Pentaerythritol Tetranitrate (PETN) & Pentolite by Environmental Microbes

Biodegradation of 2, 4, 6 Trinitrotoulene (TNT), Pentaerythritol Tetranitrate (PETN) & Pentolite by Environmental Microbes

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

Noble Elizabeth Georgie

Department of Biotechnology & Environmental Biology School of Applied Sciences RMIT University

August 2011

DECLARATION

I certify that except where acknowledgement has been made, the work contained in this thesis is that of the authors alone. The work has not been published either as part or whole to fulfil the requirements of any other academic award. The content of the thesis is the result of work, which has been carried out since the commencement date of the research program; and any editorial work paid or unpaid, carried out by a third party is given due acknowledgement

…………………………………..

Noble Elizabeth Georgie

31/08/2011

ACKNOWLEDGMENTS

I would like to thank my supervisor A/Professor Peter M. Smooker for all his guidance, support, advice and encouragement throughout the course of my study.

I would also like to thank Dr Steven Kotsonis (Orica Mining Services), my second supervisor for all his suggestions and support and for the opportunity to work with

Orica for both my PhD and as an external contractor.

A massive thank you to Dr Clint Brearley (Orica Mining Services), for all the support and encouragement throughout the course of my study. Thank you especially, for taking the time to edit and proof-read my thesis. It would not have been possible without your help.

Thank you Dr Kaiyan Liu (Grace from Orica Mining services) for all the help and guidance.

A special thank you to Steven Constantinos (Orica Mining Services) for always being my support, a shoulder to cry on when things got tough and a constant source of encouragement.

I would like to thank all the lab members in the Biotech lab for their unending support. Thank you to Natalie Kikidopoulos, Aya Taki, Rinu Thomas, Emily Gan

iii and Nahla Al-Mansour. Thank you to all the member of other labs especially

Saikrithika Gandhi, Shanthana Gouda Admane, Shruthi Saptarshi, Sandip

Kamath and Parsa Tehranchian

I would like to thank all the academic and technical staff for always offering their help and being supportive.

I would like to thank my friends here in Melbourne who has been just as loving as my family: Binu and Joe Elambasseril, Vinu, Kartik, Nikki, and Lavan; who with their presence and words of encouragement helped through some very hard times.

I would like to thank my family despite being on the other side of the world have always encouraged and supported me over the years. My mother Valsa Georgie for her unending love and support; my father, Georgie Alexander for letting me come half-way across the world to pursue my studies and his constant support throughout it. My sister, Susan and brother in-law, Robbie for their words of encouragement and wisdom. My husband, Jobin for all the prayers, support and patience.

Lastly, I owe everything to the Almighty Lord without whose grace none of this would be possible.

Noble Elizabeth Georgie

iv DEDICATION

This thesis is dedicated to my Ma and Pa who have always supported me throughout my studies both in India and in Australia. I have reached where I am today only because of their guidance, moral support and never ending love.

This thesis is also dedicated to my grandmother, who had big plans for me right from the start. I wish you were here to see what I have achieved.

v TABLE OF CONTENTS

Title page i Declaration ii Acknowledgements iii Dedication v Table of Contents vi List of Figures x List of Tables xii List of Abbreviations xiii Abstract 1 Chapter 1: Literature Review/ Introduction 4

1.1 Explosives 4 1.2 Types of explosives 4 1.3 Development and background of explosives 6 1.3.1 Nitrate ester explosive 6 1.3.2 Nitroaromatic explosive 7 1.3.3 Nitramine Explosives 8 1.4 Toxicity of explosives 8 1.5 Environmental issues surrounding the use of explosives and their role in environmental pollution 12 1.6 Methods of soil remediation and decontamination 13 1.6.1 Incineration 13 1.6.2 Composting 14 1.7 Biodegradation of Explosives 16 1.7.1 Biodegradation of Nitrate ester explosives 17 1.7.2 Biodegradation of Nitroaromatic explosives 18 1.7.3Biodegradatio of Nitroamine Explosives 18 1.8 TNT Degradation 19 1.8.1 Alkaline hydrolysis of TNT 19 1.8.2 Thermal decomposition of TNT 19 1.8.3 Fenton Oxidation of TNT 19 1.8.4 Photocatalysis 20 1.8.5 Biodegradation of TNT 20 1.8.5.1 Microbial metabolism of TNT 30 1.8.5.2 Aerobic Metabolism of TNT by Bacteria 30 1.8.5.3 Anaerobic Metabolism of TNT by Bacteria 31 1.8.5.4 Fungal Metabolism of TNT 32 1.9Degradation of PETN 33 1.9.1 Degradation of PETN on metal surfaces 33

vi 1.9.2 Degradation of PETN by Granular Iron 33 1.9.3 Microbial metabolism of PETN 34 1.9.4 Fungal metabolism of PETN and GTN 37 Aims 39

Chapter 2: Materials and Methods 40

2.1 General Procedures 40 2.2.1 Antibiotics, Media and Chemicals 40 2.2.2 Enzyme stocks 48 2.2.3 Microbiological Methods 49 2.2.3.1Bacterial Growth Conditions 49 2.2.3.2 Storage of bacterial strains 50 2.2.3.3 Estimation of Bacterial Cell Concentration 50 2.2.3.4 Bacterial profiles 51 2.3 DNA Molecular Techniques 51 2.3.1 Genomic DNA extraction 51 2.3.2 Quantification of DNA concentration 52 2.3.3 Polymerase Chain Reaction (PCR) 52 2.3.3.1 Primer Design 52 2.3.3.2 General PCR Amplification 53 2.3.3.3 Gradient PCR 53 2.3.3.4 Touchdown PCR 54 2.3.3.5 DNA Sequencing Reactions 54 2.3.3.5.1 Sodium-Acetate Ethanol Clean –up 55 2.3.4 Agarose Gel Electrophoresis 55 2.3.4.1 Agarose Gel Preparation 55 2.3.4.2 Sample preparation 55 2.3.4.3 Electrophoresis 56 2.3.4.4 Staining of Agarose Gels 56 2.3.4.5 Visualisation of DNA 56 2.3.4.6 Purification of DNA from PCR amplification 56 2.3.4.7 Purification of DNA fragments from Agarose Gel (Gel extraction) 57 2.3.4.8 Purification of PCR products for sequencing 58 2.3.4.9 Electroporation 58 2.3.4.9.1 Preparation of electro competent cells 58 2.4. Protein assays 58

