.22 pm,
Millipore, St. Louis, Mo.). The final concentration of Penicillin-G and D-cycloserine
was 3340 U/ml, and 0.1 g/liter of liquid, respectively. The methanogenic substrates
provided were methanol, formate, and acetate at final concentrations of 7 g each per liter
of liquid. These substrates were autoclaved. The bottles were then sealed with Teflon-

coated butyl rubber stoppers and aluminum crimps, and shaken thoroughly to completely

37

disperse the PCB’s. The controls were autoclaved twice at 121°C for l h, with an
interval of 5 h before PCB’s were added.

For the BESA experiment, the same preparation procedure described above was
followed with the following modifications: the experimental vessels were 60 ml serum
bottles containing 10 g PCB-free dry sediments. The liquid portion was proportionally
reduced based on the weight of sediment. Antibiotics were added at 0 time at the
concentration described above and at 8 weeks at half that concentration. The PCB used
was 3 ,4-2-CB, the methanogenic substrate provided was methanol only (7 g/ liter of
liquid). BESA (Aldrich Inc. , Milwaukee, WI) was filter-sterilized and added at a final
concentration of 2 mM; the same concentration of BESA was supplemented at 3 and 6
weeks.

Transfer. Cultures to be transferred were shaken for 30 min and the slurry was
then allowed to settle for about 30 min. Supernatant (25 ml) was transferred into each
l60-ml serum bottle containing 25 ml of Barker’s medium, 25 ml of sediment extract,
and 25 g of preincubated PCB-free Hudson River sediments. PCB, titanium citrate, and
the antibiotics were then added as described above.

Analysis of methane production. Methane production was determined by gas
chromatography using a flame ionization detector. Headspace gas was analyzed for
determination of methane production after shaking the culture and before sampling the
slurry for PCB analysis.

Other procedures. The incubation conditions, sampling, PCB analysis and data

summation were as previously described (23).

38
RESULTS

Dechlorination of Aroclor 1242 by the initial methanogenic culture. N o
dechlorination nor methane production was observed in either the autoclaved control or
the cultures amended with antibiotics without any substrate (the negative control,
henceforth). However, dechlorination was observed in the cultures amended with both
antibiotics and methanogenic substrates (the methanogenic culture, henceforth). After
8 weeks of incubation (Fig. l), the major congeners that decreased were peak 19 (3.4-2-
CB/2,3,4-CB/2,3-3-CB/2,5-2,6-CB), 35 (mainly 2,4,5-4—CB), 36 (2,5-3,4-CB/3,4,5-2-
CB), 37 (2,4-3,4-CB/2,3,6-2,5-CB/2,4,5-2,6-CB), 39 (mainly 2,3-3,4-CB/2,3,4-4-CB)
and 40 (2,4,5-2,5-CB/2,3,5-2,4—CB), with concomitant increases mainly in peak 5 (2-3-
CB), 9 (2,5-2-CB/4—4-CB), 15 (2,5-3-CB) and 16 (2,4-3-CB). In terms of average
chlorines removed from each biphenyl molecule, the dechlorination activity of the
methanogenic culture was low. After 8 weeks of incubation, only 0.22 chlorines per
biphenyl (Aroclor 1242) were removed however, for some specific congeners, the
dechlorination activity was not low. As shown in Fig. 1, after 8 weeks of incubation,
little change in peak 28 (23-25-CB) was observed and therefore this peak was used as an
internal reference. Molar ratios of several selected peaks to peak 28 were plotted (Fig.
2) to show changes in the representative congeners after 8 weeks of incubation. The
ratios for the methanogenic culture after 8 weeks incubation were compared to those at
0 time which was defined as 100% (Fig. 3). After 8 weeks of incubation, peak 19, 35,
36, 37, and 39 decreased 53, 70, 78, 65, and 64%, respectively, with peaks 5, 15, and
16 increased 263, 156, and 234, respectively.

The observation of decreases in peak 36 and peak 37 with concomitant increases

39

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Fig. 1. Mole percentage of Aroclor 1242 represented by each chromatographic peak after
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between the 0 time and 8 weeks. For a complete list of the PCB congeners associate
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Fig. 4. Changes in Peak 15, 16, 36, and 37 after 8 weeks of incubation with (A) the
methanogenic culture, and (B) the negative control group (with antibiotics, without the
methanogenic substrates). (C) the proposed dechlorination pathway for congener peak

36, and 37. Error bars are the standard deviation (SD) of duplicate samples; where not
shown, the error bars are smaller than the symbols.

43
in peak 15 and peak 16 (Fig. 4A, 4B) suggested that 2,5-3-CB (peak 15) and 2,4-3-CB

(peak 16) were the dechlorination products of 2,5-3,4-CB (in peak 36) and 2,4—3,4-CB
(in peak 37) (Fig. 4C). These represent para dechlorination, and this dechlorination
activity was part of that lost from the heat- and ethanol-treated cultures (27).

Comparison of the dechlorination between the methanogenic culture and the
heat- and ethanol-treated cultures. The dechlorination activity of the methanogenic
culture was more limited than that of the heat- and ethanol-treated cultures (the treated
culture, henceforth). After 8 weeks of incubation, only 0.22 chlorines were removed
from each biphenyl molecule by the methanogenic culture, while 0.67 chlorines per
biphenyl molecule were removed by the treated cultures.

The dechlorination pattern of Aroclor 1242 shown by the methanogenic culture was
distinctly different from that of heat- and ethanol-treated cultures. The former pattern
was characterized by accumulation of some meta-substituted congeners such as 2-3—CB
(peak 5), 2,5-3-CB (peak 15), and 2,4-3-CB (peak 16) (Fig. 1). In contrast, the treated
cultures accumulated some para-substituted congeners (Fig. 5) such as 2-4-CB/2,3-CB
(peak 6), 2,4-2-CB (peak 10), 2,4—4-CB (peak 18), and 2,4—2,4-CB (peak 25) in addition
to 2-CB (peak 1) and 2-2-CB/2,6-CB (peak 3) which were the primary products in the
untreated culture (27 ). Additionally, one of the major characteristics of the
chromatographic pattern of the treated cultures was that the abundance of peaks 35 , 36,
and 37 remained constant. However, in the methanogenic culture, these three peaks
decreased significantly (GC chromatogram not shown).

Other major differences between the dechlorination activities of the methanogenic

culture and the treated cultures were as follows: peak 9 (2,5-2-CB and 4-4-CB), which

 

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Fig. 5. Changes in mole percentage of Aroclor 1242 (represented by each chro-

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microorganisms from the Hudson River sediments.

45

represents the highest mole percentage of congeners in Aroclor 1242, completely
disappeared in treated cultures with a major increase in peak 3 (2-2-CB), whereas in the
methanogenic culture (Fig. 1), this peak did not decrease. Rather, it increased due to
accumulation of the dechlorination products. Likewise, Peak 25 (2,4-2,4-CB) decreased
in the methanogenic culture (Fig. 1), whereas in heat- and ethanol-treatments they
increased slightly (Fig. 5).

Results of BESA experiment. 2-Bromoethanesulfonic acid (BESA) was used to
examine the role methanogens play in the dechlorination. Considering that BESA is a
potential electron acceptor for some anaerobic bacteria, it is possible that it might
compete with PCB’s for electrons and hence inhibit dechlorination. To distinguish if
inhibition on dechlorination by BESA was due to competition for electrons or due to
inhibition of methanogens, one group was amended with BESA, without addition of
methanogenic substrate and antibiotics, to serve as a control. No methane production
was observed in this amendment group while dechlorination did occur (Fig. 6).
However, the same concentration of BESA inhibited both methane production and the
dechlorination activity in the methanogenic culture (Fig. 6). Results are summarized in
Table 1.

Dechlorination activity of the transferred cultures. In the initial cultural batch
one group was amended with the same methanogenic substrates but without addition of
the antibiotics. Dechlorination was also observed in this group (Fig 7). Both the
methanogenic culture and the group amended with the same methanogenic substrates but
without the antibiotics were transferred. Dechlorination activity increased in the

subsequent transfer of the methanogenic culture, while little dechlorination was observed

46

 

 

 

 

 

 

 

 

 

 

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47

 

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Fig. 7. Dechlorination by the initial cultures and the subsequent transfers of both the
methanogenic culture (with both methanogenic substrates and antibiotics) and the group
amended with methanogenic substrates but without antibiotics. The dechlorination
activities are represented by cumulation of the dechlorination product, peak 16
(normalized to peak 28), after 4 weeks incubation. Error bars are the standard deviation
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a an I “"1 7 Mr“ I
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O 1 0“"“m9 Pew“ ‘ ‘ Decreasing Peaks ‘
—4 I ‘ . u I I l ' -‘ . . . u e I I I l
0 10 20 30 40 50 50. 70 8° 90 o 10 20 so 40 so so 70 so so
Peck Number Peak Number

Fig. 8. Mole percentage of Aroclor 1242 represented by each chromatographic peak after
4 weeks of incubation with the transferred methanogenic cultures. For a complete list
of the PCB congeners associate with each peak see reference (23).

49

Table 1. Effects of BESA on PCB Dechlorination
and Methane Production

 

Amendments

Dechlori-
nation

CH‘

 

1' 377"
Antibiotics/no-substrate

Antibiotics/substrate

Antibiotocs/substrate/BESA

genial.

BESA only,
no antibiotics/substrate

 

+ Dechlorination/CH4 production

- No dechlorination/CH4 production

50

in the cultures transferred from the group without addition of the antibiotics (Fig 7).
Ortha dechlorination activity exhibited by the transferred cultures. After 4
weeks of incubation, 2-CB (peak 1) and 2—2-CB/2,6-CB (peak 3) in the transferred

methanogenic cultures decreased (Fig. 8), indicating that ortho dechlorination occurred.

DISCUSSION

Methane production is typically observed in our PCB-dechlorination assays using
PCB-free Hudson River sediments and microorganisms eluted from PCB contaminated
sediments (21, 22, 27), suggesting that methane formation is the terminal step of the
anaerobic food chain. In this anaerobic food chain, fermentative microorganisms
converted the sediment organic matter into substrates for methanogens. Therefore,
addition of eubacteria-inhibiting antibiotics should directly inhibit eubacteria, and hence
methanogens. This was confirmed by the negative control group in which addition of
penicillin—G and D—cycloserine, the eubacteria-inhibiting antibiotics, resulted in no
dechlorination nor methane production. When eubacteria are inhibited with antibiotics,
only archaebacteria could survive. There are three groups of archaebacteria: extreme
thermophiles, extreme halophiles, and methanogens. Our culture conditions were not
favorable for either extreme halophiles or extreme thermophiles, therefore, when
eubacteria were inhibited and methanogenic substrates were provided, the enriched
microorganisms were most probably methanogenic bacteria.

Penicillin-G and D-cycloserine specifically inhibit cell wall synthesis of eubacteria
and by different mechanisms (26). Both antibiotics do not inhibit archaebacteria with the

exception that Methanococcus vannielii is sensitive to D-cycloserine (6). Zinder et al.

51
(29) used combination of penicillin (3340 U/ml) and D-cycloserine (0.1 g/ml) to inhibit

eubacteria in the isolation of a thermophilic strain of Methanosarcina; the antibiotics
successfully eliminated all eubacteria. In our experiment the same concentration of the
antibiotics was used. Since the cultural temperature for Zinder’s enrichment was 55°C
while in our culture it was only 25°C, the antibiotics should be effective longer in our
culture than in that of Zinder’s because these antibiotics are more labile as temperature
increases. The effects of the antibiotics were evidenced by two observations: first,
addition of antibiotics without providing methanogenic substrate (the negative control)
resulted in no dechlorination activity nor methane production, indicating that the
eubacteria were effectively inhibited. Second, the original cultures of Barker’s medium
with methanogenic substrates showed dechlorination activity in presence and absence of
antibiotics. The dechlorination activity in the cultures with antibiotics increased in its
subsequent transfer, while the dechlorination activity in the original cultures without
antibiotics decreased with time (data not shown) and was lost from the subsequent
transfer. This may be because in the absence of antibiotics microorganisms responsible
for the dechlorination activity were outcompeted by some non-dechlorinating eubacteria
stimulated by the methanogenic substrates.

Both dechlorination activity and methane production were inhibited when BESA
was introduced to the methanogenic culture. BESA also showed inhibitory effects on
dechlorination by pasteurized cultures containing no methanogens, and the cultures
concomitantly turned black indicating formation of some metal sulfide (data not shown).
Additionally, these cultures gave—off sulfide smell when they were sampled while being

flushed with Nz-COZ. This observation suggested that BESA was probably used as an

52
electron acceptor, and that the inhibitory effects might due to the competition of BESA

with PCBs for electrons. Similar inhibitory effects of BESA on PCB dechlorination (21)
and on anaerobic dechlorination of other compounds (17) has been reported. For the
reason mentioned above, in this experiment a control group was set up to determine
whether the inhibitory effects of BESA on dechlorination by the methanogenic culture
were due to the inhibition on methanogens or due to the possible competition for
elections. As shown in Table l, in this control group no methane production was
observed while PCB dechlorination did occur. This ensured that BESA at this
concentration effectively inhibited methanogens without inhibiting dechlorination by other
microorganisms. This control proved that inhibition of BESA on the dechlorination by
the methanogenic culture was due to the direct inhibition on methanogens instead of
competition with PCB for electrons. The high concentration of the added methanol (218
mM) should also rule out the possibility of competition by BESA for electron, because
this concentration was high enough to effectively scavenge BESA in case BESA was used
as electron acceptor. These results confirmed that it was most probably methanogens that
was responsible for the dechlorination activity in the culture.

The dechlorination activity of the methanogenic culture was limited to some specific
congeners. However, it is unclear whether the actual dechlorination capacity of
methanogens in the natural environment is limited or not. IMethanogens depend on
eubacteria not only for substrates, but also some nutrients and growth factors (5 , 19).
In our methanogenic culture eubacteria were inhibited, consequently, even though
methanogenic substrates were provided, those methanogens requiring some nutrients

or/ and growth factors from eubacteria still could not grow. Moreover, in order to

 

C01

53

enhance the suppressive effects of antibiotics on eubacteria, no trace metals nor vitamins
were added to our methanogenic medium, and this also certainly eliminated some
fastidious methanogens. With the harsh selective pressure, only a portion of
methanogenic bacteria was expected to be enriched. Additionally, inhibition of
eubacteria might also result in eliminating some possible cooperation from eubacteria.
For example, lecithinase was produced when two Pseudomonas strains grown together,
while neither was able to produce the active enzyme alone (3). It is possible that with
nutrients, growth factors, and some cooperation from eubacteria, methanogens might
have a more broad dechlorination spectrum instead of the narrow one. Nevertheless, the
actual dechlorination capacity of methanogenic bacteria in natural environment remains
to be elucidated. On the other hand, different microbial populations may share a similar
dechlorination pattern. Our results do not rule out the possibility that under other
enrichment conditions some other microorganism(s) may also exhibit a similar
dechlorination pattern to the one reported here.

The transferred methanogenic cultures exhibited the ortho dechlorination activity
on specific congeners. Dort and Bedard (12) reported ortho dechlorination of 2,3,5,6—
tetrachlorobiphenyl (2,3 ,5 ,6-CB) by microorganisms from methanogenic pond (Wood
Pond, Lenox, Mass.) sediment. However, ortho dechlorination by microorganisms from
Hudson River sediment has never been observed. 2-CB, 2-2-CB, and 2,6—CB are the
major products of PCB dechlorination by Hudson River microorganisms (23). Our
results demonstrate that the Hudson River contains microorganisms with the potential for
complete dechlorination of PCBs to biphenyl.

May (1%.) et a1 observed a very similar para dechlorination activity with a

54

enrichment amended with 20 mM sulfate. The enrichment expressed no dechlorination
activity until after the sulfate was depleted. The pattern suggested that para chlorine
removal only with congener containing para and meta chlorines that were adjacently
positioned. The enrichment was than subcultured on solid media containing no sulfate
but sterilized river sediment. The PCB—dechlorinating colonies that had been grown on
solid medium for a second or third time were not methanogenic in the sediment slurries,
and the dechlorination activity concomitantly changed from mainly para-removal to meta-
removal. The concomitant disappear of methanogensis and the congener-specific para
dechlorination activity provided another supportive evidence to correlate methanogens to
this para dechlorination activity.

Different environmental PCB dechlorination patterns have been reported (7, 8, 9,
10) and also observed under laboratory experimental conditions (1 , 27). The
dechlorination pattern by the methanogenic enrichment was close to pattern H (8, 11).
The different dechlorination patterns observed have been explained by Abramowitz et al.
(1), Brown et al. (9), and Quensen et al. (23) as due to different microbial dechlorinating
populations that may exist in the Hudson River sediment. Results of the heat- and
ethanol-treatments support the suggestion that anaerobic sporeformers are at least one of
the PCB-dechlorinating physiological groups (27). Results of the methanogen experiment
reported here provide additional evidence for different dechlorinating populations. Thus,
in addition to anaerobic sporeformers, methanogenic bacteria are also probably among

the physiological groups capable of anaerobic PCB dechlorination.

55
REFERENCES

l. Abramowicz, D. A., M. J. Brennan, and H. M. Van Dort. 1989. Microbial
dechlorination of PCBs. I. Aroclor mixtures. p.49-59. In Research and development

program for the destruction of PCBs. GE Corporate Research and Development,
Schenectady, N. Y.

2. Balch, W. E., and R. S. Wolfe. 1979. Specificity and biological distribution of
coenzyme M (2-mercaptoethanesulfonic acid). J. Bacteriol. 137:256-263.

3. Bates, J. L., and P. V. Liu. 1963. Complementation of lecithinase activities in
closely related pseudomonas: its taxonomic implication. J. Bacteriol. 86:585-592.

