anaerobic degradation of 2,4,6- trinitrotoluene (tnt)

127 Pages·2010·5.15 MB·English

ANAEROBIC DEGRADATION OF 2,4,6- TRINITROTOLUENE (TNT):
MOLECULAR ANALYSIS OF ACTIVE DEGRADERS AND METABOLIC
PATHWAYS
by
ERIN MAUREEN GALLAGHER
A Dissertation submitted to the
Graduate School-New Brunswick
Rutgers, The State University of New Jersey
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Graduate Program in Environmental Sciences
written under the direction of
Professor Lily Young
and approved by
________________________
________________________
________________________
________________________
New Brunswick, New Jersey
January 2010
ABSTRACT OF THE DISSERTATION
Anaerobic Degradation of 2,4,6- Trinitrotoluene (TNT): Molecular Analysis of Active
Degraders and Metabolic Pathways
By ERIN MAUREEN GALLAGHER
Dissertation Director:
Professor Lily Young

Nitroaromatic compounds have been historically used as dyes, explosives and
pesticides. The disposal of these products has caused widespread contamination of both
soil and groundwater. 2,4,6-trinitrotoluene (TNT) has been known as the worst of these
contaminants because of the mutagenic properties of the compound, its persistence in the
environment, and the wide range of sites that are contaminated with TNT. It is not only
harmful to humans but it is also harmful to organisms in the lower trophic levels of
ecosystems, and can affect the primary production of phytoplankton in the oceans.
The following series of experiments looks to determine ways that 2,4,6-
trinitrotoluene could be biologically degraded in contaminated anaerobic environments.
The experiments use dilution culturing, molecular techniques, and chemical analysis.
Cultures were made from 3 different geographical sites (Arthur Kill, Norfolk Harbor, and
an unexploded ordnance site in Hawaii) under both sulfidogenic and methanogenic
conditions. The experiments demonstrate that there are bacteria present in the
environment that could degrade TNT and the use of stable-isotope probing (SIP) in
dilution cultures allowed the bacteria that are able to initially degrade TNT to be
identified. The stable-isotope fed dilution cultures produced samples that were used for
ii
chemical analysis to determine pieces of the biological degradation pathway. These
experiments determined that 1) there are bacteria that are able to utilize both the carbon
and/or nitrogen present in TNT for growth and 2) toluene, methylphloroglucinol,
benzoate or a benzoate derivative, and a cresol are all metabolites of anaerobic TNT
degradation.
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Preface
There is limited information regarding the microorganisms responsible for
anaerobic 2,4,6-trinitrotoluene degradation and mechanism by which it is metabolized.
Both sulfate reducing and methanogenic enrichment cultures from various locales were
established and examined to address these questions. Molecular methods and classical
culturing techniques were used in attempts to characterize microbes capable of anaerobic
2,4,6-trinitrotoluene metabolism. Bacteria actively degrading 2,4,6-trinitrotoluene and
the anaerobic pathway utilized by these microorganisms are identified and described
herein.

iv
Dedication
This work is dedicated to my amazing family and friends, especially Mom and Joe -it
would not have been possible without all of your love, support, patience, and
understanding.
The things that will destroy us are: politics without principle; pleasure without
conscience; wealth without work; knowledge without character; business without
morality; science without humanity; and worship without sacrifice.
– Mahatma Gandhi
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Table of Contents
Abstract of the Dissertation ii
Preface iv
Dedication v
Table of Contents vi
List of Tables ix
List of Figures x
Chapter 1: Introduction 1
1.1 Environmental Contamination 1
1.2 Chemical Structure of 2,4,6-Trinitrotoluene (TNT) 3
1.3 Toxicity of 2,4,6-Trinitrotoluene (TNT) 4
1.4 Remediation Options for TNT 7
1.4.1 Phytoremediation 8
1.4.2 Biological Remediation 11
1.5 Conclusions 13
1.6 Figures 16
1.7 References 23
Chapter 2: 13C-Carrier DNA Shortens the Incubation Time Needed
to Detect Benzoate-Utilizing Denitrifying Bacteria by
Stable-Isotope Probing (SIP) 27
2.1 Introduction 28
2.2 Materials and Methods 29
2.3 Results 32
2.4 Discussion 34
vi
2.5 Acknowledgements 38
2.6 Figures 39
2.7 References 43
Chapter 3: Detecting 2,4,6-Trinitotoluene Utilizing bacteria in Norfolk Harbor
Sediments by 15N and 13C Stable Isotope Probing 46
3.1 Introduction 47
3.2 Materials and Methods 49
3.3 Results 52
3.4 Discussion 55
3.5 Acknowledgements 58
3.6 Figures 59
3.7 References 65
Chapter 4: Identifying Bacteria Capable of 13C-TNT Label Incorporation and
Metabolites under Sulfate Reducing and Methanogenic Conditions 67
4.1 Introduction 68
4.2 Materials and Methods 70
4.3 Results 75
4.4 Discussion 82
4.5 Acknowledgements 88
4.6 Figures 89
4.7 References 104
Chapter 5: Conclusions and Future Work 108
5.1 Conclusions 108
5.2 Biological Degradation of TNT 109
vii
5.3 Limits of Detection in Stable Isotope Probing 111
5.4 Figures 113
5.5 References 114
Appendix
Curriculum vita 115
viii
List of Tables
1.1 Mass Recovery of TNT after 50 days of Composting 17
1.2 Microorganisms Reported to Degrade TNT Anaerobically 20
1.3 Removal Rates of TNT Using Phytoremediation 22
4.1 Clones Identified in 13C-TNT Utilization 95
4.2 TNT Metabolites by HPLC Analysis 96
4.3 13C-TNT and 13C-labeled Metabolites Molecular Weights 99
4.4 TNT Metabolites by LCMS Analysis 100
4.5 Appearance of Metabolites and Labeled TRFs Over Time 103

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List of Figures
1.1 Chemical Structures of TNT Metabolites 16
1.2 Bacterial Activity Profiles in TNT Phytoremediation 18
1.3 TNT Concentrations using Phytoremediation 19
1.4 TNT Anaerobic Degradation Pathway 21
2.1 13C H. salinarium Carrier DNA Quality Control Amplifications 39
2.2 SIP nosZ Amplification of LEO-15 and Norfolk Harbor Samples 40
2.3 T-RFLP of 13C-Benzoate Fed Communities 41
2.4 Change in SIP T-RFLP Fragments Over Time 42
3.1 Gradient Separation of Light and Heavy Carbon and Nitrogen 59
3.2 15N-SIP Quality Control Amplifications 60
3.3 T-RFLP of 15N-TNT Fed Sulfate Reducer at Time 2, 7, 14, 21 and 28 61
3.4 T-RFLP of Common Utilizers with 13C- and 15N-TNT Fed Cultures 62
3.5 Maximum Likelihood Tree of Initial 15N-TNT Utilizers 63
3.6 Change of DNA Band Height in Cesium Chloride Gradients with Varied
Concentrations of Ethidium Bromide 64
4.1 Arthur Kill Methanogenic TRFLP Fingerprints 89
4.2 Arthur Kill Sulfate Reducing TRFLP Fingerprints 90
4.3 Norfolk Harbor Methanogenic TRFLP Fingerprints 91
4.4 Norfolk Harbor Sulfate Reducing TRFLP Fingerprints 92
4.5 Hawaii UXO Methanogenic TRFLP Fingerprints 93
4.6 Hawaii UXO Sulfate Reducing TRFLP Fingerprints 94
4.7 HPLC Chromatograms Showing TNT Metabolites 97
x