Chapter 3: Isolation, Identification and characterisation of bacteria capable of degrading TNT and PETN 59

3.1 Introduction 59 3.1.1 Co-metabolism 61 3.1.2 Co-substrates 62 3.1.2.1 Surfactants 63 3.1.2.1.1 Trition X-100 63 3.1.2.1.2 Tween-80 63

vii 3.2 Materials and Methods 64 3.2.1 Microcosm 65 3.2.2 Cell Passages 66 3.2.3 Isolation of TNT and PETN degraders 66 3.2.3.1 TNT and PETN overlays 66 3.2.4 Utilization of TNT/ PETN as a nitrogen source 67 3.2.4.1 TNT Preliminary passages 67 3.2.4.2 PETN Preliminary passages 67 3.2.4.2.1 Passages of strains from Site A and B 67 3.2.4.2.2 Passages from strains from Site C and D 68 3.3 Identification of bacterial isolates 69 3.3.1 Phenotypic characteristics 69 3.3.2 Gram reaction determination 69 3.3.3 Biochemical tests 69 3.3.3.1 API® 20 NE Test 70 3.4 Co-Metabolism Degradation studies of TNT 70 3.4.1 Co-metabolism Degradation Experiments 70 3.5 Results 70 3.5.1 Details of soil and water samples from the contaminated. sites A, B, C and D. 70 3.5.2 Microbial load from Sites A, B, C and D 72 3.5.3Microbial content of soil samples from Site C and D 72 3.5.4 TNT Preliminary passages 73 3.5.5 PETN Preliminary passages 74 3.5.6 Phenotypic characteristics 76 3.5.7 Gram reaction 77 3.5.8 Biochemical tests 78 3.5.9 Co-metabolism experimental results 81 3.6 Discussion 83

Chapter 4: Molecular identification and characterisation of Isolates on TNT and PETN 88

4.1 Background 88 4.2 Materials and Methods 89 4.2.1 Genomic DNA extraction 90 4.2.2 Boiling method 90 4.2.3 Colony PCR 90 4.2.4 DNA extraction using commercially available kits 90 4.3 Results 92 4.3.1 Genomic DNA extraction 92 4.3.2 Boiling method 92 4.3.3 Colony PCR 93 4.3.4 DNA extraction using commercially available kits 93 4.3.5 Experiments evaluating the presence of onr and ner genes in Strains 106 4.3.5.1 Enterobacter sp 106 4.3.5.2 Agrobacterium 107 4.3.6 Experiments using Degenerate primers 110 4.4 Discussion 115

viii

Chapter 5: Nitrite Activity Assays 120

5.1 Introduction 120 5.1.1 Colorimetric Griess Assay 120 5.2 Materials and Methods 121 5.2.1 Strains 121 5.2.2 Culture conditions 121 5.2.3 Harvesting of culture 121 5.2.4 Nitrite concentration assays 121 5.2.4.1 Resting Cell Activity Assay 123 5.2.4.2 Cell-Free Lysate Activity Assay 124 5.2.4.2.1 Sonication 124 5.2.4.2.2 Nitrate ester reductase activity assay 125 5.2.4.2.3 Nitrite Utilisation Assay 125 5.2.4.2.4 Protein Assay 125 5.3 Results: Characterisation of Isolates 126 5.3.1 Resting cell activity 126 5.3.2 Cell-free lysate activity 131 5.3.3 Comparison of resting cell and cell-free lysate activities 137 5.4 Discussion 140

References 144 Appendices

Appendix I: 16S rDNA Sequences 164 Appendix II: CLUSTAL 2.0.12 multiple sequence alignment 168 Appendix III: Resting cell activity of the six GTN and PETN-degrading Isolates 171 Appendix IV: Cofactor preference of the Six GTN/PETN-degrading isolates in cell-free lysate using GTN/ PETN as substrate 172

LIST OF FIGURES

Figure 1.1 Structures of some important explosive compounds. 11 Figure 1.2 Microbial transformations of nitrate esters. 22 Figure 1.3 Microbial transformation of TNT. 23 Figure 1.4 Thermolysis of TNT 24 Figure 4.1 Visualised gel picture of PCR using template extracted by phenol- chloroform method 95 Figure 4.2 Visualised gel picture of PCR using template extracted by boiling method 96 Figure 4.3 Visualised gel picture of Colony PCR 97 Figure 4.4 Visualised gel picture of DNA extracted using commercial DNA kit 98 Figure 4.5a Visualised gel picture of a 16s rDNA PCR for PETN degrading isolates from microcosm 2 extracted with the commercial DNA extraction kit. 99 Figure 4.5b Visualised gel picture of a 16s rDNA PCR for PETN degrading isolates from microcosm 2 extracted with the commercial DNA extraction kit 100 Figure 4.6 Phylogram describing the distance between strains isolated from contaminated sites. 105 Figure 4.7 Gradient PCR with sample 2 using onr primers R1 and F1 108 Figure 4.8 Purified PCR products amplified using onr R1 and F1. 109 Figure 4.9 Visualised gel picture of PCR using ner primers on Agrobacterium sp. (positive control). 111 Figure 4.10 Visualised gel picture of PCR using ner primers on Agrobacterium sp. (positive control) along with other isolates. 112 Figure 4.11 Visualised gel picture of PCR using degenerate primers on samples 114