4. Berry, D. F., A. J. Francis, and J. -M. Bollag. 1987. Microbial metabolism of
homocyclic and heterocyclic aromatic compounds under anaerobic conditions. Microbiol.
Rev. 51: 43-59.

5. Bhatnagar, L., M. K. Jain, and J. G. Zeikus. 1991. p. 251-270. Methanogenic
bacteria. In Variations in autotrophic life. Academic Press, Orlando, FL.

6. Bfick, A., and O. Kandler. 1985. Antibiotic Sensitivity of Archaebacteria. P.525-
544. In C. R. Woese and R. S. Wolfe (ed.), The bacteria, vol. VIII. Academic Press,
Orlando, Fl.

7. Brown, J. F., D. L. Bedard, M. J. Brennan, J. C. Carnahan, H. Feng, and R.
E. Wagner. 1987. Polychlorinated biphenyl dechlorination in aquatic sediments. Science
236:709-712.

8. Brown, J. F., and R. W. Lawton. 1989. Identification of PCB transformations
occurring in nature. p.85-93. In Research and development program for the destruction
of PCBs. GE Corporate Research and Development, Schenectady, N. Y.

9. Brown, J. F., R. E. Wagner, D. L. Bedard, M. J. Brennan, J. C. Carnahan, R.
J. May, and T. J. Tofflemire. 1984. PCB transformations in upper Hudson sediments.
Northeast. Environ. Sci. 3:167-179.

10. Brown, J. F., R. E. Wagner, H. Feng, D. L. Bedard, M. J. Brennan, J. C.
Camahan, and R. J. May. 1987. Environmental dechlorination of PCBs. Environ.
Toxicol. Chem. 6:579-593.

11. Brown, J. F. Personal communication.

12. Dort, H. M. V., and D. L. Bedard. 1991. Reductive ortho and meta dechlorination
of a polychlorinated biphenyl congener by anaerobic microorganisms. Appl. Environ.
Microbiol. 57:1576—1578.

56

13. Evans, W. C., and G. Fuchs. 1988. Anaerobic degradation of aromatic compounds.
Ann. Rev. Microbial. 42:289-317.

14. Fathepure, B. Z., and S. A. Boyd. 1988. Dependence of tetrachloroethylene
dechlorination on methanogenic substrate consumption by Methanosarcina sp. strain
DCM. Appl. Environ. Microbiol. 54:2976—2980.

15. Fathepure, B. Z., J. M. Tiedje, and S. A. Boyd. 1987. Reductive dechlorination
of 4—chlaroresorcinol by anaerobic microorganisms. Environ. Toxicol. Chem. 6:929-934.

16. Freedman, D. L., and J. M. Gossett. 1991. Biodegradation of dichloroethane and
its utilization as a growth substrate under methanogenic conditions. Appl. Environ.
Microbiol. 57:2847-2857.

l7. Genthner, S. B. R., W. A. Price, and P. H. Pritchard. 1989. Anaerobic
degradation of chloroaromatic compounds in aquatic sediments under a variety of
enrichment conditions. Appl. Environ. Microbiol. 55: 1466-1471.

18. Holliger, C., G. Schraa, E. Stupperich, A. J. M. Stams, and A. J. B. Zehnder.
1992. Evidence for the Involvement of corrinoids and factor F430 in the reductive
dechlorination of 1,2-dichloroethane by Methanosarcina barkeri. J . Bacterial. 174:4427-
4434.

19. Man, R. A., and M. R. Smith. The methanogenic bacteria. p.948-977. In M. P.
Starr, H. Stolp., H. G. Tn’iper, A. Ballows, and H. G. Schlegel (ed.), The prokaryotes,
vol. II. Springer-Verlag, New York.

19a. May, H. D., A. W. Boyle, W. A. Price 11, and C. K. Blake. 1992. Subculturing
of a polychlorinated biphenyl-dechlorinating anaerobic enrichment on solid media. Appl.
Environ. Microbiol. 58:4051-4054.

20. Mikesell, M. D., and S. A. Boyd. 1990. Dechlorination of chloroform by
Methanosarcina strains. Appl. Environ. Microbiol. 56:1198-1201.

21. Morris, P. J., W. W. Mohn, J. F. Quensen III, J. M. Tiedje, and S. A. Boyd.
1992. Establishment of a polychlorinated biphenyl-degrading enrichment culture with
predominantly meta dechlorination. Appl. Environ. Microbiol. 58:3088-3094.

22. Nios, L., and T. M. Vogel. 1990. Effects of organic substrates on dechlorination
of Aroclor 1242 in anaerobic sediments. Appl. Environ. Microbiol. 56:2612-2617.

23. Quensen, J. F., III, S. A. Boyd, and J. M. Tiedje. 1990. Dechlorination of four
commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic microorganisms
from sediments. Appl. Environ. Microbiol. 56:2360-2369.

57

24. Shelton, D. R., and J. M. Tiedje. 1984. General method for determining anaerobic
biodegradation potential. Appl. Environ. Microbiol. 47 :850-857.

25. Tiedje, J. M., S. A. Boyd, and B. Z. Fathepure. 1987. Anaerobic degradation of
chlorinated aromatic hydrocarbons. In G. Pierce (ed.), Developments in industrial
microbiology, vol. 27. Journal of Industrial Microbiology. Suppl. No. l.

26. Ward, J. B. 1987. Biosynthesis od peptidaglycan: points of attack by wall inhibitors.
p. 1-43. In D. J. Tipper (ed.), Antibiotic inhibitors of bacterial cell wall biosynthesis.
Pergamon press Oxford, England.

27. Ye, D., J. F. Quensen III, J. M. Tiedje, and S. A. Boyd. 1992. Anaerobic
dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-treated
microorganisms from sediments. Appl. Environ. Microbiol. 58:1110—1114.

28. Zehnder, A. J. B., and K. Wuhrmann. 1976. Titanium (III) Citrate as a nontoxic
oxidation-reduction buffering system for the culture of obligate anaerobes. Science
194:1165-1166.

29. Zinder. S. H., and R. A. Mah. 1979. Isolation and characterization of a
thermophilic strain of Methanosarcina unable to use Hz-COZ for methanogesis. Appl.
Environ. Microbiol. 38:996-1008.

Chapter 3

Anaerobic Dechlorination of Polychlorobiphenyls (Aroclor 1242)

by Pasteurized and Ethanol-Treated Microorganisms

from Hudson River Sediments

58

59

ABSTRACT

A polychlorobiphenyl (PCB)-dechlorinating inoculum eluted from upper Hudson
River sediments was treated with either heat or ethanol or both. The treated cultures
retained the ability to dechlorinate PCBs (Aroclor 1242) under strictly anaerobic
conditions. The dechlorination activity was maintained in serial cultures inoculated with
transfers of 1% inoculum when the transferred inoculum was treated each time in the
same manner. No methane production was detected in any treated culture, although
dechlorination of PCBs in the untreated cultures was always accompanied by methane
production. All treated cultures preferentially removed meta chlorines yielding a
dechlorination pattern characterized by accumulation of certain ortho- and para-
substituted congeners such as 2-4-chlorobiphenyl (2-4—CB), 2,4—2-CB, and 2,4-4-CB. In
contrast, the untreated cultures showed more extensive dechlorination activities, which
almost completely removed both meta and para chlorines from Aroclor 1242. These
results suggest that microorganisms responsible for the dechlorination of PCBs in the
upper Hudson River sediments can be grouped into two populations according to their
responses to the heat and ethanol treatments. Microorganisms surviving the heat and
ethanol treatments preferentially remove meta chlorines, while microorganisms lost from
the enrichment mainly contribute to the para dechlorination activity. These results
indicate that anaerobic sporeformers are at least one of the physiological groups
responsible for the reductive dechlorination of PCBs. The selection of a dechlorinating
population by such treatments may be an important step in isolation of PCB-

dechlorinating microorganisms.

60
INTRODUCTION

During the past 20 years intensive studies on microbial degradation of
polychlorobiphenyls (PCBs) have been reported (for reviews, see references 1 and 15).
These include studies with soil communities, isolates from nature, and recombinant
organisms. The results indicate that the microbial degradation of PCBs leading to
biphenyl ring cleavage is generally limited to lightly chlorinated congeners, leaving
highly chlorinated congeners unaltered (1, 15).

Recently, the microbially mediated anaerobic reductive dechlorination of PCBs has
been described. This process converts a large array of highly chlorinated PCBs into
lesser chlorinated congeners, predominately ortho—substituted mono- and
dichlorobiphenyls (2, 3 , 13 , 14). This is especially noteworthy because the
dechlorination products are less toxic and more readily degraded by aerobic
microorganisms. However, despite its environmental significance, research on the
anaerobic reductive dechlorination of PCBs has, until now, failed to identify any
physiological group(s) of dechlorinating microorganisms. Therefore, a PCB-
dechlorinating inoculum was treated with either heat or ethanol or both to determine

whether anaerobic sporeformers are responsible for PCB dechlorination.

MATERIALS AND METHODS
Inoculum preparation. Sediments were collected in September, 1989 from the
upper Hudson River near Hudson River Falls, N.Y. (site H7 in reference 3) and shipped
under anaerobic conditions to the laboratory as described previously (13). To make the

inoculum, sediments were transferred into sterile Erlenmeyer flasks and mixed with an

61
equal volume of reduced anaerobic mineral medium (RAMM) (16) while flushing with

filter-sterilized 02—free N2-C02 (80:20 vol/vol) using a Hungate apparatus. The flasks
were then sealed and incubated at 25°C in the dark for two weeks to allow the facultative
anaerobic microorganisms to consume residual oxygen. Anaerobic conditions were
indicated by the detection of methane. The slurry was vigorously shaken by hand for 3
min and then allowed to settle for 30 min. Supernatant containing the eluted
microorganisms was used as the inoculum.

Pasteurization. Inoculum was anaerobically introduced into sterile N2-COZ
(80:20, vol/vol)-flushed anaerobic culture tubes (Bellco Glass Inc. , Vineland, N .J.) (20
ml per 28-ml tube). The tubes were then sealed with teflon-coated butyl rubber stoppers
(West Co., Phoenixville, Pa.) and incubated in a water bath at 80, 85, or 90°C. The
temperature increase was monitored with another tube containing the same amount of
inoculum and a thermometer. When the thermometer reached 78, 83, or 88°C (for
pasteurization at 80, 85 , or 90°C, respectively) (7), the tubes were incubated for 15 min
(80 and 85°C) or 10 min (90°C) and then immediately cooled to room temperature.

Treatment with ethanol or combination of ethanol and heat. A 70-ml inoculum
was anaerobically transferred into a sterile N2-C02 (80:20, vol/vol)-flushed l60—ml
serum bottle and mixed with an equal volume of autoclaved ethanol (200 proof,
dehydrated; Quantum Chemical Co., Tuscola, 111.); the bottle was then sealed. The
mixture was incubated at room temperature (20°C) for 35 min and occasionally shaken
by hand. The serum bottle was then centrifuged at 3,000 x g for 30 min at 20°C. The
total time for the inoculum to contact ethanol was about 1 h. After the serum bottle was

centrifuged, the liquid portion was removed and the remainder was washed three times

62
with autoclaved 0.9% N aCl to remove the residual ethanol. Another 140 ml of inoculum

without ethanol was centrifuged, and the supernatant was anaerobically filter sterilized
(0.22 pm; Millipore Co., Bedford, Mass), and then used to re-suspend the ethanol-
treated inoculum. Oxygen contamination of the re-suspended inoculum did not occur as
indicated by resazurin test.

For the combined treatment, the inoculum was first treated with ethanol, then
anaerobically transferred to the culture tubes and heated at 80°C as described above.

Preparation of experimental vessels. Assay vessels were prepared as described
previously (13) with the following modifications. A preincubation procedure was used
to ensure anaerobic conditions in the assay vessels prior to initiation of the actual
dechlorination assay. For this, serum bottles (60 ml) received 10 g of the PCB-free dry
sediment and 10 ml of inoculum prepared from site H7 sediment. The PCB-free
sediment was collected from the Hudson River at River Mile 205 (13). The H7
inoculum was added to moisten the dry sediment and to introduce additional
microorganisms to reduce the preincubation time. After methane was detected in the
head space, the bottles were autoclaved at 121°C for l h on three consecutive days, with
incubation at 37°C between each autoclaving.

After the third autoclaving the following were added to each preincubated serum
bottle while it was being flushed with filter-sterilized 02-free N2-C02 (80:20, vol/vol)
with a Hungate apparatus: 10 ml of inoculum, 10 ml of RAMM, 100 pl of 10%
autoclaved solution of cysteine, and 80 pl of 10% (wt/vol) Aroclor 1242 (Monsanto Co. ,
St. Louis, Mo.) in acetone. The final concentration of cystine was 330 mg/liter of

liquid, and the final total PCB concentration was 800 ug/ g of sediment (dry weight).

63
The bottles were then sealed with teflon-coated butyl rubber stoppers and aluminum

crimps and shaken thoroughly to completely mix the PCBs. The controls were
autoclaved twice at 121°C for l h, with an interval of 5 h before PCBs were added.

The experiments were performed three times: in May, July, and September (1990).
A flow diagram (Fig. 1) summarizes the experimental procedure. The inoculum for the
three experiments was established in April 1990. In the May and July experiments, the
treated cultures were only pasteurized at 80°C for 15 min. However, in the September
experiment, all treatments were performed; heating at 80°C or 85°C for 15 min,
treatment with 50% ethanol for 1 h, and treatment with the combination of heat and
ethanol. Cultures from experiments initiated in the September were serially transferred,
and the transferred inocula were treated each time.

All treatments were in triplicate except those in the May experiment and the 90°C
pasteurized transfer (the third serial culture of the September experiment), which were
done in duplicate.

Transfer. Cultures that were to be transferred (heat treated and heat plus ethanol
treated), were shaken for 30 min and then the slurry was allowed to settle for about 30
min. The supernatant was used as the inoculum and transferred directly or treated again
in the same manner as described above.

For the 1% transfer of the culture treated with the combination of ethanol and heat,
4 ml of the inoculum was treated again in the same manner as described above except
that the vessel was a 10-ml serum battle, the time exposed to ethanol was 1 h, the

centrifugation was performed at 8,000 x g for 10 min, and the heating temperature was

 

1 1
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65
increased to 85°C.

The experimental vessels for the transferred cultures were prepared as described
above except that only 0.3 ml of the treated inoculum was inoculated to each bottle
containing 29.7 ml of RAMM to make a 1% transfer.

Other procedures. The incubation conditions, sampling, extraction, and analysis

procedures, and data summation have been described previously (13).

RESULTS

Dechlorination of Aroclor by heat- and ethanol-treated cultures. In all three
experiments (May, July, and September), the treated cultures showed dechlorination
activities (Fig. 2, data for two experiments shown). Higher dechlorination rates for the
treated cultures occurred before 4 weeks and then slowed between 4 and 8 weeks. After
8 weeks, no further dechlorination was observed. Despite the fact that the treatments
were different and the same inoculum was used over a 5-month period, the treated
cultures shared a common dechlorination pattern, as illustrated in Figure 3B. As
dechlorination proceeded, the levels of most highly chlorinated congeners decreased, with
concomitant major increases in 2-chlorobiphenyl (2-CB) (peak 1), 2-2-CB and 2,6-CB
(peak 3), 2-4-CB and 2,3—CB (peak 6), 2,4—2-CB (peak 10), 2,4—4-CB (peak 18) and 2,4-
2,4-CB (peak 25) as dechlorination products.

Dechlorination of PCBs in the untreated cultures was always accompanied by
methane production. No methane was detected in any treated culture.

The acetone used as a carrier for PCB additions is a potential substrate. However,

it does not appear to be directly important to the dechlorinating microorganisms because

66

 

 

 

 

 

 

 

 

 

 

    

 

 

 

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Incubation Time (Weeks)

Fig. 2. Dechlorination of Aroclor 1242 by heat and/or ethanol treated microorganisms
eluted from the Hudson River sediments by experiments performed in (A) September,
and (B) May. Error bars are the SD of triplicate (A) or duplicate (B) samples, where
not shown, the error bars are smaller than the symbols.

67

 

 

 

 

 

 

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Peak Number

Fig. 3. Male percentage of Aroclor 1242 represented by each chromatographic peak after
12 weeks incubation in the May experiment with (A) Autoclaved (B) Treated (80°C, 15
min), and (C) Untreated microorganisms eluted from the Hudson River sediments. For
a complete list of the PCB congeners associate with each peak see (13).

68
we failed to enhance PCB dechlorination by using higher levels of acetone (data not

shown).

Dechlorination of Aroclor 1242 by the untreated cultures. In contrast to the
stable dechlorination activities of the treated cultures, the dechlorination activities of the
untreated cultures varied among the three experiments. The highest dechlorination
activity was observed in the May experiment when the inoculum was relatively fresh
(Fig. 2B) compared to July (not shown) and September (Fig. 2A). In the May
experiment the major dechlorination products were 2-CB and 2,6-CB and/or 2-2-CB;
these congeners accounted for 79 % of the total PCBs recovered after 12 weeks
incubation. In the July and September experiments, lesser amounts of these
dechlorination products were formed (23 % for July; 22 % for September). The same
ortho-substituted products plus some ortho- and para-substituted products (2-4-CB, 2,4-2-
CB, 2,4-4—CB) tended to accumulate.

Comparison of the dechlorination patterns between treated and untreated
cultures. The common dechlorination pattern shared by all treated cultures in the three
experiments (May, July and September) is very different from that of the untreated
culture in the May experiment (Fig. 3). The essential difference is that the treated
cultures accumulated dechlorination intermediates containing para-substituted chlorine(s)
(Fig. 3B) such as 2-4-CB and 2,3-CB (peak 6), 2,4-2-CB (peak 10), 2,64CB (peak 12),
2,5-4-CB (peak 17), 2,4-4-CB (peak 18), and 2,4—2,4-CB (peak 25), in addition to the
2—CB (peak 1) and 2-2-CB and 2,6—CB (peak 2) which were the primary products in the
untreated (May) culture (Fig. 3C). Additionally, the chromatographic patterns of the

treated and the untreated cultures are easy to distinguish by the apparent difference in the

69
heights of peaks 35, 36 and 37 (the responses of electron capture detector for these peaks

is high), which were evident in the treated cultures but not in the untreated cultures (gas
chromatograms not shown).

The dechlorination patterns obtained with the treated inocula were similar to those
of the untreated inocula in the July and September experiments.

Analysis of the homolog distribution (Fig. 4) showed that at 4 weeks the
distribution in both treated and untreated cultures was similar Wig. 4A). After 4 weeks,
dechlorination slowed in the treated cultures, but continued in the untreated culture,
resulting in mono- and dichlorinated biphenyls as the final products (Fig. 4B). At 4
weeks, the mole percentage of monochlorinated biphenyls in both cultures was almost
equal. From 4 weeks to 12 weeks, the mole percentage of monochlorinated biphenyls
in the treated cultures remained the same, while in the untreated cultures it increased
from 9 to 33%. The increase in monochlorinated biphenyls is mainly due to 2-CB.
Figure 4B also illustrates that after 12 weeks incubation, the tetra-, penta-, and
hexachlorinated congeners have decreased much more in the untreated culture than in the
treated culture. Although the mole percentage of dichlorinated congeners in both cultures
was comparable, their compositions were different. In the untreated culture, the major
dichlorinated biphenyls were 2—2-CB and 2,6-CB (peak 3), while in the treated cultures
the major dichlorinated biphenyls were 2-2-CB and 2,6-CB (peak 3) plus 2-4—CB and 2,3-
CB (peak 6) (Fig. 3).

Dechlorination of Aroclor 1242 by transferred cultures. The treated culture in
the May experiment was transferred at concentrations of 25, 10, and 1%. All transferred

cultures showed similar dechlorination activities (data not shown). Two treated cultures

70

60

 

 

 

 

 

 

 

50 .' A D Untreated
- Autoclaved
A 2221 treated
40 ~ 7
30 -
E 20 -1
Cl)
8
Q) 10 ‘ Y
n“ HIE
‘D l
0 so - B A
2

 

 

 

 

1
.i I g ;
IE r .1 a _._r

mono di tri tetra penta hexa

Homolog Group of PCBs Aroclor 1242

 

 

 

 

 

 

Fig. 4. Homolog distribution of Aroclor 1242 after (A) 4 and (B) 12 weeks of incubation
with treated (80°C, 15 min) and untreated microorganisms (May experiment) eluted from
the Hudson River sediments. The hepta-, octa-, nona- and decachlorinated homologs are
not plotted because their male percentages in Aroclor 1242 are too low. Error bars are
the SD of duplicate samples, where not shown, the error bars are smaller than the
symbols.

71

 

 

 

 

 

 

 

 

101‘

3.0-

Average Chlorines Per Biphenyl

2 5. O—OAutoclaved

° 0—0 Untreated

0—0 85°C, 15 min

v—V90°C, 10 min

EI—CI 507. ethanol, 1 h/B5°C, 15 min

 

 

2.0

 

o a i 6 a 1‘0 1'2 14

’ Incubation Time (Weeks)

Fig. 5. Dechlorination of Aroclor 1242 by the cultures transferred from the September
experiment (1% transfer). (A) The second serial culture (85°C, 15 min). (B) The third
serial culture (90°C, 10 min). Error bars are the SD of triplicate (A) or duplicate (B)
samples, where not shown, the error bars are smaller than the symbols.

72

from the September experiment were transferred to the second serial culture at
concentration of 1% following treatment of the inocula; one was originally pasteurized
at 85°C, and the other was originally treated with the combination of ethanol and heat.
When the inoculum from the combined treatment was treated again, the temperature was
increased from 80 to 85°C. The transferred pasteurized cultures exhibited dechlorination
activities similar to the original cultures, whereas the transferred cultures that received
the combined treatment showed a lag time of 12 weeks (Fig. 5A). After 4 weeks, the
inoculum taken from the second serial culture (Fig. 5A) was heated at 90°C and
transferred (1%) to a third serial culture. The dechlorination activity was still evident

(Fig. 5B) even though the pasteurization temperature was raised to 90°C.

DISCUSSION

Heat- and ethanol-treated microorganisms eluted from upper Hudson River
sediments were able to dechlorinate Aroclor 1242 under anaerobic conditions, as
evidenced by a decrease in the highly chlorinated congeners and a corresponding
accumulation of lesser- chlorinated congeners.

In previous studies in which PCB dechlorination was observed, the cultures also
produced methane. This correlation, along with the observed dechlorination of other
compounds in methanogenic samples (6, 8, 17) and by pure cultures of methanogens (5,
12), has led to speculation that methanogens may be required or responsible for
dechlorination of PCBs. However, the pasteurized cultures produced no methane while
dechlorinating the PCBs, indicating that methanogens or methanogensis is not required

for PCB dechlorination.

73
Gottschalk et al. (7) pointed out that heating at 70°C or 80°C for 10 min is

generally regarded as sufficient for elimination of non-sporeformers, including most
thermophilic nonsporeformers. Koransky et al. (11) and Johnston et a1. (10) reported
that treatment with 50% ethanol for 1 hour is also an effective technique for selective
isolation of sporeforming bacteria from mixed cultures. In our experiments, the
dechlorinating microorganisms survived both heat and ethanol treatments, and the
selective conditions we used were even more strict, including heating at 85°C for 15 min
or at 90°C for 10 min, timing from temperatures 3°C higher than those Gottschalk et al.
(7) recommended, and combining both heat and ethanol treatments. The dechlorination
does not appear to be catalyzed by thermophiles, because in our temperature profile
experiment, no dechlorination by the pasteurized cultures was observed when the bottles
were incubated at 37, 50, or 65°C (data not shown). Furthermore, the dechlorinating
microorganisms withstood not only high temperatures but also 50% ethanol for 1 h,
which should eliminate thermophiles. Spores were observed microscopically in the
untreated and treated cultures, and were more prevalent in the latter. Because the
dechlorination does not appear to be due to thermophiles and the vegetative cells of
nonthermophilic microorganisms should have been eliminated by the heat and ethanol
treatments, the dechlorinating microorganisms are most likely sporeformers.

The treated cultures had a common meta preferential dechlorimrtian activity
regardless of the type of treatment. This activity was not only resistant to the heat and
ethanol treatments but also stable in the inoculum over time and through serial transfers.
This indicates that the responsible microbial population was stably maintained. This

stability is consistent with the survival capability of sporeformers.

74

Comparison of the dechlorination patterns showed that the major difference between
the treated and untreated cultures was that the treated cultures lost some of the para
dechlorination activity present in the fresh untreated inoculum (Fig. 3). Besides this, in
contrast to the stable meta preferential dechlorination activities in the treated inoculum,
the para dechlorination activity of the untreated inoculum changed with time. As the
inoculum aged, more para dechlorination activity was lost, and the dechlorination pattern
of the untreated culture approached that of the treated one. The differences in surviving
the heat and ethanol treatments and in maintaining the activity over time between meta
and para dechlorination activities may reflect the physiological characteristics of the
different responsible microbial populations.

It seems that Hudson River microorganisms responsible for dechlorination of
Aroclor 1242 can be grouped into two populations according to their responses to the
heat and ethanol treatments: one group can survive such treatments, while the other will
be eliminated. The surviving papulation is responsible for the meta preferential
dechlorination activities, whereas the eliminated population mainly contributes to para
dechlorination activities. The untreated cultures in the May experiment consisted of both
populations and thus had both activities.

Even though the eliminated population could not withstand the heat and ethanol
treatments, it might include some sporeformers as well, because spores normally show
widely varying levels of heat and ethanol sensitivity. For example, some bacterial spores
can survive at 100°C or more for several hours (9), while some are known to be almost
as heat sensitive as the vegetative cell (10).

In the May experiment, when the fresh inoculum was used, dechlorination of

75

Aroclor 1242 by the Hudson River microorganisms in the untreated culture occurred
almost exclusively at the meta and para positions (Fig. 3), as previously observed by
Quensen et al. (13). This dechlorination pattern is very close to the pattern C of Brown
et al. (24), while the dechlorination pattern of the treated cultures is close to pattern M
(1a). Quensen et al. ( 13) and Abramowicz et al. (1a) suggested that dechlorination
pattern C is the result of two separate and partially complementary dechlorination
activities. One is pattern Q, characterized by para dechlorination activities; the other is
pattern M, which is meta preferential. They also suggested that two PCB-dechlorinating
populations may exist in the Hudson River sediments. The results of this study support
this suggestion. Culture conditions and the particular batch of sediment used may both
contribute to determining whether one or both dechlorinations are expressed in a
particular experiment.

According to our experimental results, it appears that the anaerobic sporeformers
are at least one of the physiological groups responsible for the reductive dechlorination

of PCBs.

76
REFERENCES

1. Abramowicz, D. A. 1990. Aerobic and anaerobic biodegradation of PCBs: a
review. Crit. Rev. Biotechnol. 10:241-251.

1a. Abramowicz, D.A., M. J. Brennan, and H.M. Van Dart. 1989. Microbial
dechlorination of PCBs. I. Aroclor mixtures, P. 49-59. In Research and development
program for the destruction of PCBs. GE Corporate Research and Development,
Schenectady, N.Y.

2. Brown, J. F., D. L. Bedard, M. J. Brennan, J. C. Carnahan, H. Feng, and R.
E. Wagner. 1987 . Polychlorinated biphenyl dechlorination in aquatic sediments.
Science 236:709-712.

3. Brown, J. F., R. E. Wagner, D. L. Bedard, M. J. Brennan, J. C. Carnahan,
R. J. May, and T. J. Tofflemire. 1984. PCB transformations in upper Hudson
sediments. Northeast. Environ. Sci. 23:167-179.

4. Brown, J. F., R. E. Wagner, H. Feng, D. L. Bedard, M. J. Brennan, J. C.
Carnahan, and R. J. May. 1987 . Environmental dechlorination of PCBs. Environ.
Toxicol. Chem. 6:579-593.

5. Fathepure, B. Z., and S. A. Boyd. 1988. Dependence of tetrachloroethylene
dechlorination on methanogenic substrate consumption by Methanosarcina sp. strain
DCM. Appl. Environ. Microbiol. 54:2976-2980.

6. Fathepure, B. Z., J. M. Tiedje, and S. A. Boyd. 1987. Reductive dechlorination
of 4-chlororesorcinol by anaerobic microorganisms. Environ. Toxicol. Chem. 6:929-
934.

7 .Gottschalk, G., J. R. Andreesen, and H. Hippe. 1981. The genus Closm'dium
(nonmedical aspects). p.1767-1803. In M. P. Starr, H. Stolp., H. G. Trfiper, A.
Ballows, and H. G. Schlegel (ed.),The prokaryotes, vol. II. Springer-Verlag, New
York.

8. Horowitz, A., J. M. Suflita, and J. M. Tiedje. 1983. Reductive dehalogenations
of halobenzoates by anaerobic lake sediment microorganisms. Appl. Environ.
Microbiol. 45:1459-1465.

9. Ingram, M. 1969. Sporeformers as food spoilage organisms. p. 549-610. In G.
W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press, Inc. , New York.

10. Johnston, R., S. Harmon, and D. Kautter. 1964. Methods to facilitate the
isolation of Clostridium botulinum type B. J. Bacteriol. 88:1521-1522.

77

11. Koransky, J. R., S. D. Allen, and V. R. Dowell, Jr. 1978. Use of ethanol for
selective isolation of sporeforming microorganisms. Appl. Environ. Microbiol.
35:762-765.

12. Mikesell, M. D., and S. A. Boyd. 1990. Dechlorination of chloroform by
Methanosarcina strains. Appl. Environ. Microbiol. 56:1198-1201.

13. Quensen, J. F., III, S. A. Boyd, and J. M. Tiedje. 1990. Dechlorination of
four commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic
microorganisms from sediments. Appl. Environ. Microbiol. 56:2360-2369.

14. Quensen, J. F., III, J. M. Tiedje, and S. A. Boyd. 1988. Reductive
dechlorination of polychlorinated biphenyls by anaerobic microorganisms from
sediments. Science 242:752-754.

15. Rachkind-Dubinsky, M. L., G. S. Sayler, and J. W. Blackburn. 1987.
Microbiological decomposition of chlorinated aromatic compounds, p. 142-152.
Marcel Dekker, Inc., New York.

16. Shelton, D. R., and J. M. Tiedje. 1984. General method for determining
anaerobic biodegradation potential. Appl. Environ. Microbiol. 47 :850-857.

17. Tiedje, J. M., S. A. Boyd, and B. Z. Fathepure. 1987. Anaerobic degradation
of chlorinated aromatic hydrocarbons. In G. Pierce (ed.), Developments in industrial
microbiology, vol. 27. Journal of Industrial Microbiology. Suppl. No. 1.

Chapter 4

Anaerobic Dechlorination of Polychlorobiphenyls (PCB’s)

by Pasteurized Microorganisms as Affected by Molybdate,

2-Bromoethanesulfonic Acid (BESA), and Sulfate

78

79

ABSTRACT

Anaerobic dechlorination of Aroclor 1242 by the pasteurized cultures was
completely suppressed during 12 weeks of incubation when either 5 mM molybdate, 20
mM sulfate, or 25 mM 2-bromoethane sulfonic acid (BESA) was added at 0 time and the
same amounts of molybdate and sulfate, and half as much BESA were re-fed either at
2, 4, 6, and 8 weeks (molybdate and BESA) or 4, and 8 weeks (sulfate). Effects of
these anions on dechlorination at different concentrations were screened. BESA and
sulfate completely inhibited dechlorination at their lowest screened concentrations, 1 mM
and 2 mM, respectively. Molybdate partially inhibited the dechlorination at 2, 4, and
8 mM, and completely inhibited the dechlorination at 16 mM. Methanogens were
eliminated by the pasteurization as evidenced by the fact that no methane was detected
in any pasteurized culture. Since methanogens were eliminated, BESA probably inhibited
the dechlorination by acting as a sulfuroxy anion. It seems that the inhibition of
dechlorination by molybdate, sulfate, and BESA is not due to general toxicity but due to
the effects on sulfidogens. Since molybdate should not inhibit microorganisms competing
with sulfidogens, while sulfate should not inhibit microorganisms depending on
sulfidogens, it appeared that microorganisms capable of anaerobic dechlorination of PCBs
in the pasteurized cultures were probably sulfidogens. Molybdate probably directly
inhibited sulfidogens, while both sulfate and BESA probably inhibited the dechlorination
by competing with PCB as electron acceptors. Only sporeforming bacteria were expected
to survive the harsh pasteurization, and among sulfidogens the only known genus forming

spores is Desulfotomaculum. Therefore, the experimental results suggest that

80

microorganisms responsible for the dechlorination of PCBs in our pasteurized enrichment
are probably Desulfotomaculum-like sporeforming sulfate-reducing bacteria. However,
isolation of the microorganism(s) and demonstration of PCB dechlorination activity with

the isolatc(s) are required in order to make a final conclusion.

INTRODUCTION

Characterization of the PCB dechlorination activities in an undefined microbial
community is an important step in the identification of the responsible microbial
population(s). Information from these studies can provide a basis for further enrichment,
and may facilitate the identification and isolation of microorganisms capable of PCB
dechlorination.

Microbial group specific inhibitors can sometimes be used to distinguish which
group is responsible for a specific activity (18). We previously investigated the effects
of molybdate (a specific inhibitor of sulfate-reducing bacteria), 2-bromoethanesulfonic
acid (BESA, a specific inhibitor of methanogenic bacteria) on the anaerobic
dechlorination of PCBs in non-pasteurized cultures (Chapter 3). We also tested the effect
of sulfate on PCB dechlorination by the same cultures. All of these anions inhibited both
dechlorination and methanogensis. But because the non-pasteurized inoculum contained
a high diversity of microorganisms, the results alone did not specifically define the
microbial group responsible for PCB dechlorination activity.

Pasteurization of the inoculum used above eliminates methanogens which play an
important role in anaerobic microbial processes (2, 29), but a partial dechlorination

activity was still retained (28). Pasteurization also decreased the microbial diversity in

81

the system since only spore-forming bacteria maybe expected to survive the treatment.
Therefore, the effects of molybdate, BESA, and sulfate on PCB dechlorination in
pasteurized cultures were investigated to further characterize the dechlorination activity
in the pasteurized culture, and to try to correlate the activity to a certain type(s) of

microorganism(s) .

MATERIALS AND METHODS

Inoculum and pasteurization. Sediments were collected from the upper Hudson
River, N.Y. (site H7 in reference 3). Inoculum preparation and pasteurization were as
described elsewhere (28). The inoculum for the general inhibition experiment was
prepared directly from Hudson River sediment and was pasteurized at 85°C for 15 min
(timing began when the thermometer in the monitor tube reached 83°C). The inoculum
for the concentration profiles experiment was taken from a PCB dechlorinating culture
previously pasteurized at 90°C for 15 min and this culture exhibited retained a meta-
preferential dechlorination activity. The inoculum from this culture was re-pasteurized
at 90°C for 10 min prior to use.