x Figure 5.1 GTN-degrading activities of resting cells of isolates cultured on LB and mM9+PETN. 128 Figure 5.2 PETN-degrading activities of resting cells of isolates cultured on LB and mM9+PETN 129 Figure 5.3 GTN and PETN-degrading activity of resting cells of isolates cultured on mM9=PETN 130 Figure 5.4 PETN-degrading acitvity of cell-free lysates in the presence of NADPH for isolates cultured on LB and mM9+PETN 132 Figure 5.5 GTN-degrading activities of cell-free lysates in the presence of NADH for isolates cultured in LB and mM9+PETN 133 Figure 5.6 GTN-degrading activity of cell-free lysates in the presence of NADPH for isolates cultured in LB and mM9+PETN. 134 Figure 5.7 PETN and GTN-degrading activities of cell-free lysates in the presence of NADPH cultured in mM9+PETN 135 Figure 5.8 GTN-degrading activities of cell-free lysates in the presence of NADH/ NAD(P)H for isolates cultured in mM9+PETN media. 136 Figure 5.9a PETN-degrading activities of resting cells and cell-free lysates of all isolates cultured on LB and mM9+PETN media 138 Figure 5.9b GTN-degrading activities of resting cells and cell-free lysates of all isolates cultured on LB and mM9+PETN media. 139

xi LIST OF TABLES

Table 1 Microbial strains exhibiting the phenomenon of co-metabolism 61 Table 2 Compounds subjected to co-metabolism and products formed 62 Table 3 Effects of surfactants on biodegradation of aromatic compounds 64 Table 4 Details of soil/water samples used for microbial analysis 71 Table 5 Microbial load of contaminated soil and water samples 72 Table 6 Microbial content of samples obtained from Site C and Site D 73 Table 7 Results of HPLC-UV analysis of TNT passages from Site A and B 74 Table 8 Results of HPLC-UV analysis of PETN passages from Site A and B75 Table 9 Results of HPLC-UV analysis of PETN passages from Site C and D75 Table 10 Phenotypic characterization of colonies of bacterial isolates 76 Table 11 Gram reaction of isolated strains 78 Table 12 Biochemical tests performed with isolates TNT-1, Pa-3, ST2, ST4, ST7 79 Table 13 Biochemical attributes of Achromobacter xylosoxidans from analysis with API® test kit (20 NE) 80 Table 14 Results of co-metabolism experiment using Tween-80 as co-substrate with TNT 82 Table 15 Results of co-metabolism experiment using Triton X-100 as co- susbtrate with TNT. 82 Table 16 Results of co-metabolism experiment using Tween-80 as co-susbtrate with PETN. 82 Table 17 Results of co-metabolism experiment using Triton X-100 as co- susbtrate with PETN. 83 Table 18 Universal primers 616V and 1492R 89 Table 19 16S rDNA BLAST results showing the top 5 matches for the TNT and PETN degrading isolates 101 Table 20 Primers designed from onr gene 106 Table 21 Primers designed to amplify nerA from Agrobacterium sp. 110 Table 22 Degenerate Primers 113

xii ABBREVIATIONS

A absorbance 2 ADNT 2 amino dinitrotoluene 4 ADNT 4 amino dinitrotoluene ADNTs aminodinitrotoluenes AZTs tetranitroazooxytoluenes BLAST Basic Local Alignment Search Tool bp base pair(s) cfu colony-forming units CL20 2,4,6,8,10,12-hexonitrohexazaisowurtzitane Da Dalton (DANTs diaminomono-nitrotoluenes DNA deoxyribose nucleic acid dNTP dinucleotide triphosphate EDTA ethylenediaminetetraacetic acid EGDN ethylene glycol dinitrate g gravitational force GDN Glycerol dinitrate GTN glycerol trinitrate, nitroglycerine HADNTs hydroxylamino-dinitrotoluenes HMX high melting explosive, octahydro-1,3,5,7- tetranitro-1,3,5.7-tetrazocine HPLC high performance liquid chromatography IPTG isopropyl-β-D-thiogalactopyranoside kb kilobase pair(s) LB Luria Bertani broth M mol/L NADH reduced nicotinamide adenine dinucleotide NADPH reduced nicotinamide adenine dinucleotide phosphate NAD(P)H NADH and NADPH

xiii NEM N-ethylmaleimide OD600 optical density at 600 nm OYE old yellow enzyme PCR Polymerase Chain Reaction PETN Pentaerithritol Tetranitrate rDNA ribosomal DNA RDX Royal Demolition Explosive, hexahydro-1,3,5- trinitro-1,3,5-triazine RNA ribose nucleic acid sp. species TAE Tris-acetate-EDTA TE Tris-EDTA Tm melting temperature TAT triaminotoluene TNT 2,4,6-trinitrotoluene U unit of enzyme activity (μmol/min) X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactoside

xiv ABSTRACT

The nitroaromatic compound 2, 4, 6-trinitrotoluene (TNT) and the nitrate esters glycerol trinitrate (GTN) and pentaerythritol tetranitrate (PETN) are high explosives that have been produced on a massive scale for use in industrial and military applications. Through many years of production and use of these explosives, large quantities of toxic wastes have been generated, which has resulted in extensive contamination of soil and groundwater. These organic nitrate contaminants are highly recalcitrant and not normally found in the environment, thus classifying these compounds as xenobiotics. During this study, 36 bacterial species were isolated from contaminated soil and water that were able to utilize either TNT or PETN as a sole source of nitrogen. These were identified as Arthrobacter sp. (Pa-3, Pb-5, ST12,