Preparation of the experimental batch. 60—ml serum bottles were used as the
experimental vessels for the general inhibition experiment. Each serum bottle received
10 g of PCB-free Hudson River sediments. The bottles containing sediments were
evacuated and refilled with N2 in a glove box lock, then flushed with Nz-COZ (80:20,
vol/vol) with a Hungate apparatus, and received 10 ml of RAMM (21) containing 0.1%
methanol and 10% freshly prepared non-pasteurized inoculum. The serum bottles were

then incubated at 37°C for 1 week. After methane was detected, the bottles were

82

autoclaved for 1 h on 3 consecutive days with incubation at 37°C between each
autoclaving. After the third autoclaving, 10 ml of the pasteurized inoculum, 10 ml of
sterile RAMM, 100 p1 of 10% autoclaved solution of cysteine, and 80 pl of 10% PCBs
(Aroclor 1242, Monsanto Co. , St. Louis, M0.) in acetone were added. The stock
solutions of molybdate (sodium salt), sulfate (sodium sulfate), and BESA were bubbled
with N2, autoclaved and then introduced. The initial concentration of molybdate and
BESA were 5 mM, and 50 mM, respectively. The same amounts of molybdate and half
as much BESA were re-fed at 2, 4, 6, and 8 weeks. The initial concentration of sulfate
was 20 mM, and the same amount was added at 4 and 8 weeks. The controls were
autoclaved twice, 1 h each time with an interval of incubation at 37°C for 5 h before
PCBs were added. The samples were shaken for 1 h and then incubated at 25°C in dark.

For the concentration profiles experiment, the experimental vessels were 28-ml
serum tubes. The experimental batch of the concentration profiles experiment was
prepared in the same way as that of the general inhibition experiment, except that the 28-
ml serum tube received 1 g of sediment, 4.5 ml of RAMM, and 0.5 ml of inoculum, and
5 pl of 10% Aroclor 1242 in acetone. The cultures were incubated at 30°C instead of
25°C, and molybdate, sulfate, as well as BESA were added only at the beginning of the
experiment.

Analysis of methane, sulfate, and molybdate. Methane was assayed on a gas
chromatograph (V arian 3700) with a flame ionization detector.

Before being sampled for molybdate and sulfate, the culture tubes were vortexed
for 3 min. After the sediments settled, and while the tubes were flushed with N2, 1 ml

of the liquid portion was taken, acidified with 1 N HCl, and bubbled with N2 for 5 min

83
to drive out H28. The samples were then centrifuged, filtered (0.22 pm, Millipore Co. ,

Bedford, Mass.), and analyzed for sulfate and molybdate. Sulfate and molybdate were
analyzed with a high pressure ion chromatograph (Dionex) equipped with a HPIC AS4A
column (20 cm) and a conductivity detector. The mobile phase was 1.7 mM
NaHCO3/1.8 mM Na2CO3, and the flow rate was 2.3 ml/min.

PCB analysis and data summation. These procedures were as previously

described (20).

RESULTS

Results of the general inhibition experiment. Dechlorination was completely
inhibited by molybdate, sulfate, and BESA (Fig. 1) during 12 weeks of incubation. The
color of the cultures amended with either sulfate or BESA turned dark black after two
weeks of incubation. Additionally, these cultures gave a strong sulfide smell when they
were sampled while being flushed with N 2-C02. In contrast, those cultures amended
with molybdate turned yellowish, and there was no sulfide odor when these cultures were
sampled. N o methane production was detected in any of these pasteurized cultures as
previously reported (28), indicating that methanogens were eliminated by the
pasteurization.

Concentration effects of molybdate, BESA, and sulfate on PCB dechlorination
by the pasteurized cultures. Both BESA and sulfate completely inhibited dechlorination
at all concentrations tested. The lowest concentrations for BESA and sulfate were 1 mM
and 2 mM, respectively (Fig. 2). Molybdate partially inhibited dechlorination at 2, 4 and

8 mM, and completely inhibited dechlorination at 16 mM (Fig. 2).

84

 

 

 

 

‘ -—-ta— Autoclaved

I —-o— Non-amendment
2.0 " —-0— Molybdate
—-t—- BESA

- —II— Sulfate

1-5 I T I r I I
O 2 4 6 8 10 12

Incubation Time (Weeks)

 

Total Chlorines Per Biphenyl

 

 

Fig. 1. Dechlorination of Aroclor 1242 by the pasteurized microorganisms as affected
by molybdate, sulfate, and BESA. The microorganisms were eluted from Hudson River
sediments before the pasteurization. Error bars are the standard deviation (SD) of
triplicate samples; where not shown, the error bars are smaller than the symbols.

85

Efi'ects of Sulfate, Molybdate, and BESA on
Dechlorination by the Pasteurized Culture -

 

 

 

 

 

 

 

'3'.
55
a 3.3 .1 I Autoclaved
55 No Amendment
04 E 3 g 3 i U M004
8 m 3‘1- “ 33 fl 3 1 ’ [2 BESA

o 33 -‘. I 2 '
.‘3 3 :3 :3 p 3 = f
r: 3 3. 3
g :3: 3 3 5 3 g
0 2-9- 3 35 3 3
<1) 33 f 33 ;
no 3 3 l 3 v
3 3 33 3 5

33 3 . 33 3: 4

4 2.7-

1 2 4 8 15
Concentration (mM)

Fig. 2. Effects of molybdate, sulfate and BESA at different concentrations on
dechlorination by the pasteurized microorganisms. Error ‘bars are the standard deviation
(SD) of triplicate samples; where not shown, the error bars are smaller than the symbols.

86
Consumption of both sulfate and molybdate was confirmed by chromatographic

analysis. After 4 weeks of incubation, sulfate and molybdate in the 4 mM amendment
groups decreased 23.1, and 27.7% , respectively. Differences in the color of the cultures
amended with different concentrations of molybdate, BESA, and sulfate were also
observed. Compared with the non-amended cultures, the color of the cultures amended
with 8 or 16 mM sulfate or 16 mM BESA turned black, while the molybdate-amended
cultures exhibited a slight yellowish color. These colors were consistent with those
observed in the general inhibition experiment and in our similar experiment with the non-
pasteurized cultures (Chapter 3). No methane production in any culture in this

experiment was detected.

DISCUSSION

Molybdate, sulfate, and BESA all inhibited dechlorination of PCBs by the
pasteurized cultures. In the general inhibition experiment, after 12 weeks of incubation,
dechlorination was still completely blocked by all of the three anions at the experimental
concentrations. However, it was unclear whether molybdate, sulfate, or BESA at their
experimental concentrations might have had general toxic effects. Furthermore, in order
to examine the sensitivity of the dechlorination process to the inhibitors , it was necessary
to lower the experimental concentrations. Therefore, concentration profiles of
molybdate, BESA, and sulfate were screened. As shown in Fig. 2, the dechlorination
by the pasteurized cultures was more sensitive to both BESA and sulfate than to
molybdate, since both BESA and sulfate completely inhibited dechlorination at their

lowest experimental concentrations, 1 mM, and 2 mM, respectively, while molybdate did

87

not completely inhibit dechlorination at 8 mM.

BESA is a specific inhibitor of methanogens (1, 18). However, methanogens were
eliminated by the pasteurization as evidenced by the fact that no methane production was
detected in any of the pasteurized culture. Therefore, BESA probably functioned as a
sulfuroxy anion due to its sulfonic moiety, which is a potential electron acceptor for
sulfate-reducing bacteria (SRB). The black color and these cultures and the sulfide smell
support this hypothesis. This, coupled with the facts that molybdate is a specific
inhibitor of SRB and sulfate is a electron acceptor of SRB, suggests that in the
pasteurized cultures the effects of BESA, molybdate, and sulfate are all related to SRB.

In order to facilitate the discussion, we here divide all microorganisms in the
pasteurized cultures into two groups based on their relationship to SRB: one group is
composed of microorganisms not related to SRB and is designated as Group I, whereas
Group II consists of SRB and those microorganisms depending on or competing with
SRB. Microorganisms capable of dechlorinating PCBs (DCM, henceforth) in the
pasteurized cultures may belong to either group or both.

As mentioned above, in the pasteurized culture the effects of molybdate, sulfate,
and BESA are all related to SRB, while Group I only consists of microorganisms not
related to SRB. Therefore, if there were DCM in Group 1, general toxicity is the only
possible explanation.

It appears that inhibition of dechlorination by BESA was not due to toxicity.
Sparling et al. (23) examined the effect of 25 mM BESA on a wide variety of
microorganisms, including the anaerobic/facultative anaerobic spore-formers Clostridia

and Bacillus, and found that BESA had no significant side effect. Other investigators

88
also found that 25 mM BESA had no undue influence upon bacterial growth of eight

different types of anaerobes (18). The addition of 2 and 5 mM BESA to salt marsh
sediments caused moderate but discernible decrease in the rate of sulfate reduction (16
and 22 % , respectively) when compared with sediment: lacking inhibitor, this was
probably because SRB used the sulfonic acid moiety as an electron acceptor in preference
to sulfate (6). In our experiment, BESA completely blocked dechlorination by the
pasteurized cultures at concentration of 1 mM, which is much lower than 25 mM.

It is unlikely that inhibition of dechlorination by 2 mM molybdate was due to a
general toxic effect. Molybdate is generally regarded as a specific inhibitor of SRB (24)
and thus widely used in microbiology studies due to its specificity (18). Molybdate at
high concentrations may have a toxic effect on some microorganisms since it can bind
free sulfide ions to form a molybdosulfide complex (25), and this could influence
microorganisms requiring sulfide ions (18) e.g. methanogens (2, 29). However, this
toxic effect depends on experimental concentration. It is reported that 2 20 mM inhibit
microorganisms that require sulfide such as methanogens (9, 22), and also inhibited PCB
dechlorination in non-pasteurized cultures (Chapter 3). However, Lovely and Klug (l 1)
reported that a concentration at ~ 2 mM did not inhibit methanogensis in sediments. In
our experiment 2 mM molybdate partially inhibited the dechlorination. The effective
concentration in our cultures should have been even lower because some molybdate
should adsorb onto clay surfaces (19) present in the sediment slurries. Additionally,
molybdate is a component of nitrogenase and nitrate reductase, therefore, any anaerobic
nitrogen-fixers (such as some Closm'dia) and denitrifiers present would consume some

of the molybdate, further reducing its effective concentration. Therefore, at this low

89

concentration, general toxic effect is unlikely.

In summary, it is possible that some microorganism(s) in group I might be inhibited
by either 1 mM BESA, 2 mM sulfate, or 16 mM molybdate, and the sensitive
microorganism(s) happened to be the DCM. However, it is improbable that it(they)
was(were) completely suppressed by all three of these anions. Therefore, it appeared
that Group I did not contain DCM. Hence all DCM in the pasteurized culture were in
Group II.

Microorganisms in Group H can be subdivided into three categories: (i) those
competing with SRB, (ii) those dependent on SRB, and (iii) SRB. The DCM may belong
to one or all three categories. It is possible to distinguish which category they belong
to based on the effects of sulfate, molybdate, and BESA.

Sulfuroxy anions may inhibit anaerobic dechlorination by competing for electrons
if the DCM are either SRB or SRB-competing microorganisms. Inhibition of
dechlorination by sulfuroxy anions have been reported in many studies (7, ll, l4, l6,
17). When the DCM are SRB, sulfuroxy anions may inhibit dechlorination by diverting
electron flow to sulfuroxy-anion—reduction instead of dechlorination (10); if the DCM are
not SRB but SRB-competing microorganisms, sulfuroxy anions may also inhibit
dechlorination by diverting the electron flow away from the DCM to SRB, however, in
this case, relief of the inhibition by molybdate is expected. For example, Madsen and
Aamand (12) observed that dechlorination of chlorophenol by an enriched culture was
inhibited by sulfur oxyanions, but this inhibition could be relieved by molybdate. The
dechlorinating microorganism (DCB-2) in the enrichment was later isolated and identified

as Clostn'dium sp. (13). Since DCB-2 is not SRB, the inhibition was obviously due to

9O
competition between DCB-2 and SRB for electrons. As a second example, Gibson and

Suflita reported stimulation of the dechlorination of 2,4,5 ,-trichlorophenoxyacetic acid
by molybdate in a methanogenic aquifer amended with sulfate (8).

In our experiment, if the DCM were SRB—competing microorganisms, addition of
molybdate should enhance PCB dechlorination. Since molybdate inhibited dechlorination
by the pasteurized cultures, the results indicate that SRB-competing microorganism in the
pasteurized cultures are not among DCM.

The possibility that the DCM depend on SRB is inconsistent with the fact that
sulfate completely inhibited dechlorination at all experimental concentrations. RAMM
medium does not contain sulfate, and the sediment used in the culture was freshwater
sediment (Hudson River sediment) containing little low sulfate. Therefore, the culture
conditions are sulfate-limited, and addition of sulfate should stimulate growth of SRB and
thus benefit SRB-dependent microorganisms. If any DCM were SRB-dependent,
stimulation, instead of complete inhibition, would be expected. Integration of the results
of the BESA, molybdate, and sulfate experiments suggests that both SRB-dependent and
SRB-competing microorganisms are not responsible for the dechlorination activity in the
pasteurized cultures.

One exception to the above discussion is that the SRB were not the DCM but were
part of an obligate syntrophy associated with the DCM. Then both molybdate and sulfate
would inhibit dechlorination activity. Molybdate would inhibit dechlorination by
inhibiting SRB, while sulfate would inhibit dechlorination by driving SRB from
fermentation to anaerobic respiration, and hence disrupting the obligate syntrophy

relationship. This could be true if SRB functioned as the only H2-producer and the DCM

91

an H2-consumer. This is possible, btft not likely. Our cultures contained a mixed
community, and many anaerobic spore-formers such as some Clostridium (27) are well-
known versatile Hz-producers. Therefore, if the DCM are Hz-consumers, inhibition of
SRB would not be expected to completely block the dechlorination, since other H2
sources should be readily available.

From the above line of reasoning, it appears that the DCM in the pasteurized
cultures is (are) probably SRB. The inhibitory effects of the amendments on PCB
dechlorination can be explained by molybdate’s directly inhibition of SRB, while sulfate
and BESA inhibited dechlorination by competing with PCB as electron acceptors. This
is similar to the experiment reported by Linkfield and Tiedje (10). In that case
dechlorination by a sulfidogen, D. tiedjei, was inhibited by sulfuroxy anions, suggesting
that when grown with sulfuroxy anions, D. tiedjei prefers these sulfuroxy anions as
electron acceptors, and may divert the flow of electron from dechlorination to sulfuroxy
anion reduction (10). However, whether sulfate inhibits dechlorination by sulfate-
reducers depends on the culture conditions. After the culture medium had been
modified, dechlorination by D. tiediei occurred in the presence of sulfate (4). The above
discussion is summarized by Table 1.

The hypothesis that sulfate and BESA inhibited dechlorination by competing for
electrons is consistent with the observation of the dark black color which probably
indicated the formation of sulfide, and the sulfide smell of the sulfate- and BESA-
amended cultures. Consumption of sulfate was confirmed by HPIC analysis. Despite
that it was unclear whether the consumption was dissimulatory or assimilatory, it

nevertheless provided another possible evidence. However, we did observe inhibition

92

Table 1. The Possible Interpretation for the Results

 

Possible dechlorinating microorganism(s)

 

 

 

Inhibited

by Depending on SRB* Competing with SRB

SRB

Sulfate No Yes Yes
Molybdate Yes No Yes
BESA" No/Yes Yes/No Yes
* Sulfate-reducing bacteria
* The effects of BESA on sulfate-reducing bacteria (SRB) may vary. It may

enhance/ inhibit SRB and hence may promote/ suppress microorganisms depending—
on/competing-with SRB depending on the culture conditions (e. g. balance between
the available electron donor and sulfate concentration of the culture).

93

of SRB by molybdate in our similar experiment with untreated cultures. In that
experiment, supplementation with sulfate (to inhibit methanogens by promoting SRB)
inhibited methanogensis, but failed to inhibit methanogensis in presence of molybdate
(Chapter 3).

The inhibitory effects resulting from competition for electrons may depend on the
balance between the sulfuroxy anions and the available electron donors. In some studies
addition of electron donors to deplete available sulfate allowed dechlorination to start (10,
17). May et al (17) reported that when 20 mM sulfate was added to anaerobic sediment
slurries spiked with PCBs, dechlorination was not observed until after 15 weeks when
sulfate was depleted. In our experiment the culture medium was pre-incubated to ensure
a strictly anaerobic condition, and was not supplemented with electron donor(s). The
prc-incubation procedure should coMe some electron donor(s). Additionally,
pasteurization should eliminate some fermentative microorganisms, decreasing the
potential of the microbial community to utilize electron donors from the sediment
contained in the culture. These differences may explain why concentrations of BESA and
sulfate as low as 1, and 2 mM completely inhibited dechlorination by the pasteurized
cultures.

Our pasteurization conditions should eliminate non-sporeformers, and the possibility
of thermophiles can also be ruled out as previously discussed (28). Therefore, the DCM
in the pasteurized cultures should be sporeformers. Among SRB the only known genus
forming spores is Desulfotomaculum. Therefore, these results suggest that
microorganisms responsible for dechlorination activity in our pasteurized cultures are

most probably Desulfotomaculum-like sporeforming SRB. Attempt to isolate the

94

responsible dechlorinating microorganism(s) did not succeed. To current knowledge
genus Desulfotomaculum is diverse (5), the dechlorinating microorganism(s) in our
pasteurized cultures may be very different from those have been described. However,
isolation of the dechlorinating Desulfotomaculum-like sporeforming SRB (pure strain(s)
or in obligatory syntrophy) and demonstration of the dechlorination by the isolate(s) are
required in order to make a final conclusion. On the other hand, we do not extrapolate
this proposition to other enrichments. Sporeforming bacteria are a diverse group, and
different enrichments may have different sporeforming populations depending on
inoculum source, pasteurization technique, and culture conditions. The proposition that
the DCM in our pasteurized cultures is(are) Desulfotomaculum—like sporeforming SRB
does not rule out the possibility that other sporefomer(s) may also have PCB

dechlorination capability.

95
REFERENCES

1. Balch, W. E., and R. S. Wolfe. 1979. Specificity and biological distribution of
coenzyme M (2-mercaptoethanesulfonic acid). J . Bacteriol. 137 :256-263.

2. Bhatnagar, L., M. K. Jain, and J. G. Zeikus. 1991. p. 251-270. Methanogenic
bacteria. In Variations in autotrophic life. Academic Press, Orlando, FL.

3. Brown, J. F., R. E. Wagner, D. L. Bedard, M. J. Brennan, J. C. Carnahan, R.
J. May, and T. J. Tofflemire. 1984. PCB transformations in upper Hudson sediments.
Northeast. Environ. Sci. 3:167-179.

4. Cole, J ames. Personal communication.

5. Devereux, R., M. Delaney, F. Widdel, and D. A. Stahl. 1989. Natural relationships
among sulfate-reducing bacteria. J . Bacteriol. 171:6689-6695.

6. Dicker, H. J. and D. W. smith. 1985. Effects of organic amendments on sulfate
reduction activity, H2 consumption, and H2 production in salt marsh sediments.
Microbial. Ecol. 11:299-315.

7. Genthner, S. B. R., W. A. Price, and P. H. Pritchard. 1989. Anaerobic
degradation of chloroaromatic compounds in aquatic sediments under a variety of
enrichment conditions. Appl. Environ. Microbiol. 55:1466-1471.

8. Gibson, S. a, and J. M. Suflita. 1990. Anaerobic biodegradation of 2,4,5-
trichlorophenoxyacetic acid in samples from a methanogenic aquifer: stimulation by
short-chain organic acid and alcohols. Appl. Environ. Microbiol. 56:1825-1832.

9. Jones, J. G., B. M. Simon, and S. Gardner. 1982. Factors affecting methanogensis
and associated anaerobic processes in the sediments of a stratified eutrophic lake, I . Gen.
Microbiol. 128:1-11.

10. Linkfield, T. G., and J. M. Tiedje. 1990. Characterization of the requirements and
substrates for reductive dehalogenation by strain DCB-l. J. Ind. Microbiol. 5:9-15.

11. Lovley, D. R., and M. J. Klug. 1983. Sulfate reducers outcompete methanogens
at fresh water sulfate concentrations. Appl. Environ. Microbiol. 45:187-192.

12. Madsen, T., and J. Aamand. 1991. Effects of sulfuroxy anions on degradation of
pentachlorophenol by methanogenic enrichment culture. Appl. Env. Microbiol. 57:
2453-2458.

13. Madson, T., and D. Licht. 1992. Isolation and characterization of an anaerobic
chlorophenol-transformating bacterium. Appl. Env. Microbiol. 58:2874-287 8.

96

4. May, H. D., A. W. Boyle, W. A. Price, II, and C. K. Blake. 1992. Dechlorination
of PCBs with Hudson River bacterial cultures under anaerobic conditions: effect of
sulfate, sulfite and nitrate. 1992. Annu. Meet. Am. Soc. Microbiol.

15. May, H. D., A. W. Boyle, W. A. Price 11, and C. K. Blake. 1992. Subculturing
of a polychlorinated biphenyl-dechlorinating anaerobic enrichment on solid media. Appl.
Environ. Microbiol. 58:4051-4054.

16. Mohn, W. W., and J. M. Tiedje. 1992. Microbial reductive dechlorination.
Microbiol. Rev. 56:482-507.

17. Morris, P. J., W. W. Mohn, J. F. Quensen III, J. M. Tiedje, and S. A. Boyd.
1992. Establishment of a polychlorinated biphenyl-degrading enrichment culture with
predominantly meta dechlorination. Appl. Environ. Microbiol. 58:3088-3094.

18. Oremland, R. S., and D. G. Capone. 1988. Use of "specific" inhibitor in
biogeochemistry and microbial ecology. In K. C. Marshall (ed.), Advances in microbial
ecology. vol. 10. Plenum Publishing Co.

19. Phelan, P. J., and Mattigod, s. v. 1934. Adsoption of Molybdate anion (M0042'
)by sodium-saturated kaolinite. Clas, Clay Minerals 32:45-48.

20. Quensen, J. F., III, S. A. Boyd, and J. M. Tiedje. 1990. Dechlorination of four
commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic microorganisms
from sediments. Appl. Environ. Microbiol. 56:2360—2369.

21. Shelton, D. R., and J. M. Tiedje. 1984. General method for determining anaerobic
biodegradation potential. Appl. Environ. Microbiol. 47:850-857.

22. Smith, L. R. and M. J. Klug. 1981. Electron domors utilized by sulfate-reducing
bacteria in eutrophic lake sediments. Appl. Environ. Microbiol. 42: 1 16-121.

23. Sparling, R. and Daniels L. 1987. The specificity of growth inhibition of
methanogenic bacteria by bromoethanesulfonate. Can. J. Microbiol. 33:1132-1136.

24. Taylor, B. F., and R. S. Oremland. 1979. Depletion of adenosine triphosphate in
Desulfovibio by oxyanions of group VI elements. Curr.Microbiol. 3: 101-103

25. Tonsager, S. R., and B. A. Averill. 1980. Difficulties in the analysis of acid-labile
sulfide in Mo-S and Mo-Fe-S systems, Anal. Biochem. 102:13-15.

26. Widdel, F. 1988. Micribiology and Ecology of sulfate- and sulfur-reducing bacteria.
1:. 469-585. In A. J. B. Zehnder (ed.), Biology of anaerobic microorganisms. John Wiley
8:. Sons, Inc., New York.

97

27. Wood, W. A. 1961. Fermentation of carbohydrate and related compounds. p.59-149.
In I. C. Gunsalus and R.Y. Stanier (ed.), The bacteria, Vol 2. Academic Press, New
York.

28. Ye, D., J. F. Quensen III, J. M. Tiedje, and S. A. Boyd. 1992. Anaerobic
dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-treated
microorganisms from sediments. Appl. Environ. Microbiol. 58:1110—1114.

29. Zeikus, J. G.. 1977. The biology of methanogenic bacteria. Bacteriol. Rev. 41:514-
541.

Chapter 5

Differentiation of Three Classes of Congener—specific

Dechlorination Activity on Aroclor 1242

98

99

ABSTRACT

Two classes of congener specific microbial dechlorination activity on Aroclor 1242

in Hudson River sediments have been reported previously, one mainly removes meta
chlorines, while the other mainly removes para chlorines. Results of temperature
experiments presented here indicate three (one meta, two para) classes of dechlorination
activity on Aroclor 1242 by microorganisms from the Hudson River sediments. The
previously recognized para dechlorination activity (Pattern Q activity) actually consists
of two activities: one that dechlorinates para-substituted congeners without meta chlorines

(p0), and the other mainly dechlorinates para chlorines from congeners containing meta

chlorines (pm). The p a dechlorination activity was lost from the untreated cultures when

the temperature was raised from 25°C to 30°C perhaps because the responsible

microorganisms were unable to successfully compete for nutrients, and this para

dechlorination activity was nutrient—dependent. The pm dechlorination activity is very

similar to that exhibited by our methanogenic enrichment, which is very close to

environmental dechlorination process H seen in the tidal Hudson. Decrease in the lag
time of the meta-preferential dechlorination by heat shock was observed probably due to
the activation effects of heat shock, and this observation supports the previous suggestion
that anaerobic sporeformers are among the meta-preferential dechlorinating
microorganisms. Compared with the two para dechlorination activities,the meta-
preferential dechlorination is a fast precess. Despite the fact that the pa activity lost at
30°C, 30°C was more favorable than 25°C for both meta-preferential and the pm

dechlorination activities.

 

100
INTRODUCTION

Temperature is an important factor affecting biodegradation (8, 9). Temperature
is one of the selective factors in determining the dominant populations and resultant
metabolic activities in dynamic microbial communities (9, 11, 17). In anaerobic
microbial dechlorination research, isolation of pure strains capable of aryl dechlorination
is difficult often due to the complex trophic relationship among the involved microbial
populations. As a result, indirect methods with different selective pressures including
incubation temperature have been used to differentiate the involved microorganisms and
their dechlorination capacities (10, 19, 20). Kohring et al. (10) screened anaerobic
biodegradation of 2,4-dichlorophenol (2,4-DCP) in freshwater lake sediments at different
temperatures and observed two peak activities in different temperature ranges. Based on
this observation they proposed that at least two different microorganisms were involved
in the transformation of 2,4-DCP to 4-CP. Wu et al. (19) examined the effect of
temperature on anaerobic transformation of 2,3,4,6-tetrachlorobiphenyl (2,3,4,6,-CB) in
methanogenic pond sediment slurries, and observed that the fastest dechlorination
occurred at 30°C. A higher rate of dechlorination was observed at 50, 55, and 60°C
than at 37, 40, and 45°C, indicating two dechlorinating communities.

Tiedje et al. (16) investigated dechlorination of Aroclor 1242 at 12, 25, 37, 45, and
60°C by microorganisms eluted from Hudson River sediments, and found that the optimal
temperature for the dechlorination activity was 25°C. No dechlorination activity was
observed at 37°C and above, suggesting that the microorganisms involved have similar
optimal and tolerant temperature ranges. Despite the fact that only one peak activity was

found , however, two dechlorination activities have been reported (one is meta-

101

preferential, the other is mainly para-removal) (2), and different dechlorination activities
usually reflect the involvement of different microorganisms which may have different
temperature preferences.

Heat- and ethanol-treatments were used to eliminate microorganisms from a mixed
PCB dechlorinating population. The resulting simplified p0pulation retained a meta-
preferential dechlorination activity (20), providing a way to separate microorganisms
capable of meta dechlorination from the mixed community. It thus became possible to
characterize the temperature requirement of the meta-preferential dechlorination activity.
By comparing the dechlorination at different temperatures by both the pasteurized and
the untreated cultures, information about the temperature requirement of the para
dechlorination activity can also be obtained. Because both incubation temperature and
pasteurization are powerful selective forces, the combination of both should more
effectively ”fractionate" the diverse microbial populations in the community, providing
information about the composition of the mixed community. The results presented here
indicate three distinct PCB (Aroclor 1242) dechlorination activities by microorganisms

from Hudson River sediments.

MATERIALS AND METHODS
Inoculum preparation and pasteurization. Sediments were collected from the
upper Hudson River near Hudson River Falls, N.Y. , and shipped under anaerobic condi-
tions to the laboratory as previously described (13). Inoculum preparation and the
Pasteurization technique were as previously described (20) except that the pasteurization

temperature was 85°C, the pasteurization time was 15 min, and the pasteurization was

102

timed after the temperature in the monitor tube reached 83°C.

Experimental batch preparation. Anaerobic culture tubes (28-ml, Bellco Glass
Inc., Vineland, N .J .) containing PCB-free dry Hudson River sediment (1 g per tube)
were sealed with rubber stoppers in an anaerobic chamber. The PCB-free sediment was
collected from the Hudson River at river mile 205‘. The sediment was air-dried and
sieved with a 2-mm-mesh screen. The sediment was then moistened with 1 ml of
Reduced Anaerobic Mineral Medium (RAMM) (15) containing 0.1% methanol and 10%
inoculum. The tubes were then pre-incubated (20). After formation of methane was
confirmed, all tubes were autoclaved at 121°C for 1 h on 3 consecutive days with
incubation at 37°C between each autoclaving.

After the third autoclaving the following were added to each preincubated culture
tube while flushing with filter-sterilized (0.22 pm, Millipore Co. , Bedford, Mass.) 02-
free N2_-C02 (80:20, vol/vol) using a Hungate apparatus: 1 ml of inoculum, 2 ml of
RAMM, 6 pl of 10% (wt/vol) Aroclor 1242 (Monsanto Co., St. Louis, Mo.) in acetone.
The final concentration of PCBs was 600 ug/ g sediment (dry weight). Autoclaved 10%
solution of cysteine was added at a final concentration of 250' mg/L. The tubes were
then sealed with Teflonocoated butyl rubber stoppers (West Co. , Phoenixville, Pa.) and
aluminum crimps, and the contents were mixed with a Vortex mixer to completely
disperse the PC35. The controls were autoclaved twice at 121°C for 1 h with an interval
of 5 h before PCBs were added. For the pasteurized treated cultures the inocula was
pasteurized before being introduced into the incubation tubes.

Incubation. The tubes were incubated in the dark at 10, 25 , 30, 37, 50, and 65°C.

To confirm the existence of 02 free conditions of the different temperatures, triplicate

103

monitor tubes containing 5 ml of RAMM, cystine (250 mg/L) and resazurin, without any
sediments, were prepared and incubated at each temperature along with the experimental
tubes.

Analysis of methane production. Headspace gas of the samples was analyzed for
determination of methane production'before the samples were extracted for PCB analysis.
Methane was detected by gas chromatography using a flame ionization detector.

Sampling, extraction, analysis, and data summation. These were as previously

described (13).

RESULTS

Anaerobic conditions over the experimental temperature range. During 12
weeks of incubation all 02-monitor tubes retained their black color indicating no 02
contamination except for one of the triplicates incubated at 50°C which turned pink after
7 weeks of incubation. Inspection showed that there was a tiny breach at the mouth of
the serum tube, and this was probably the reason for the oxygen contamination in this
tube. These results confirmed that all cultures were under strictly anaerobic conditions
at the different incubation temperatures.

Temperature profiles of the dechlorination activities. Dechlorination only
occurred in those cultures incubated at either 25 or 30°C, no dechlorination was observed
when cultures were incubated at 37, 50, 65, and 10°C (Fig.1). As previously described
(20), no methane production was observed by the pasteurized cultures, while all unn'eated
cultures produced methane. The highest methane production occurred at 25°C (Fig. 2).

Temperature profiles for the dechlorination activities of both the treated and the untreated

104

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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bars are the standard deviation (SD) of triplicate samples; where not shown, the error
bars are smaller than the symbols.

105

 

 

 

 

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106

 

 

 

 

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107

 

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108

 

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109

cultures are illustrated in Fig. 3.

Differences in the dechlorination patterns at 25°C and 30°C. There was no
difference in dechlorination pattern shown by the pasteurized cultures at 25 or 30°C.
However, when incubated at 30°C, the untreated cultures lost some dechlorination
activity on 2-4-CB/2,3-CB (peak 6), 2,4-2-CB(peak 10), 2,6-4-CB (peak 12), and 2,4—4—
CB (peak 18). As shown in Fig 4, comparison between untreated cultures incubated at
25°C and 30°C showed that the 30° cultures accumulated more congeners associated with
peaks 6, 10, 12, and 18, and less congeners associated with peaks 1 (2-CB) and peak 3
(2-2/2—6-CB), which are the para dechlorination products of peaks 6, 10, 12, and 18.
The congeners associated with peaks 6, 10, 12, and 18 are para-substituted without meta
chlorines and with at least one ortho chlorine attached to the biphenyl rings. Despite the
loss of this para dechlorination activity, however, 0.24 more chlorines were removed
from each biphenyl molecule after 4 weeks when the untreated cultures were incubated
at 30°C than when they were incubated at 25°C (Fig. 1A). Similar enhancement effect
was also observed with the pasteurized cultures (Fig. 1B). There was a difference
between the dechlorination extension after 8 weeks of incubation between at 25 and at
30°C by the untreated cultures (Fig. 1A), this was due to losing the para dechlorination
activity at 30°C.

Differences in the 30°C dechlorination patterns between pasteurized and
untreated cultures. Comparison of the 30°C dechlorination patterns between the
pasteurized and the untreated cultures showed that the pasteurized cultures lost some
dechlorination activities on congener peaks 35 (2,4,5-4-CB), 36 (2,5-3,4-CB), 37 (2,4-

3,4-CB/2,4,5-2,6-CB/2,3,6-2,5—CB), 39 (2,3-3,4—CB/2,3,4-4-CB), 19 (3,4-2—CB/2,3,4—

110
CB/2,3-3-CB/2,5-2,6-CB), and 29 (2,3—2,4-CB/2,3,6-3—CB/3,4-4-CB) (Fig.5). These

are all para-substituted congeners with meta chlorines.
Differences in the 25°C dechlorination patterns between pasteurized and
untreated cultures. We previously reported the differences in the dechlorination
patterns between the untreated and the pasteurized cultures at 25°C (20), and in the
present experiment identical results were obtained. After 12 weeks of incubation, the
unueated cultures showed both meta and para dechlorination activities while the
pasteurized cultures lost some para dechlorination activity and thus, showed a meta-
preferential dechlorination activity. The para dechlorination activities lost from the
pasteurized cultures were actually the activities lost from the untreated cultures when
temperature changed from 25°C to 30°C, plus those para activities remaining at 30°C
but eliminated by not being able to withstand the pasteurization
Decrease in the lag time of the dechlorination by heat shock. In our previous
exPeriment (the July experiment mentioned in Chapter 3) decrease in the lag time of the
dechlorination by the heated cultures was observed (Fig. 6). After 2 weeks of incubation
dechlorination occurred in the pasteurized cultures but not the untreated cultures.
Timecourse of different dechlorination activities. Analysis on the dechlorination
Patterns showed that during 4 weeks of incubation with either pasteurized or untreated
cillltures, only meta dechlorination activity was observed and this dechlorination was a
I'Iilpid processes. The two kinds of para dechlorination activities were observed in the
uIltreated cultures only after 4 weeks of incubation, and reached the maximum at 12

Weeks.

111

 

 

 

 

 

 

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g iAutoclaved
E: 3.2 "’--"
m ' Untreated
h ' O
a
9..
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a)
E
3 SOC/15min
£1 ..
U 2.8
:9, A
5‘3
2.6 I I I I
O 1 2 3 4

Incubation Time (Weeks)

Fig - 6. Dechlorination of Aroclor 1242 by microorganisms from Hudson River
s'5=<1iments. Error bars are the SD of triplicate samples; where not shown, the error
bars are smaller than the symbols.