ST13, ST14, ST17, ST23,ST26), Pseudomonas sp. (ST1, ST2, ST15, ST18,

ST19,ST22), Enterobacter sp. (ST3, ST5, ST6, ST8 , ST11), Klebsiella sp. (ST4,

ST10, ST16, ST20, ST21, ST28, ST29), Microbacterium sp. (ST7, Pa-4, Pb-6,ST9,

ST24, ST25 ST27,ST30), and Achromobacter xylosoxidans (TNT1, TNT2). Six representative isolates were selected from the 36 isolates for further analysis of growth and enzyme activity. The isolates were found to catalyse the NAD(P)H- dependent reductive cleavage/breakdown of GTN and PETN in a manner that was similar to a class of previously described oxidoreuctase flavoproteins. These include

Old Yellow Enzyme (OYE), PETN reductase, GTN reductase, xenobiotic reductases

A and B and N-ethyl maleimide reductas. This is the first report of nitroaromatic degradation by Achromobacter xylosoxidans as per the author’s knowledge.

Six isolates from this study were evaluated by PCR using different primers for the presence of the previously described genes, onr and nerA, (or homologues thereof), however; similar genes were not successfully amplified perhaps due to the divergence of these genes from published sequence resulting in PCR products not similar to the described genes.

The contamination of the environment by organic nitrate compounds is of major concern because of the scale on which explosives have been manufactured, used and tested. The remediation of organic compounds is of ecological interest so as to resolve soil and water contamination. With the turn of the century, the f ocus has shifted to biological methods of remediating contaminated soil and water.

Chemical and physical methods that are currently used for the remediation of contaminated sites, such as incineration, can be expensive and can also lead to the formation of by-products that are more recalcitrant than the parent compounds.

Biological methods for remediation of explosive-contaminated sites on the other hand can be more cost-effective and significantly reduce toxicity of the soil following treatment since microorganisms known to degrade specific explosives are utilised in the treatment; as has been demonstrated at large-scale using composting for example. Bioremediation using characterized microorganisms to degrade specific explosive contaminants, as well as the enzymes produced by them, also holds the potential for rapid and reproducible explosive remediation.

The aim of this study was to isolate bacteria able to degrade TNT and PETN and to characterize these bacteria and the enzymes produced by them. Thirty-six bacteria were isolated from contaminated soil and water that were able to utilize either TNT or PETN as a sole source of nitrogen. Identification was based on 16S rRNA gene sequences and biochemical testing. Studies investigating co-metabolism of the explosives in the presence of Tween-80 was performed. Triton X-100 was also evaluated as co-metabolic substrate. It was demonstrated that the addition of Tween-

80 as a co-substrate assisted in the co-metabolism of explosives and did improve the growth and therefore the degradation of the explosive. However, Triton X-100 did not result in co-metabolism of the explosives since an improvement in growth was not observed.

The information acquired in this study can assist in the further characterization, optimization and production of explosive-degrading enzymes.

CHAPTER 1

INTRODUCTION

1.1 Explosives

Explosives are chemicals or mixtures of chemicals that when initiated, lead to the release of a large amount of energy. Once initiated, explosives rapidly decompose to form a stable material along with the liberation of a large amount of heat and expanding gases. An explosion is created due to the expansion of the gases, which exerts high pressure on the surrounding regions.

Explosives generally contain high amounts of nitrogen and oxygen mixtures that lead to the formation of gaseous products such as carbon dioxide, carbon monoxide, oxygen, nitrogen and water vapour. Most explosives, unlike chemicals, tend to detonate rather than burn. A detonation is a rapid chemical reaction (>1 km/s) that uses the oxygen present in the material (i.e. the explosive) rather than in the air. During a detonation, gases that expand rapidly are produced by the chemical reaction, which releases energy as pressure and heat.

1.2 Types of explosives

Explosives may be broadly classified into two major types: Low explosives and High explosives. Low explosives undergo deflagration rather than detonation, which means that combustion is propagated by thermal conductivity that results in the burning of the material at a slower rate. The slower rate of burning creates less pressure as compared to detonation.

High explosives detonate creating more pressure and burn much faster, detonating almost instantaneously. Detonation is supersonic such that it explodes at a rate faster than the speed of sound (Meyers and Shanley, 1990) and thus is propagated through shock compression.

Low explosives include gunpowder or black powder, which has been used as an explosive, but its principal use is that of a propellant. High explosives include 2, 4, 6- Trinitrotouluene (TNT); one of the most commonly used explosives for military munitions, civilian mining and quarrying activities. http://www.globalsecurity.org/military/systems/munitions/explosives- nitroaromatics.htm, 27/1/2011)

Glycerol trinitrate (GTN), also known as nitroglycerin, is classified under the nitrate ester explosives along with Pentaerythritol tetranitrate (PETN). Both these explosives were first characterized as a medicine to treat angina pectoris and are still in use to this day.

Pentolite is a 50/50 mixture of TNT and PETN and has civilian and military applications. It is the main composition used in primers for mining and quarrying and also boosters for seismic exploration, which is subsurface mapping using sound waves.

Royal demolition explosive or RDX is also known as Hexahydro-1, 3, 5- trinitro-1, 3, 5-triazine and/or Cyclonite and was the first nitramine explosive to be developed. It is the most important military explosive in the US. Other nitramines include HMX (high melting explosive; octahydro-1, 3, 5, 7- tetranitro-1, 3, 5, 7 tetrazocine), nitroguanidine and tetryl.