1 12
DISCUSSION

No dechlorination of Aroclor 1242 by microorganisms from Hudson River
sediments was observed at temperatures of 37°C and above, consistent with results
reported by Tiedje et al. (16) but in contrast to those of Wu et al. (19) for the
dechlorination of 2,3,4,6-CB in methanogenic pond (Wood Pond, Le.nox, Mass.)
sediment. It appears that the microorganisms capable of 2,3 ,4,6-CB dechlorination at
thermophilic temperatures reported by Wu et al. (19) were not present in the Hudson
River inocula used in our experiments.

Two classes of dechlorination activities on Aroclor 1242 in the Hudson River
sediments have been reported previously (2). In the present study, comparison of the

dechlorination patterns between the pasteurized and the untreated cultures at different
temperatures showed three classes of dechlorination activity on Aroclor 1242 by
microorganisms from the Hudson River sediments. The previously (2) recognized para
dechlorination activity (Pattern Q) actually consists of two activities: one on para-
substimted congeners without meta chorines and with at least one ortho chlorine attached
to the biphenyl ring (designated as p0 activity), and the other on para chlorines from
c011geners containing meta chlorines (pm activity). The dechlorination activities exhibited
by the untreated cultures incubated at 30°C is actually composed of both the meta-
Preferential dechlorination activity (m, activity) and the pm activity.

Both the pa and the pm dechlorination activities could not survive the pasteurization,
hOwever, the pa activity was more unstable than the pm activity as by the observation that

it Was lost from the untreated cultures when incubated at 30°C. A temperature increase

from 25°C to 30°C may directly inhibit the microorganisms responsible for the pa

113

activity. However, competition between the microorganisms may be a more reasonable
explanation. Microbial metabolic rate changes as temperature increases, and those more
stimulated by the increasing temperature may out compete more slow-growing
populations. This is especially in nutrient-limiting cases like our culture conditions in
which the medium contains no substrate except the limited sediment organic matter.
In our previous experiment (20), and here again, we observed that the para
dechlorination activity of the inoculum changed with time; as the inoculum aged, some
para (mainly the pa) dechlorination activities was lost (Fig. 7). It was also noted that
timely transfers significantly increased the para (mainly the p 0) dechlorination activities
(Fig. 8). Both observations support the idea that the pa activity is nutrient—dependent.
This is further supported by the effects of preincubation on dechlorination. We have
observed that non-preincubated cultures showed all three dechlorination activities (Pattern
C) (2), while those preincubated lost the pa activity probably because during the
preincubation procedure some nutrients in the medium were consumed (unpublished
data). These results not only suggest that the pa activity was nutrient-dependent, but also
that the relationship between microorganisms capable of the m, and the p 0 dechlorination
is most likely competitive. Abramowitz et al. (2) reported that addition of cysteine
hydrochloride at 1 mg/ liter resulted in para-dominated dechlorination activity (pattern Q).
The exact cause of this effect is unclear but it does provide additional evidence that
nutrients play a key role in determining the expression of dechlorination activities.
Considering these points it seems reasonable that at. 30°C the pa activity was lost from
the untreated cultures because microorganisms responsible for this activity lost

competition for nutrients.

114

 

 

 

 

 

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NU
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l Decreasing peaks

Mole Percent
I I
M ._e ...e
O O O O
lllllllllllhlllllllllllllllllllll

I
or
o

 

 

 

Fig. 7. Mole percentage of Aroclor 1242 represented by each chromatographic peak
after 12 weeks incubation at 25°C with freshly prepared and aged Hudson River
inoculum. For peak assignments, see reference (13).

Mole Percent Mole Percent

Mole Percent

115

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1'7 iiiiiii I IIIIIIIII l IIIIIIIII I IIIIIIIII I IIIIIIIII l UUUUUUUUU i IIIIIIIII I IIIIIIIII I IIIIIIII
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0 10 20 30 4O 50 50 70 80 90
‘ IIIIIIIII l’ TTTTTTTTT F IIIIIIII I IIIIIIIII I IIIIIIIII I UUUUUUUUU l IIIIIIIII I fififififififi I IIIIIIIII
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- Ongmal culture -
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O 10 20 30 40 50 60 70 80 90

Fig. 8. Mole percentage of Aroclor 1242 represented by each chromatographic peak
after 12 weeks incubation at 25°C with original cultures and the transferred (25%)
cultures. For peak assignments, see reference (13).

116

In contrast to the unstable p0 dechlorination activity, the m, activity was not only
resistant to the heat- and ethanol-treatments, but also stable over time and through serial
transfers (20). Moreover, after being heated at 85°C for 15 min, the m, activity was not
eliminated, instead, it was stimulated. There are two possible explanations: i) beat
eliminated some competitors of the dechlorinating microorganisms, and ii) heat
stimulated spore germination. The first possibility is not likely as evidenced by the
following facts: first, such stimulative effect was never observed in the ethanol-treated
cultures (including same batch). Microorganisms eliminated by heat should also be
eliminated by the ethanol-treatment, and if the stimulative effect was due to elimination
of competing microorganisms, in the same experimental batch the stimulative effect
should also be observed in the ethanol-treated cultures. Second, the activation effect
varied from one inoculum batch to another, probably depending on whether most
sporeforming bacteria in the inoculum were in the form of vegetative cells or bacteria
spores. It appears that the stimulative effect is probably because that heat shock
stimulates spore germination (14). The activation results provided additional evidences
supporting the previous conclusion (20) that anaerobic sporeformers are among the
physiological groups responsible for the meta-preferential dechlorination activity in the
pasteurized cultures. Morris et al. (12) also suggested that meta dechlorinating
microorganisms may be more robust or more broadly distributed among microbial
groups. '

The pm dechlorination activity was very similar to the dechlorination activity
expressed by our methanogenic enrichment. This dechlorination is very close to the

environmental alteration process H seen in the tidal Hudson (7, 7a). -

117

While the pa activity was eliminated when cultures were incubated at 30°C, the
rates of both the m2 and the pm dechlorination accelerated when temperature increased
from 25°C to 30°C. In general, higher temperatures that do not kill the microorganisms
result in higher metabolic activities (3). Enzymes usually have Q10 value near 2 (3), that
is, when temperature increased 10°C within the tolerance range, the activity is roughly
doubled. As to microbial population, for example, it was reported that sulfate reducers
in marsh sediment had a Q10 of 3.5 for sulfate reduction (1).

At 30°C enhancement of the m, activity did not suppress the pm activity in our
nutrient-limiting cultural conditions, suggesting that the microorganisms involved in these
two dechlorination activities either had different nutrient requirements, or had a
somewhat cooperation relationship. It is well known that methanogens depend on
fermentative bacteria for substrates, and that the consumption of the fermentation
products by methanogens is beneficial, or even necessary for fermentative bacteria (4,
5).

When both meta and para dechlorination expressed, meta dechlorination activity
always occurred first. Compared with the two para activities, the m, dechlorination
process usually completed within a short time, suggesting that this dechlorination is a
rapid process. Boyle et al. (6) also reported a strict meta dechlorination of 2,3,6-CB by
the cultures enriched from Hudson River sediments and concluded that it was a rapid
dechlorination. There are three possible explanations for the different timecourse
between the meta and para dechlorination activities. (i) The para-removal
microorganisms were relatively slow-growing, while microorganisms responsible for the

m, activity were fast-growing. (ii) The initial population size of - the para-removal

118

microorganisms was smaller, and hence it took time to increase the population size to a
threshold level necessary for detectable activity, while microorganisms comprising the
m, activity were dominant in the inoculum. (iii) accumulation of para-substituted
congeners from the meta dechlorination induced the production of the para-dechlorination
enzyme(s). However, the para-dominant dechlorination activity (pattern Q) indicated that
accumulation of para-substituted congeners was not required for the para dechlorination
activity, and thus ruled out this possibility. It seems that both the first and the second
explanations are possible.

Tiedje et al. reported dechlorination at 12°C. Wu et al. also observed
dechlorination of 2,3,4,6-CB starting from 4°C. In this experiment those cultures
incubated at 10°C showed little dechlorination activity within 16 weeks. ’ Prolonged
incubation time may be needed at these lower temperatures.

Results of the temperature experiment demonstrated and partially characterized
three anaerobic PCB dechlorination activities by microorganisms from Hudson River
sediments. These results indicate that isolation of different microorganisms capable of
anaerobic PCB dechlorination and enhancement of different in situ microbial

dechlorination activities are possible.

1 19
REFERENCES

l. Abdollahi, H., and D. B. Nedwell. 1979. seasonal temperature as a factor
influencing bacteria sulfate reduction in a saltmarsh sediment. Microb. Ecol.5:73-79.

2. Abramowicz, D. A., M. J. Brennan, and H. M. Van Dort. 1989. Microbial
dechlorination of PCBs. I. Aroclor mixtures. p.49-59. In Research and development

program for the destruction of PCBs. GE Corporate Research and Development,
Schenectady, N. Y.

3. Atlas, R. M., and R. Bartha. 1987. Microbial Ecology. The Benjamin/Cummings
Publishing Company, Inc. Menlo Park, CA.

4. Berry, D. F., a. J. Francis, and J.-M. Bollag. 1987. Microbial metabolism of
homocyclic and heterocyclic aromatic compounds under anaerobic conditions. Microbiol.
Rev. 51:43-59.

5. Bhatnagar, L., M. K. Jain, and J. G. Zeikus. 1991. Methanogenic bacteria. p.251-
270. In Variations in autotrophic life. Academic. Orlando, FL.

6. Boyle, A. W., W. A. Price 11, and H. D. May. 1992. Rapid and sustained meta
dechlorination of 2,3 ,6—trichlotobiphenyl under Anaerobic conditions. Q-29, p. 340.
Abstr. 928t Gen. Meet. Am. Soc. Microbiol. 1992, American Society for Microbiology,
Washington, DC.

7. Brown, J. F., and R. W. Lawton. 1989. Identification of PCB transformations
occurring in nature. p.85-93. In Research and development program for the destruction
of PCBs. GE Corporate Research and Development, Schenectady, N. Y.

7a. Brown, J. F. . Personnel communication.

8. Cooney, J. J. 1984. The fate of petroleum pollutants in freshwater ecosystem, p.399-
433. In Atlas, R. M. (ed.) Petroleum Microbiology. Macmillan Publishing Co. New
York.

9. Dort, H. M. V., and D. L. Bedard. 1991. Reductive ortho and meta dechlorination
of a polychlorinated biphenyl congener by anaerobic microorganisms. Appl. Environ.
Microbiol. 57:1576-1578.

10. Kohring, G-W., J. E. Rogers, and J. Wiegel. 1989. Anaerobic biodegradation of
2,4—dichlorophenol in freshwater lake sediments at different temperatures. Appl. Environ.
Microbiol. 55:348-353.

11. Leahy, J. G., and R. R. Colwell. 1990. Microbial degradation of hydrocarbons in
the environment. Microbiol. Rev. 54:305-315.

120

12. Morris, P. J., W. W. Mohn, J. F. Quensen 111, J. M. Tiedje, and S. A. Boyd.
1992. Establishment of a polychlorinated biphenyl-degrading enrichment culture with
predominantly meta dechlorination. Appl. Environ. Microbiol. 58:3088-3094.

13. Quensen, J. F., III, S. A. Boyd, and J. M. Tiedje. 1990. Dechlorination of four
commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic microorganisms
from sediments. Appl. Environ. Microbiol. 56:2360-2369.

14. Russell, A. D. 1982. The destruction of Bacterial Spores. Academic Press, New
York.

15. Shelton, D. R., and J. M. Tiedje. 1984. General method for determining anaerobic
biodegradation potential. Appl. Environ. Microbiol. 47 :850-857.

16. Tiedje, J. M., J. F. Quensen 111, J. Choc-Sanford, J. P. Schimel, and S. A.
Boyd. 1992. Microbial reductive dechlorination of PCB’s. Session 15. In Pacific basin
conference on hazardous wastes, 1992 Proceeding, Bangkok, Thailand. East-West center,
Honolulu, Hawaii.

17. Visser, F. A., J. B. V. Lier, A. J. L. Macario, and E. C. D. Macario. 1991.
Diversity and population dynamics of methanogenic bacteria in a granular consortium.
Appl. Environ. Microbiol. 57:1728-1734.

18. Ward, J. B. 1987. Biosynthesis of peptidaglycan: points of attack by wall inhibitors.
p. 1-43. In D. J. Tipper (ed.), Antibiotic inhibitors of bacterial cell wall biosynthesis.
Pergamon press Oxford, England.

19. Wu, Q.,D. Bedard, and J. Wiegel. 1992. Effect of temperature on the anaerobic
transformation of 2,3 ,4,6-tetrachlorobiphenyl in methanogenic pond sediment slurries.
Q-25, p.339. Abstr. 92st Gen. Meet. Am. Soc. Microbiol. 1992, American Society for
Microbiology, Washington, DC.

20. Ye, D., J. F. Quensen [[1, J. M. Tiedje, and S. A. Boyd. 1992. Anaerobic
dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-treated
microorganisms from sediments. Appl. Environ. Microbiol. 58:1110-1114.

SUMMARY

Addition of molybdate, BESA, and sulfate inhibited concomitantly PCB
dechlorination and methane formation, suggesting that either sulfate-reducing bacteria or
methanogenic bacteria, or both, were involved in anaerobic PCB dechlorination.

Further investigations were then conducted to examine whether methanogenic
bacteria are capable of PCB dechlorination. The results showed that methanogenic
bacteria are probably among the physiological groups capable of anaerobic PCB
dechlorination. This conclusion is based on the following observations: (i) In sediments
containing Aroclor 1242, addition of eubacteria-inhibiting antibiotics, which should
directly inhibit fermentative bacteria and thereby indirectly inhibit methanogens, resulted
in no dechlorination activity nor methane production. However, when substrates for
methanogenic bacteria were provided along with the antibiotics (to free methanogens
from dependence on eubacteria), concomitant methane production and dechlorination of
PCB’s were observed. (ii) Both methane production and decthrination in the culture
amended with antibiotics plus methanogenic substrates were inhibited by BESA, the
specific inhibitor of methanogenic bacteria. The inhibitory effects of BESA on the
dechlorination was due to the direct inhibition on methanogens because BESA at the
experimental concentration did not inhibit PCB dechlorination by other microorganisms.

The dechlorination of Aroclor 1242 by the methanogenic culture was mainly para-
removal, and was distinctly different from, and more limited than, that observed with

untreated or pasteurized inocula. This dechlorination process was very similar to the

121

122

environmental dechlorination process H.

Anaerobic sporeformers are another physiological group responsible for the
reductive dechlorination of PCBs. The pasteurized and ethanol-treated cultures retained
a partial dechlorination activity, suggesting that the responsible microorganisms survived
both pasteurization and the ethanol treatment. The dechlorinating microorganisms were
neither methanogens nor thermophiles since no methane production was detected in any
treated culture, and no dechlorination was observed when the cultures were incubated at
37°C and above. Additionally, the dechlorinating microorganisms withstood not only
high temperatures but also 50% ethanol for l h, which should eliminate thermophiles.
Besides the above results, the dechlorination activity of the treated cultures was stable
in the inoculum over time and through serial transfers, indicating that the responsible
microbial population was stably maintained, and this stability is consistent with the
survival capability of sporeformers.

The treated cultures had a common dechlorination activity regardless of the type
of treatment. This activity preferentially removed meta chlorines yielding a
dechlorination pattern characterized by accumulation of certain ortho- and para-
substituted congeners. In contrast, the untreated cultures showed more extensive
dechlorination activities, which almost completely removed both meta and para chlorines
from Aroclor 1242. These results suggest that microorganisms surviving the heat and
ethanol treatments preferentially remove meta chlorines, while the para dechlorination
activities are mainly contributed from microorganisms eliminated by such treatments.

The dechlorinating microorganisms in the pasteurized and ethanol-treated cultures

are probably Desulfotomaculum-like sulfate-reducing bacteria. The dechlorination by the

123
pasteurized cultures was inhibited by both molybdate, BESA, and sulfate. As discussed

in Chapter 4, it appeared that the inhibition was not due to general toxicity but due to the
effects on sulfidogens. Molybdate probably directly inhibited sulfidogens, while both
sulfate and BESA probably inhibited the dechlorination by competing with PCB as
electron acceptors. Only sporeforming bacteria are expected to survive the harsh
pasteurization, and among sulfidogens the only known genus forming spores is
Desulfotomaculum. Therefore, microorganisms responsible for the dechlorination of
PCBs in our pasteurized cultures are probably Desulfotomaculum-like sulfate-reducing
bacteria. However, isolation of the microorganism(s) and demonstration of PCB
dechlorination activity with the isolate(s) are required in order to make a final
conclusion. On the other hand, anaerobic sporeformers are a diverse group, different
enrichments may have different sporeforming populations depending on inoculum source,
pasteurization technique, and culture conditions. The proposition that the dechlorinating
microorganisms in our pasteurized cultures are Desulfotomaculum-like sulfate-reducers
does not rule out the possibility that other sporefomer(s) may also have PCB
dechlorination capability.

Temperature experiments demonstrated and partially characterized three classes of
dechlorination activity. One was meta-preferential, while the other two were mainly
para-removal. The major difference between the two para dechlorination activities is
that one removes para chlorines from congeners without meta chlorines (the pa activity)
whereas the other mainly removes para chlorines from congeners containing meta
chlorines (the pm activity). The pm activity is very similar to that exhibited by the

methanogenic cultures, and this dechlorination process is very close to the environmental

124

dechlorination process H. In contrast to the stable meta dechlorination activity, both the
pa and the pm activities could not survive the pasteurization. The pa activity was more
unstable than the pm activity and appeared to be nutrient—dependent.