Ammonium nitrate is not an explosive itself, but mixtures with fuel oil (known as ANFO) form the basis of all bulk explosives used for mining and quarrying. Unlike molecular explosives such as TNT that posses the fuel and oxygen required for detonation within one molecule, ammonium nitrate-

based explosives contain the fuel (e.g. diesel) and oxygen (i.e. nitrate ion) in separate molecules.

1.3 Development and background of explosives

An account of all the explosives that have been formulated over the years is described below starting with gunpowder, the first explosive developed, to the more recent formulation of explosives. The structures of some of the important explosives are shown in Figure 1.1

The Chinese at the start of the millennium formulated the first explosive; it was named “black powder” but is now usually called gunpowder. Gunpowder, a mixture of potassium nitrate (saltpetre), sulphur and charcoal was primarily used as fuel for fireworks and primitive cannons by the Chinese. It is believed that the Chinese adopted their primitive cannons to develop the first gun complete with a metal barrel, gunpowder and a projectile by the 12th century ("http://www.silk-road.com/artl/gun.shtml, 27/1/11). The massive military potential of gunpowder was brought into the west when Roger Bacon not only named the ingredients and the proportions but went on to describe the explosive properties of the mixture. The first recorded use of gunpowder for rock blasting was in 1627. The first recorded use of gunpowder for mining purposes in the United States was in 1773, where it was used for blasting purposes in a copper mine in Connecticut. http://www.explosives.org/HistoryofExplosives.htm).

1.3.1 Nitrate ester explosive

Nitroglycerin, an explosive liquid obtained by nitrating glycerol, discovered by chemist Ascanio Sobrero in 1847. Glycerol trinitrate (GTN) and Pentaerythritol tetranitrate (PETN) are the two important nitrate ester

explosives, characterized by the O-NO2 bond. GTN and PETN were first

characterized as medicines and are still used in the treatment of angina pectoris.

The Swedish inventor Alfred Nobel invented the first detonating blasting cap, which made the use of gunpowder to trigger the explosion of GTN. Since GTN was unpredictable as an explosive. At times it did not explode when detonated or exploded when it was not desired. This issue was solved when the blasting cap was used along with GTN. Alfred Nobel went on to invent dynamite in 1866 by mixing nitroglycerine with kiselguhr making it a much more stable explosive. The erratic behaviour of GTN was also reduced by allowing the adsorption of nitroglycerin into a solid (kiselguhr). Hence, GTN (or dynamite) in this form was much safer to use and easier to detonate. Glycerol trinitrate (GTN) is a colourless to pale yellow, viscous liquid.The

molecular formula is C3H5(O.NO2)3. The molecular weight is and the vapour pressure is 0.00035 - 0.002 hPa at 20 °C. The water solubility of GTN is 1.25 - 1.95g/I at 20 ºCand the log Kow is 1.62 - 1.77. ( Rosenblatt et al.,

1991).

PETN was developed in Germany in 1894 and came to be used extensively during World War II. Being a very powerful and highly sensitive explosive it was much too dangerous to be used on its own since it was very easily detonated. To harness this characteristic but still harvest the powerful nature of the explosive, it is often used as a detonator as well as being combined with other explosives (e.g. Semtex = PETN/RDX, Pentolite = PETN/TNT). Pentaerythritol tetranitrate (PETN) is white solid substance with a molecular

weight of 316.15. The molecular formula is C5H8N4O12. The solubility in water is 43 mg/L at 25ºC; it is soluble in benzene and sparingly soluble in alcohol,

-10 ether, etc. The vapour pressure at 25ºC is 1.035 × 10 mm Hg and the Log Kow is 1.62. (HASDB, 2000)

1.3.2 Nitroaromatic explosive

TNT was first synthesized by the German chemist Joseph Wilbrand in 1863; however it was not used as an explosive until 1891. TNT, a nitroaromatic

explosive, is synthesized in a stepwise process in which toluene is nitrated with sulphuric and nitric acids forming mono and dinitrotoluene. The mono- and dinitrotoluene is then fully nitrated with nitric acid and oleum. 2, 4, 6,

Trinitrotoluene is a yellow coloured solid. The formula formula is C7H5N3O6. The molecular weight is 227.13 and the solubilty in water is 130mg/L; it is soluble in acetone, benzene, alcohol and ether. The vapour pressure at 20ºC is -4 1.99x10 mmHg and the Log Kow is 1.60. (HSDB, 1990; Budaveri et al, 1989)

Another commercially important nitroaromatic explosive is picric acid or 2, 4, 6-trinitrophenol. It is synthesized by nitrating phenol. Picric acid was used as a dye until 1871 when its explosive properties were discovered. This explosive was encased in shell casings and used for military purposes. Picric acid is more stable than GTN and so was used in shell casings. A limitation however is the reactivity of picric acid with the metal in the shell casings. The picrate thus formed was susceptible to undesired detonation by shock or friction.

1.3.3 Nitramine Explosives

The most recently introduced class of organic nitrate explosives are the nitramine explosives. The first nitramine explosive developed was hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine, which was first synthesized by Hans Hemming in 1899. It was patented as an explosive in 1920 and further developments to the explosive by the War Department in Woolwich, U.K. resulted in the name RDX or Royal Demolition Explosive being added. It was measured to be equally as powerful as GTN and PETN but not as sensitive. RDX is the most widely used explosive in recent times.

In 1930, another nitramine explosive was synthesized which was much more stable than RDX: octahydro-1, 3, 5, 7-tetranitro-1, 3, and 5, 7-tetrazocine (High Melting Explosive or HMX). Yet another nitramine explosive has

been synthesized recently which is even more powerful than HMX: 2, 4, 6, 8, 10, 12-hexonitrohezaisowurtizane (CL20 or HNIW).