In presence of 3 ~ 5 mM sulfate, a distinct dechlorination activity characterized by
the ability to attack 2,4—4-chlorobiphenyl (2,4—4-CB) and 2,4,6-2-CB was observed. This
activity is part of the pa activity.

Ortho dechlorination of 2-CB and 2-2-CB/2,6-CB by the transferred methanogenic
cultures was observed, suggesting that the Hudson River contains microorganisms with
the potential for complete dechlorination of PCBs to biphenyl.

The different dechlorination activities observed under different cultural conditions
are summarized in Table 1. These observed dechlorination activities, together with other
reported dechlorination activities, indicate that diverse microorganisms in Hudson River
sediments may have the capability to remove chlorines from certain positions of specific
PCB congeners. The presence or absence of these active microorganisms may determine

the actual PCB-dechlorination pattern observed in-situ.

Table l. The Observed Dechlorination Activities in This Study

 

Activity

Culture condition

 

remove meta chlorines
ethanol

remove para chlorines with
adjacent meta chlorines

mainly attack 2,4,4-CB/2,4,6,-2-CB

remove ortha chlorines from
2-CB, 2-2-CB/2-6-CB

inocula treated with heat or/ and

methanogenic

inpresence of 3~5 mM sulfate

methanogenic

 

Appendix

126
I. Effects of More Selective Pasteurization Treatments

1) Different pasteurization time (90"C)

The inoculum for the lst serial culture was prepared with the upper Hudson River
sediment, was originally pasteurized at 85°C for 15 min and then was serially transferred
using a 1% inoculum. The inoculum for all subsequent transfers was re-pasteurized.
The pasteurization temperature and time were changed from 85°C/ 15 min to 90°C/ 10 min
from the 3rd serial transfer. A lag time for dechlorination activity of 4 weeks was
observed in the 3rd serial culture, however, no such a lag time was observed (data not
shown) in the all subsequent transfers. The inoculum from the 5th serial transfer was
pasteurized at 90°C for 10, 15, 20, 25, and 30 min to increase selective pressure. The
transfers heated for 10, 15, and 20 min retained the dechlorination activity. In contrast,
no dechlorination was observed in those cultures heated for 25 and 30 min even after 6
months of incubation (Fig. 1).

Besides the cultures heated at 85°C for 15 min in the lst serial culture observed
above, there were other two experimental groups, one was pasteurized at 90°C for 10
min, the other was pasteurized at 100°C for 10 min. Only one of the 90°C-treated
triplicates showed dechlorination activity, and that was after an 8 week lag time (data not
shown). The more active PCB dechlorination by the 5th serial culture compared to the
lst serial culture indicates that the heat-resistant dechlorinating microorganisms were
enriched through the serial transfers.

2) Pasteurization at temperatures above 90°C

In one batch of the BESA experiments reported in Chapter 1, one of the triplicate

autoclaved controls showed dechlorination activity (Fig. 2). After the contamination was

127

 

3.5

 

 

 

Total Chlorines per Biphenyl

 

 

 

2.5 I
0 4

Incubation Time (weeks)

00-1

10 min
15 min
20 min
25 min
30 min
Autoclaved

hs)
ENDEEIII]

mont

(6)

\\
I
\\

\
\

\
\

Total Chlorines per Biphenyl

\
\

\
III/I

III/II
\

\
\

 

Fig. l. Dethlorination of Aroclor 1242 by microorganisms pasteurized at 90°C for
different time after (A) 4 and 8 weeks, and (B) 6 months of incubation. Error bars are

the standard derivation (SD) of duplicate samples, where not shown, the error bars are
smaller than the symbols.

128

 

Autoclaved-1

 

 

 

Autoclaved-3

2.2"

Totnl Chlorines per Biphenyl

 

 

 

2.0 . , . r . ,
0 4 8 1 2

Incubation Time (weeks)

Fig. 2. Dechlorination of Aroclor 1242 by autoclaved microorganisms eluted from the
Hudson River sediments. The experimental vessels were 160—ml serum bottles containing
25 g PCB-free sediments and 75 ml RAMM with microorganisms. The serum bottles
were autoclaved at 121°C for 1 hour.

129
carefully examined and ruled out, this unexpected result suggested that some

dechlorinating microorganism(s) was(were) particularly heat-resistant. It was this result
that prompted me that initiated the pasteurization and ethanol-treatment experiments.
Some details of the above-mentioned controls are as follows: The autoclaved controls
were l60-ml serum bottles containing 25 g PCB-free Hudson River sediment, 75 ml
RAMM (7) with eluted microorganisms, and 50 pl of 10% (wt/vol) Aroclor 1242 in
acetone. These controls were part of an experiments to study the on-set dechlorination,
and therefore, had been incubated for 4 weeks, and were autoclaved after dechlorination
was confirmed. Then 100 pl of 10% Aroclor 1242 in acetone was re-spiked to each
culture. The cultures were then incubated at 25°C in the dark, with this time designated
as 0 time. Despite the fact that the controls had been autoclaved (121°C) for 1 hour,
after an additional 3 weeks of incubation, dechlorination was observed in one of the
triplicates. (Fig. 2) (this experiment was set up in March, 1989, data of 0 time and 5
weeks were summarized in May, 8 weeks in June).

The active culture was then transferred. The inoculum concentration was 1/3 to
repeat the original inoculum size (1/3 of the total liquid portion). Inoculum from this
culture was split and re-heated at 100°C, and 121°C, respectively, for 10 min. The
pasteurization was performed in 28-ml serum tubes instead of l60-m1 serum bottles as
previously described. No dechlorination was observed during 12 weeks of incubation.
After 4 months of incubation, one of the duplicate 100°C-heated cultures showed
dechlorination activity (Fig. 3). Since the cultures were not sampled between 12 weeks
and 4 months, no information about the lag time after 12 weeks was obtained,

nevertheless the dechlorination occurred sometime between 12 weeks and 4 months.

130

C3 Autoclaved
m Pasteurized
(100°C/ 10 min)

 

total ortho metal a: para

Average Chlorines Per Biphenyl

Substituted Chlorines

Fig. 3. Dechlorination of Aroclor 1242 by microorganisms transferred from the culture
retaining the dechlorination activity after autoclaving (see Fig. 2). The inoculum was re-
pasteurized at 100°C for 10 min upon transfer, and the pasteurization was performed with
a 28-ml serum tube (refer to reference 8).

131

Effects of pasteurization temperature on the lst and the 2nd serial cultures were different.
The lst serial cultures were heated at 121°C for 1 h and one of the triplicates still
retaimd the dechlorination activity, and the lag time was only 3 weeks. However, those
2nd serial cultures re-pasteurized at 121°C for 10 min lost dechlorination activity, and
only one of the two 2nd serial cultures re-pasteurized at 100°C for 10 min retained the
dechlorination activity with a lag time longer than 12 weeks. The difference might be
because: (i) the 1 serial cultures were heated in l60-ml serum bottles, while the inoculum
for the 2nd serial cultures was heated in 28-ml serum tubes, (ii) the lst serial cultures
were heated in presence of sediments, however, for the 2nd serial cultures only the
supernatant from the lst serial culture was taken as the inoculum and only this inoculum,
not the whole culture, was pasteurized, (iii) the status of the heat-resistant
microorganisms in the lst and the 2nd serial cultures might be different (vegetative cells
or spores).

It is unclear whether pasteurization of inoculum in the 28-ml serum tubes or in the
160-ml serum bottles is important, or pasteurization of the inoculum with presence or
absence of sediment is important. However, pasteurization with the serum tubes, and
without presence of sediment should be more effective.

3) Serial dilution experiment

Inoculum taken from the 4 serial cultures was re-pasteurized at 90°C for 10 min,
and was diluted. Figure 4 shows the dechlorination after 4 weeks. After 8 weeks of
incubation, dechlorination occurred in one of the triplicates of the 10'6 dilution (Table
1). No dechlorination was observed in a dilution higher than 10‘6 as measured at 6

months (Table 2). HPLC analysis showed that in both dechlorination-positive and

132

 

 

 

 

3.5
'32
5 Autoclaved
a ll- ..
c:
t—
” ”a?
“‘ii
a”): d.) 30"
r: 3
8 53
U
E
o
E—
2.5 ' I I I ' I ‘ I .

Dilution (power of 10)

Fig. 4. Results of dilution experiment after 4 weeks of incubation. Error bars are
the standard derivation (SD) of triplicate samples; where not shown, the error bars are
smaller than the symbols.

Table 1. Results of the Dilution Experiment (8 weeks)

 

 

 

 

 

 

 

 

(total chlorines per biphenyl)
Power of 10
# of triplicates -2 -3 -4 -5 -6 -7 -8
1 2.81 2.87 2.94 2.99 3.21 3.24 3.24
2 2.98 3.03 2.88 3.23 3.26 3.28 3.27
3 3.00 2.98 2.93 3.23 2.93* 3.23 3.23
Table 2. Results of the Dilution Experiment (6 months)
(total chlorines per biphenyl)
Power of 10
# of triplicates -5 -6 -7 -8
1 2.90 3.18 3.20 3.20
2 3.11 3.19 3.18 3.18
3 3.16 2.75* 3.21 3.21
* Dechlorination ocurred

134

negative cultures, the major metabolites were butyrate and acetate. There was no
significant difference in the amount of the metabolites between the positive and negative
cultures.

In preparation of this experiment, 3 ml inoculum was mixed with 3 ml of 0.9%
NaCl containing 1 mM Na28.9H20 and 0.0001 % of resazurin. After re-pasteurization,
the inoculum was serially diluted 10—fold and 0.6 m1 of the diluted inoculum was
introduced into each 60-ml serum bottle containing 29.4 ml RAMM to make an 1%
transfer.

The number of the microorganisms in the pasteurized inoculum was counted by the
Most-Probable-Number (MPN) method with a mixed medium of RAMM and AC
medium (10); and the ratio of RAM to AC was 4:1. Each tube was inoculated with

0.1 ml inoculum. The MPN result was as follows (12 weeks):

Transfer -2 -3 -4 -5 -6 -7 -8 -9 -10 -11
(power of 10)

Dilution O l 2 3 4 5 6 7 8 9
(power of 10)

positive: 5 5 5 5 5 4 2 0 0 0

From the data, a population of 1.32 x 108 organisms was calculated for the
original inoculum, i.e. 10’6, theoretically 132 microorganisms were present. Since only
1 of 3 of the cultures was positive, this is rear the end point of dechlorination active
members. These numbers only give a roughly idea, because some microorganisms might

not grow in this medium. The reason to replace sediments with AC medium is to

135

facilitate determination of growth. These results also imply that the dominant strains are
not the active dechlorinators, and therefore this approach will not easily yield a pure

strain capable of PCB dechlorination.

II. Attempts to Stimulate PCB Dechlorination by Spore Germinants

Effects of spore germinants on dechlorination was investigated. L-alanine and a
variety of alanine analogues are common germinants, while D-alanine is the inhibitor of
L-alanine (4). In this experiment, both L-alanine and D-alanine were introduced at
concentration of 2 mM. D-alanine was used either as a negative control (amendment of
D-alanine only), or as a competitive inhibitor (amendment of L-alanine plus D-alanine).
Another group was the heat-treated group (which was heated at 80°C for 15 min); was
used as a positive control since heat stimulates spore germination.

No difference in the lag time among different groups was observed (data not
shown), indicating that most spore-forming bacteria in the inoculum may have already
been present as vegetative cells instead of bacterial spores. This conjecture is supported
by the fact that in previous experiments a difference in lag time between the heated and
untreated cultures was observed (see Chapter 5). However, no difference in these two
groups in lag time was seen this time. This may have been due to the fact that two
weeks before preparing this experimental batch, some inoculum was taken for another
experiment. At this time some RAM was added to the stored sediments. It is known
that fresh medium stimulates spore germination (3, 4).

To investigate the effects of a germinant, addition of the germinant directly to the

sediments is recommended. However, whether the presence of sediment may alter the

136

effects of the germinants is unclear. Another alternative approach way is to introduce
sporulant to the inoculum and wait for some time to allow sporulation occur before the

inoculum is used.

III. Isolation and Testing Sporeformers

Microorganisms from the pasteurized cultures were isolated on different media.
All these media contained RAMM and sediment extract (either pre-incubated (8) or not)
with different modifications. The modifications included addition of the following media,
carbon or energy sources, or growth factors at different low concentrations: AC medium,
reinforced clostridium medium, VL medium, cooked meat medium, blood agar base,
trypticase, yeast extract, malt extract, lactate, H2. all media were from Difco (9). These
were added either separately or as a combination of two or three items in different
proportions. For some cultures, 500 ppm of Aroclor 1242 was also incorporated.

The inocula used were from the following cultures: Those pasteurized at 90°C for
20 min (Section 1.1), the positive culture of the 10*5 dilution (Section 1.3), and the
culture pasteurized at 100°C for 10 min (Section 1.2). Roll tubes, anaerobic agar plates,
and deep agar tubes were employed.

After the colonies appeared, colonies of different morphologies were picked.
Microorganisms from similar morphological colonies were examined under microscope,
and those colonies containing different cell morphologies were also picked. About 140
isolates from different experimental batches were tested for the dechlorination activity.
For this, the isolates were inoculated into 60-ml serum bottles containing 10 g

preincubated PCB-free Hudson River sediment, 30 ml RAMM, 0.15 ml cysteine (final

Table 3. Results of the Isolates Experiment

137

 

 

 

** Reinforced aostrz’dium medium.

Number of Average Cl'
Samples Isolates Isolation medium per biphenyl
Autoclaved 3.23
Mix-A 5 RAMM, 51313,, AC 2.79
Mix-B 4 RAMM, SE, H2, RC" 2.73
Mix-C 6 RAMM, SE, H2, ac 2.79
Mix-D 5 RAMM, SE, 11,, pcs 2.72
* Pre-incubated sediment extract.

138

concentration was 330 mg/liter liquid), and 60 pl 10% Aroclor 1242 in acetone. No
pure isolate showed dechlorination activity, however, some cultures with combimd
isolates retained the dechlorination capacity. Results are listed in Table 3. The results
listed in table 3 are actually only one of many batches of the isolation experiments,
positive results were obtained only in this batch.

The inoculum for this batch was from the 10‘6 transfer of the dilution experiment.

Further isolation and purification are needed to finally isolate and identify either
pure strain(s) or a minimum syntrophic consortium capable of anaerobic PCB

dechlorination.

IV. Evaluation of PCB bioavailability

PCBs are nonionic organic compounds (NOCs). In soil and sediment they
distribute mainly between soil solution and soil organic matter. It is generally believed
that only the N OCs in soil solution would be accessible to microorganisms, those
partitioned into soil organic matter would be unavailable because they are separated from
the microorganisms by the organic bulk phase. Additionally, as time goes by, the NOCs
in soil can diffuse to remote sites in the soil matrix, and become entrapped in micropores
in soil aggregates, also influencing their bioavailability. The availability of NOCs to
microorganisms is, therefore, controlled by both desorption and diffusion rates of N OCs,
which is determined by both soil conditions and the nature of NOCs, that is, the partition
coefficient and mobility. When either one of these rates is slower then the dechlorination
rate, bioavailability will sooner or later be a limiting factor.

To investigate the bioavailability to microorganisms of PCBs in historically

139

contaminated soils, a bioavailability experiment was conducted. The investigated soils
were a Glens Falls, New York, drag strip soil (GFDS soil) and Saginaw, Michigan, soil
site 8RC 10-20 cm. Both soils are historically contaminated by PCBs; the GFDS soil
is contaminated by Aroclor 1242.

The experiment was performed in two batches. One batch was set up with GFDS
soil, the control PCB-free soil was from New Bedford Harbor, Mass., while the other
batch was set up with Saginaw 8RC soil, and PCB-free Hudson River sediments served
as control.

Since different soils have different physical and chemical conditions such as
structure, PH, nutrients, sulfuroxy/nitroxy anions, heavy metals, et al. , and all of these
may effect dechlorination. Therefore, any difference in dechlorination rate and extent
between the contaminated soil and the PCB-free soil can not be simply attributed to the
bioavailability. In order to account for the effects from any factor other than
bioavailability, both contaminated and the PCB-free soils were spiked with the same
concentration of late eluting congeners, 2,3,4-3,4-CB and 2,3,4,5,6-CB (15 pg/g dry
soil) as internal standards. Dechlorination of both historically contaminated and freshly
added PCBs were first normalized by the standards (calculated as the ratio to the
standards), and then the normalized rates were comparable.

There were two amendment groups for the historically contaminated soils. One
was only spiked with the PCB congener standards (group B), while the other group
(group A) received both the standards and Aroclor 1242 (300 pg/g dry soil, which is
about the same concentration of PCBs as found for the historically contaminated PCBs).

The experimental results did demonstrate that the aged PCBs were less available

140

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

c:
U
I
Y 3.0
V
“ A
(’1 c
N U
\
a: g, 2.5 ~ A
0 t?
I
m V; P I I
I m s \ \’\I
VI: 0; 2 O _ '\’\’\’\’:
m o :\:\:\:\:\
CI \ \ \
N 2‘ :~:~:~:1:3
\ \ \ \ \l
“- .c 7 I I I I
o o 1 5 _ 'TITITITI.‘
N . t F
I: E V‘I‘I‘I‘IV
.C E "I‘I‘I\IN
g. 5 '2'2'1'2'1
.5 5 1.0 ‘ xiii“
h \’\’\’\’\
o @9202
_.: «3.3.3:.
8 0 5 ;\’\’\’\’\l
O .
100
[El users/2.3.4.49
2.3,4-3,4-CB
80 ‘
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.s a? 607 B
'- A4
2 a
{'3 3
o .
a :9. 4° —
._ c. f .
c 'l
e ,0 .
W///
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0 llllll’Af -4

Fig. 5. Dechlorination of aged (GFDS soil '

. ,expenmentalgrouBandfreshl '
(NBF sorl) 2,3,4-3-CB and 2,3,4-4-CB by microorganisms frgm )Hudson y SEE:
decsedlmmmr 16 weeks of incubation. (A) normahzed' by 2,3,4-3,4-CB; (B) percentage

. Error bars are the standard derivation (SD) of duplicate samples where
not shown, the error bars are smaller than the symbols. ,

141
than the freshly spiked ones. Figure 5A shows PCB dechlorination in both freshly spiked

(NBF soil) and the historically contaminated (group B, GFDS soil) 2,3-3,4-CB/2,3,4-4-
CB (coeluting congeners) normalized by 2,3,4—3,4-CB, as an example. Comparison
between group A (with addition of fresh Aroclor 1242) and group B (without introducing
fresh PCBs) in GFDS soil also showed that the amount of dechlorinated 2,3-3,4-
CB/2,3,4-4-CB in group A is 1.56 time that in group B (data not shown).