The majority of explosives currently in use are nitramines of which RDX is most important due to its low sensitivity (and hence good stability), high power and relatively low cost.

1.4 Toxicity of explosives

Aside from the obvious physical hazards associated with explosives, toxicity to biological systems is also of concern. The vasodilatory effects of the nitrate esters GTN and PETN, and their ability to cause methaemoglobinaemia, which affects the ability of red blood cells to transport oxygen (Stokinger et al., 1982), is one example of toxic effects of nitrate esters. Although useful medically as a vasodilator at controlled dose rates, overexposure to GTN may result in negative effects caused by the action of nitric oxide (NO), a by- product of GTN metabolism (Servent et al., 1989). Low level exposure in humans has been known to cause headaches and nausea as well as occasionally causing vomiting and abdominal cramps (Beard et al., 1982). However, no chronic effects have been reported throughout the industrial production of the explosive. Certain cases of dermatitis have been recorded (Stokinger et al., 1982). Animal studies have demonstrated symptoms that include respiratory problems and decrease in blood pressure (Stokinger et al., 1982). GTN has an acute oral LD50 of 0.5-0.9 g/kg of body weight in rats (Midgley et al., 2000). Acute exposure to GTN can cause death by respiratory or cardiac arrest and GTN has been classified as a class C carcinogen. There have been few studies carried out on the toxic effects of PETN possibly due to the fact that PETN is a less efficacious vasodilator and is comparably non toxic (Rosenblatt et al., 1991). A recent study performed (Quinn et al., 2009) exposed rats to daily oral adjusted volumetric doses of PETN. Mating, body weight, feed consumption as well as the overall condition of the adults were observed and recorded. The study showed that only body weights and feed consumption were affected by treatment; however, these differences may be attributed more to the volumetric adjustments of dosage in the control and

9 high-dose groups than to PETN toxicity. No adverse effects on development or reproduction from PETN exposure were observed.This study suggests that PETN is unikely to transport or bioaccumalate in the environement based on water solubility, octonol water partition coefficient and biodegradation rates.

TNT has been classified as a mutagen by the Ames assay (an in vitro test for mutagenicity that measures the occurrence of reverse mutagenesis in Salmonella typhimurium) (Fernando et al., 1990). TNT has been shown to cause liver damage, toxic hepatitis, vomiting, dermatitis, anaemia, aplastic and haemolytic anaemia and methaemoglobinaemia in humans (Beard et al., 1982; Fernando et al., 1990; Marshall, 1932; Rosenblatt et al., 1991). TNT toxicity can also induce hepatic necrosis and cirrhosis. During World War I, workers exposed to TNT during its manufacture in ammunition plants were known as “canaries” due to the yellow colour of their skin (jaundice) caused by TNT poisoning. The oral LD50 values for TNT in rats is 0.8-1.3 g/kg, Johnson et al., 2000). Studies have demonstrated that TNT is very toxic to earthworms, the bacterium Vibrio fishceri and aquatic organisms (Drzyzga et al., 1995). A number of mutagenic studies have been performed on Salmonella strains as well as some mammalian cell lines with TNT and its degradation by-products. Most of these studies concluded that the metabolites of TNT were far more mutagenic than TNT itself. Chronic effects include dermatitis, hepatitis, haematological changes, ocular effects and cancer; consequently TNT has also been given a carcinogen classification of C (Rosenblatt et al., 1991).

Figure 1.1: Structures of some important explosive compounds. GTN and PETN are nitrate esters, picric acid and TNT are nitroaromatics and RDX, HMX and CL20 are nitramines. Reproduced from H.M.B, Seth-Smith et al, 2002.

Reports of widespread exposure to RDX have been reported in the munition industry. Chronic levels of RDX exposure have lead to seizures and unconsciousness and acute levels of exposure resulting in insomnia and irritability (Seth-Smith., 2002). The oral LD50 was measured as 0.07-0.12 g/kg bodyweight in rats. A study conducted by Kaplan reported that skin could be a route of entry for RDX, although skin absorption was not confirmed with animal studies on treating rabbits and rats topically with RDX. Conflicting results were obtained on observing carcinogenic effects on animals. Fetotoxicity and developmental effects were only observed only under maternal toxicity. RDX toxicity resulted in lower body weight due to a reduction in food intake by rats. A recent study showed that troops who became intoxicated developed grand mal seizures with associated headaches and/or amnesia on chewing an RDX containing explosive or when using it as a cooking fuel. A study on a child that ingested RDX and developed seizures further strengthened the hypothesis that RDX can travel to the central nervous system (CNS). RDX is designated as a class C carcinogen (Seth-Smith, 2002).

1.5 Environmental issues surrounding the use of explosives and their role in environmental pollution

Several explosives such as TNT, HMX and RDX are major environmental contaminants as a result of their manufacture and deployment in both industrial and military applications. The decommissioning of weapons is also a cause of environmental pollution. Most explosives are highly recalcitrant and are resistant to degradation in situ thus causing the explosives to persist in the environment for years. Unused charges remain live for extended periods of time due to the metastability of the explosives (Dario et al 2010, Meyer and Shanley 1990).