On the other hand, despite of less availability of the aged PCBs, the overall
dechlorination of the aged PCBs in GFDS soil was much greater than that of the fresh
ones in NBF soil (Fig. 5B), suggesting that soil conditions are more important than
bioavailability in determining dechlorination rate and extent. Additionally, comparison
between group A and group B of the GFDS soil showed that the dechlorination amount
of 2,3,4-3,4—CB (standard) in group A (with addition of fresh Aroclor 1242) was only
about half that in group B (data not shown), despite the fact that the initial amount of
2,3,4-3,4-CB in both groups was almost equal since the residual 2,3,4-3,4-CB in GFDS
soil is negligible (0.5% of the spiked one) and the amount of 2,3,4-3,4-CB in the
introduced Aroclor 1242 was also negligible. This result indicated that the increase in
PCB concentration inhibited dechlorination of 2,3,4-3,4-CB. The inhibitory effect was
obviously not due to the possible toxic effect of the introduced PCBs since the overall
dechlorination was much greater after introducing the PCBs (data not shown). Instead,
the inhibitory effect was most probably because the addition of extra PCBs increased the
concentration of other congeners, and consequently increased the competitors competing
for the dechlorination capacity (dechlorinating enzymes/microorganisms). This indicated

that the dechlorination conditions in GFDS were mainly capacity-limiting.

142

From the bioavailability results It can be concluded that bioavailability, soil
conditions, as well as dechlorinating capacity (dechlorinating enzymes/microorganisms)
are all factors in determining the dechlorination rates and extent. However, according
to the experimental results, it appeared that bioavailability is less important than the other
two factors. At least, as determined by this experiment, substantial dechlorination of

environmental PCB residues can occur.

V. Influences of Some Carbon and Energy Amendments on PCB Dechlorination by
Microorganisms from Hudson River Sediments

To enhance dechlorination and to enrich microorganisms able to dechlorinate PCBs,
influences of some carbon and energy sources as well as other nutrients such as yeast
extract on anaerobic dechlorination were investigated. Results are as follows:

1) Effects of acetate, acetone, yeast extract, and sediment extract on the on-set
dechlorination

This experimental batch was prepared in 160-ml serum bottles. Each bottle
contained 25 g of pre-incubated PCB-free Hudson River Sediment, 75 ml RAMM
containing the eluted microorganisms, and 50 pl of 10% Aroclor 1242 in acetone. The
final concentration of PCBs was 200 pg/ g dry sediment. The cultures were first
incubated at 25°C for 4 weeks until dechlorination activity was confirmed by sample
analysis. Amendments were made on the fifth week. Acetate, acetone, and yeast extract
amendments consisted of 1.5 ml of 10% solution of each. Sediment extract consisted of
10 ml extract prepared by autoclaving PCB-free Hudson River sediment with deionized

water (1:1, wtzvol) for 1 hour. The mixture was then shaken for another hour,

143

 

0.0-

 

l
H
H

 

 

-1.0- '.- \:E

No Amend
Acetone
Yeast Ex
Acetate

Sediment Ex
’2-0 r V r a r r ' I r

o 2 4 6' 8 to

Change in Chlorines per Biphenyl

ZIIII

 

 

 

Incubation Time (Weeks)

Pig. 6. Effects of different amendments on on-set dechlorination of Aroclor 1242 by
microorganisms from Hudson River sediments. Error bars are the standard derivation
(SD) of triplicate samples; where not shown, the error bars are smaller than the symbols.

W

(D

 

144
centrifuged and filtered. All amendment solutions were bubbled with N2-C02 and were

autoclaved in serum bottles sealed with Teflon-coated stoppers and aluminum crimp
seals.

No continued dechlorination was observed in any group including the non-
amendment group which served as the control. This was probably because the more
readily dechlorinated congeners had already been dechlorinated to a level below the
threshold for a activity. Therefore, an additional 100 pl of 10% Aroclor 1242 in acetone
was added, and that time was designated as 0 time. The cultures were incubated for
additional 8 weeks.

Both acetate and yeast extract completely inhibited dechlorimltion. In contrast,
sediment extract enhanced dechlorination (Fig. 6). Acetone at this concentration had no
effect on dechlorination.

2) Effects of butyrate and formate on the on-set dechlorination

Butyrate and formate were both added at 0 time and at 4 weeks at a final
concentration of 10 mM and 100 pM, respectively. No significant effect on
dechlorination was observed.

3) Influences of some amendments on dechlorination by the pasteurized
cultures

The amendments for this experiment were cellulose, glucose, lactate, methanol, H2,
yeast extract, peptone, and lactate plus sulfate along with three nails (5) per bottle. The
concentration of sulfate was 2 mM (final concentration), H2 was introduced with a gas
manifold; cellulose was added as shredded filter paper; the concentrations of all

amendments, except sulfate and H2, were 0.5 g/ liter liquid. All amendments were

145

 

 
  
  
   

Autoclaved

 

_§

Glucose

Cellulose

-~
~.
~

Non-amendment

Total Chlorines per Biphenyl

 

 

 

2.4 T I I
O 4 8 1 2

Incubation Time (weeks)

Fig. 7. Influences of cellulose and glucose on dechlorination of Aroclor 1242 by the
pasteurized microorganisms from Hudson River sediments. The cellulose and glucose
were added at 0 time and 4 weeks at a final concentration of 0.5 g/liter liquid. Error

bars are the standard derivation (SD) of duplicate samples; where not shown, the error
bars are smaller than the symbols.

146
autoclaved except H2 which was filter sterilized, and the nails which were flamed.

The cultures were prepared in 60-ml serum bottles containing 10 g preincubated
PCB-free Hudson River sediment, 29.4 ml RAMM, 0.3 ml inoculum, 0.15 ml cysteine
(final concentration was 330 mg/ liter liquid, 0.15 ml of stock amendments solution, and
60 pl 10% Aroclor 1242 in acetone.

The inoculum was taken from the 2nd serial pasteurized culture (Section 1.1),
therefore, the culture of this experiment was actually the 3rd serial culture (re-
pasteurized, 1% transfer). The amendments were added at 0 time and at 4 weeks except
sulfate which was only added at 0 time.

Both cellulose and glucose showed slight but discernible inhibitory effects after 4
weeks of incubation (Fig. 7), other amendments had no effects on dechlorination (data
not shown). In the experiments described in Chapter 4, sulfate inhibited dechlorination
by the pasteurized cultures at 2 mM. However, in this experiment, with addition of
lactate, no inhibitory effect by sulfate was observed (data not shown), indicating that the
sulfate effect was balanced by lactate.

Since no amendments enhanced dechlorination, it seemed that with presence of
PCB-free Hudson River sediments, carbon and electron donor were not limiting factors
for PCB dechlorination by the pasteurized cultures.

4) Investigation of solid supports for PCB culture

Ashed PCB-free Hudson river sediments and sloppy agar were used as supporting
material to replace non-ashed sediments in order to have an organic matter free solid
support. Lactate, pyruvate, acetate, formate, ethanol, and methanol were supplemented

as amendments each at two experimental concentrations: 0.5 g/ liter liquid, and 5 g/ liter

147
liquid. Hz-COZ (80:20) was also among the investigated substrates and was introduced

to 0 (equal to the atmospheric pressure) and 0.5 atrn, respectively. For the 0.5 atm
amendment the gas was introduced by needle attached to a gas manifold. The needle
hole was then immediately sealed with silicon glue. In the sloppy agar experiment,
0.125 % agar was added, while in the ashed sediment experiment, the ashed sediment was
10 g/60-ml serum bottle. The liquid portion consisted of 97.5% RAMM and 2.5% of
sediment extract containing 0.5 g/liter of yeast extract and 0.4 g/liter of cysteine.

The inoculum was taken from the 5th serial transfer of the pasteurized cultures
(Section 1.1) The inoculum was re-pasteurized at 80°C for 10 min, and the inoculum
was 5 %.

The RAMM turned pink after being introduced into the ashed sediment, despite the
fact that the ashed sediment was evacuated three times, filed with H2-C02, and stored
in an anaerobic chamber for 2 days. This may have been because, after being heated and
ashed, the metals associated with the sediments were highly oxidized, and thus consumed
the reducing power. The RAMM then had to be changed couple times, the medium then
turned colorless before inoculation.

No dechlorination was observed in any culture, suggesting that either the supporting
materia failed to effectively disperse PCBs which are highly hydrophobic, or no
experimental amendment supported growth of the dechlorinating microorganisms.

5) Effects of preincubation on PCB dechlorination

In previous experiments, the preincubation procedure (8) was always followed for
preparation of the experimental batches. The reason for the preincubation is to allow the

facultative microorganisms to consume any residual oxygen. Strictly anaerobic

148

conditions can also be ensured by detection of methane formation since methanogens only
grow under strictly anaerobic conditions.

Preincubation should also consume some sediment organic matter and nutrients, and
produce some metabolites. All of these might influence dechlorination. To investigate
the possible effects of the preincubation procedure on dechlorination, an experiment was
set up with the following amendments:

a. autoclaved control

b. preincubated serum bottles

c. non-preincubated serum bottles

d. preincubated serum bottles washed with RAMM four times (the pre/wash amendment
group, described below)

e. non-preincubated serum bottle with addition of 0.1% methanol

The purpose of amendment ”e" was to investigate if introducing 1% methanol has
any effect on dechlorination, since in the pre-incubation procedure, 0.1% of methanol
is added as electron donor to scavenge the residual oxygen.

The preincubated cultures were incubated at 37°C for 10 days and methane
production was confirmed by gas analysis. After preincubation, triplicate bottles were
taken for the pre/wash group. The liquid portion (10 ml) was withdrawn, the
preincubated sediment (10 g, dry weight) in the serum bottles was then washed four
times with 30 ml of RAMM and shaken for 10 min each time. To avoid washing out
sediment-associated nutrients, the RAMM had been added into other sediments,
autoclaved along with the bottles to be washed, and then had been shaken for 10 min

after autoclaving. After the washing process, fresh RAM and inoculum were

149

introduced. Therefore, the pre/ wash group was the same as the preincubated group
except that the RAMM was fresh RAMM, therefore, no nutrient in the RAMM was
consumed, and it did not contain metabolites from preincubation. This group was also
the same as the non-preincubated group except that the sediment was preincubated and
thus contained less organic matter due to the preincubation. Differences among the three
amendments (amendment "e” is not included) are listed in Table 4.

Results showed that addition of 0.1% methanol had no effect on dechlorination
(Fig. 8A). Compared with the non-preincubated cultures, preincubation inhibited
dechlorination (Fig. 9). The preincubated culture showed dechlorination pattern M,
while the non-preincubated culture exhibited pattern C (Fig. 10). The differences in the
dechlorination pattern between the preincubated and the non-preincubated cultures are
shown by the bottom panel in Figure 10.

Consuming sediment organic matter appears was not to be the reason that the
preincubation had side effects on dechlorination, as evidenced by the fact that the
dechlorination by the pre/ wash group was not effected, even though this group also
contained less sediment organic matter. Both accumulation of metabolites and
consumption of nutrients may contribute to the inhibition effect. The nutrients consumed
were most probably from RAMM instead of sediment, since nutrients from the sediment
in the pre/ wash group were equally consumed as those of the preincubated group.
Metabolites proved to be unlikely cause of the effect, according to the methanol
experiment (described below). Therefore, this result suggested that the effect of
preincubation was probably due to deficiency of nutrients from RAMM that resulted in

the inhibition effect. This result is also consistent with the previous observation that the

150

Table 4. Differences Among the Four- Amendment Groups"

 

 

 

 

Sediment organic matter Nutrients in RAM Metabolites
Amendments More Less More Less Yes No
Preincubated _ ' _ +
Pre/ wash _ + _
N on-preincubated + +

 

* Preincubation should consume some sediment organic matter and some nutrient, and prodce
some metabolites.

151

 

 

   

Non-preincubated

 

Total Chlorines per Biphenyl

 

 

1.0 r Y r r T Y r I 7’ Y 7 r

 

 

 

 

2.0- W

. —°— Autoclaved
.—0— Without methanol
‘ -—*-— 0.1% methanol

‘ “"0“ 0.5% Methanol

T V T ‘I’ f T f f f
1.0 I I r

O 4 8 ' 1 2

 

Total Chlorines per Biphenyl

 

 

Incubation Time (weeks)

Fig. 8. Effects of methanol on dechlorination of Aroclor 1242 by (A) non-preincubated
and (B) preincubated cultures. The 0.5 % amendments received methanol at 0 time and
4 weeks, while the 0.1% amendment only received methanol at 0 time. Error bars are
the standard derivation (SD) of triplicate samples; where not shown, the error bars are
smaller than the symbols.

152

 

 

 

S”
O
1 :

 

 

~
--
I ~.
--~
‘.

l"
O
1

—°— Autoclaved
—‘¢—" Preincubated
—0"'— Non-preincubated
" ' ’1 " "' Pre/wash

r Y I I I I Y T I Y H r

O. 4 12

 

Total Chlorines per Biphenyl

 

 

p—l
O
on

Fig. 9. Dechlorination of Aroclor 1242 by different experimental groups of the
premcubation experiment. Error bars are the standard derivation (SD) of triplicate
samples; where not shown, the error bars are smaller than the symbols.

153

 

 

 

 

 

 

 

 

 

 

 

 

2° ' 1
l = z
‘5 .. Preincubated ‘1 0
l ; o
1 1 h
to «i 1 0.:
5 ; 5 2
1 I C
a 1 .11 Arm L 1 2
O 10 20 30 40 50 CO 70 80 so
I u
1 C
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80 7O 80 90

Fig. 10. Mole percentage of Aroclor 1242 peaks after 12 weeks of incubation with the
preincubated and non-preincubated cultures. The bottom panel shows differences in male
percentage between preincubated and non-preincubated cultures after 12 weeks of
incubation. For peak assignments, please see reference 6.

154

activity that was lost from the preincubated group was also lost from aged inoculum, and
that the lose could be overcome by transferring the old culture to fresh RAMM (refer to
Chapter 5). All these results suggested that the activity loss was unstable and was
nutrient dependent.

However, whether pattern M or pattern C is expressed appears to depend not only
on the preincubation time, but also on inoculum size and the activity of the inoculum.
For example, in previous experiments, despite the standard preincubation treatment,
pattern C was often expressed. It appears that all factors affecting nutrients in the
culture may affect the expressed patterns.

6) Effects of methanol on both preincubated and non-preincubated cultures

As mentioned above, 1% methanol had no effect on dechlorination. However, as
shown in Fig. 8, addition of methanol at a concentration of 5 g/liter liquid (at 0 time and
4 weeks) completely inhibited dechlorination by the preincubated cultures and partially
inhibited dechlorination by the non-preincubated cultures. This result provided additional
evidence that the culture was nutrient-limiting. As listed in Table 2, the differences
between the preincubated and non-preincubated cultures were that the preincubated
cultures contained less sediment organic matter, less nutrient, and metabolites. Less
sediment organic matter and metabolites in the preincubated cultures do not likely
enhance the inhibition by methanol. However, addition of methanol should accelerate
consumption of nutrients. If the inhibitory effects of methanol on dechlorination is to
accelerate consumption of nutrients, then the less nutrients present in the culture, the
stronger the inhibitory effects. Results of the methanol experiment support this

postulation, since methanol has stronger inhibitory effects on dechlorination by the

 

155

preincubated cultures, and the preincubated cultures contained less nutrient due to the

preincubation procedure.

156

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1. Alexander, M. 1965. Most-Probable-Number method for microbial populations. p.
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Agronomy. Inc..

2. Brown, J. F., R. E. Wagner, D. L. Bedard, M. J. Brennan, J. C. Carnahan, R.
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3. Mair, anne and D. A. Smith. 1990. The genetics of bacterial spore germination.
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4. Norris, J. R., R. C. W. Berkeley, N. A. Logan, and A. G. O’Donnell. 1981. The
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Trl‘iper, A. Ballows, and H. G. Schlegel (ed.), The prokaryotes, vol. II. Springer-Verlag,
New York.

5. Pfenning, N. F. Widdel, and H. G. Triiper. 1981. The dissimilatory sulfate-
reducing bacteria. In M. P. Starr, H. Stolp., H. G. Trfiper, A. Ballows, and H. G.
Schlegel (ed.), The prokaryotes, vol. II. Springer-Verlag, New York.

6. Quensen, J. F., III, S. A. Boyd, and J. M. Tiedje. 1990. Dechlorination of four
commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic microorganisms
from sediments. Appl. Environ. Microbiol. 56:2360-2369.

7. Shelton, D. R., and J. M. Tiedje. 1984. General method for determining anaerobic
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8. Ye, D., J. F. Quensen III, J. M. Tiedje, and S. A. Boyd. 1992. Anaerobic
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9. Diffo Labortories. Difco Manual. 1974. 9 edition. Difco Laboratories Inc. Detroit,
Michigan.