Many countries have now begun site characterization including both residential and industrial sites (Spain, 2000). TNT, RDX and HMX have been listed as the environmental pollutants that are of greatest concern (Kruchoski, 1997). TNT and RDX are found at highest concentrations in soil and

groundwater (Simini et al., 1995, Steukart et al., 1994), and the many soil studies conducted have concluded that TNT and RDX are the major soil pollutants among explosives (Bart et al., 1997, Selim et al., 1995, Xue et al., 1995). It was found from other soil studies that nitrate esters are not found at high enough concentrations to require any form of treatment (Jergeret al., 2000), however they is also considered to be recalcitrant. This could be due to their uncharged and electron withdrawing nature which caused nitrate esters to be of low aqueous solubility and resistant to hydrolysis (Rosser et al 2001, White and Snape, 1993).

Leaching has been known to lead to the spread of the explosives. Leaching pollutes the groundwater under the contaminated soil; the contaminants are spread to other areas by carry-off by water bodies. The degree to which the explosives stay in the soil or move to contaminate groundwater depends on the solubility of the explosive and the degree to which it is absorbed by soil. TNT and RDX have been shown to have low aqueous solubility at a maximum of 100 mg/l and 38 mg/l respectively at 20 degrees (Lynch et al., 2001). TNT binds very strongly to soil whereas RDX absorbs much less tightly (Pennington et al., 2002, Singh et al., 1998). This makes RDX contamination much less contained when compared to TNT due to the ability of RDX to migrate very quickly through the soil matrix. This is despite its lower aqueous solubility. The toxic nature of these explosives in soil means that the land cannot be used for alternate purposes unless it is reclaimed or cleaned up completely. It is also important to prevent or control groundwater leaching by these pollutants.

1.6 Methods of soil remediation and decontamination

A number of methods are utilized to remediate or decontaminate soil and water contaminated with explosives. These include methods such as: • Composting • Incineration

• Bioslurry treatment • Phytoremediation • Fenton oxidation • Treatment with zero-valent iron • Reverse Osmosis • Ultrafiltration • Resin adsorbtion

The two most widely employed methods of remediation currently used, incineration and composting, will be discussed in more detail.

1.6.1 Incineration

Incineration is the most common method for the clean up of explosive contaminated soil and the most effective, but it is expensive for use with polluted soils because of the cost of soil excavation, transportation and the energy required for incineration. However, a number of contaminated sites have been remediated in the past by using this process. It is performed by the removal of soil from the contaminated site and incineration of the soil and the explosives contained within (van Ham et al., 1997). Incineration allows the efficient removal of explosive pollutants from soil but the costs of carrying out incineration as compared to composting is higher. The cost of carrying out incineration becomes an even greater limitation when a smaller mass of soil requires treatment due to economies of scale (Lewis et al, 2004). One of the other limitations of using incineration as a clean-up method is that it often requires additional disposal or further treatment of the explosive products since complete combustion rarely occurs (Garg et al., 1991). Often the combustion leads to the formation of harmful compounds such as nitrous oxide (NO), carbon monoxide (CO), hydrogen chloride (HCl) as well as dioxins which also need to be contained (van Ham et al., 1997). The costs of incineration are very high, with every ton of soil that requires remediation costing an estimate of US $800 (Funk et al., 1993). However, in spite of the associated cost, it is still the most effective method of remediation.

1.6.2 Composting

Composting is a biological method of remediation wherein resident soil microbes present in the contaminated soil degrade the contaminants. The composting soil or water is augmented with organic matter such as manure, vegetable waste, and pine chips. Using such supplements dilutes the contaminants as well as provides additional carbon sources for the microbes, which in turn increases microbial activity. The composting process requires that moisture content be controlled as well as ensuring that the soil is well aerated. It has been observed that the temperature of contaminated soil often increases during composting due the microbial activity, which results in optimal conditions for the degradation of the contaminants.

A number of studies carried out on composting of TNT and RDX contaminated soils have shown that the levels of the explosives have decreased substantially during composting (Isbister et al., 1984, Williams et al., 1992, Ziegenfuss et al., 1991). Since the studies were performed under different conditions, it has been shown the removal of explosives occurred when the soil was aerated as well as when it was left non-aerated. Most studies concluded that greater removal was seen in thermophilic conditions (55°C) when compared to composting at mesophilic conditions (35°C) (Williams et al., 1992).

The main advantages of composting are that the toxicity and mutagenicity of the composted soil and water is greatly reduced when compared to the original contaminated soil (Greist et al., 1993, Greist et al., 1995, Isbister et al., 1984). The disadvantage of using composting as a remediation process is that the indigenous bacteria found in the soil varies from site to site and thus may break down the explosives in different ways. Composting is an expensive process even though the costs associated are not as high incineration, as it requires the excavation of the contaminated soil, regular aeration etc for which an estimated cost is US $300 per ton (Jerger et al., 2000).

Both composting and incineration, the most commonly used methods for remediation, appear to remove the parent compound, but the resulting by- products of the remediation may be toxic.

There are several biologically-based approaches to remediation of which soil slurry reactors, composting and land farming have been examined comprehensively. The use of bioslurry reactors has been shown to increase treatment efficiency; however it is of a higher cost than composting. Bench- scale trials (Boopathy et al., 1994) have been developed by a number of researchers, but only a few have demonstrated full-scale trials (Lenke et al., 1998). Phytoremediation has been studied wherein plants are used to remove explosive contaminants from the soil at low cost, but this proceeds at a slow rate and the resultant contaminated plant matter still needs to be dealt with.

Non-biological methods of remediation such as treatment with zero-valent iron reduce the recalcitrance of the explosives by the reductive transformation of the electron withdrawing groups. Fenton oxidation proceeds by generating hydroxyl radicals to oxidise explosives into less toxic compounds (Barreto- Rodrigues et al., 2009; Liou et al., 2003; Oh et al., 2003; Qi-zhao, 1982; Wang & Lemley, 2002; Zoh & Stenstrom, 2002). Reverse osmosis, resin adsorption and ultrafiltration are other non-biological methods employed in remediation; however these methods are used mainly for separation of the compounds and do not modify the explosives into non-toxic or non- hazardous forms.

1.7 Biodegradation of Explosives

Microbes are the final re-processors of organic carbon in the environment. Microorganisms work by catalysing the breakdown of organic matter found

in the environment to CO2. However, there are a number of chemicals not normally present in nature until their manufacture (collectively known as xenobiotics) that can be integrated into the metabolism of microorganisms as

a carbon or nitrogen source. This can be of immense potential wherein the complete degradation of the contaminants can be carried out without human involvement. The use of naturally occurring flora for the removal of organic contaminants has been referred to as “intrinsic remediation or natural attenuation” (Lewis et al., 2004) These microorganisms derive their energy and nutrients from the compounds to be degraded which is beneficial since other chemicals need not be added to the soil or water.

The use of specific bacteria to bioremediate explosives can be a much more effective method compared to composting or incineration. Using the bacterial strains capable of degradation that have been characterised, and by providing optimal conditions, the by-products of degradation can be determined. The specific bacterial strains capable of breaking down explosives into non-toxic compounds can be selected for use. The organisms themselves or the enzymes produced by them can be used for bioremediation.

1.7.1 Biodegradation of nitrate ester explosives

The biodegradation of nitrate esters progresses by successive denitrations whereby each nitro group reacts much more slowly than the previous nitro group (Bhaumik et al., 1997; Christodoulatos et al., 1997; Wendt et al., 1978)). The degradation of GTN can sometimes lead to the formation of glycerol as a by-product (Christodoulatos et al., 1997; Meng et al., 1995), which is used as a carbon source to aid in the biodegradation of the remaining explosive. A number of bacterial strains have been isolated from contaminated soils that are capable of degrading nitrate esters; these include Pseudomonas sp., Agrobacterium radiobacter and Bacillus sp. The degradation activity has been observed under a number of conditions such as aerobic, anaerobic as well as when mixed or pure cultures are utilized.

The degradation of PETN by Enterobacter was described by Binks et al., (1996), wherein a strain of Enterobacter was isolated from explosive-

contaminated soil containing a mixed bacterial culture. The mixed culture was able to grow on PETN as a sole nitrogen source. The study conducted indicated that the growth of the mixed culture was not due to nitrogen fixation from the air since the control cultures set up did not support growth. The growth of E. cloacae PB2 along with PETN resulted in a decrease in the concentration of PETN in the medium. The strain is also able to degrade GTN by using it as a nitrogen source. The enzyme responsible for degradation is PETN reductase which is a homologue of the old yellow enzyme (OYE); it catalyses the reduction of two of the four nitro groups of PETN to alcohol groups. Similar denitrations are seen with GTN. Pathways showing these denitrations are demonstrated in Figure 1.2.

1.7.2 Biodegradation of Nitroaromatic Explosives

The microbial action on nitroaromatic compounds such as TNT and picric acid proceeds by the reduction of nitro groups, reducing them to nitroso, hydroxylamino and amino groups or by direct hydride transfer to the ring structure (Spain, 1995 -Fig 1.3). The amino derivatives are very stable and adsorb to the soil quite strongly inhibiting further breakdown of the by- products in the environment. The hydroxylamino and amino derivatives often dimerize to form azo and azoxy dimers which are resistant to metabolism and are much more toxic and recalcitrant than the parent compound.

In the mechanism of TNT degradation, the removal of N from the TNT ring via the formation of Meisenheimer complex explains the aerobic degradation of TNT by bacteria (Figure 1.3). These studies report active bacterial strains such as Rhodococcus, Nocardiodes and Mycobacterium (Behrend et al., 1999; Lenke et al., 1998; Vorbeck et al., 1994). A genetically engineered Pseudomonas sp that is capable of utilizing TNT as a carbon and nitrogen source that shows TNT mineralization and ring cleavage has been created. Often several dead- end products were observed indicating that more than one reaction was occurring.

Fungal degradation of TNT by Phanaerochaete chrysosporium has been shown to involve both ring cleavage and mineralization (Fernando et al., 1991); it is also able to liberate carbon dioxide from TNT during metabolism.

1.7.3 Biodegradation of Nitroamine Explosives

RDX is much easier to biodegrade as compared to TNT due to its lack of aromaticity, and it has been shown to undergo several types of reactions. The degradation of RDX results in different end products depending on whether aerobic or anaerobic conditions were maintained. Studies show that under aerobic conditions, nitrite accumulation occurs whereas under anaerobic conditions, nitro group reduction occurs forming nitroso intermediates, which break down further as degradation progresses.

Relatively few studies reporting bacterial strains capable of degrading HMX have been published, although there have been a number of recent studies reporting the degradation of CL-20 mostly under anoxic conditions by microbial cultures including Rhodococcus, Pseudomonas, Ochrobactrum, Mycrobacerium and Ralstonia.

1.8 TNT Degradation

1.8.1 Alkaline hydrolysis of TNT

TNT has been shown to be degraded by alkaline hydrolysis. Batch alkaline hydrolysis has been described as an alternate method of breaking down RDX (Behki et al., 1993). Nitroaromatics have been known to be unstable in alkaline media and quickly degraded to non-definable degradation products, followed by the condensation and liberation of nitrite.

1.8.2 Thermal decomposition of TNT

A number of studies have been carried out to determine how thermal decomposition proceeds since it is fundamentally important in the explosives

field. The determination of the kinetics and mechanisms involved in thermal decomposition form an integral part in the characterisation of TNT. Evidence from previously performed studies show that one or more of the following steps take place during the thermolysis of TNT (and other polynitro

compounds) : a) C-NO2 homolysis, b) isomerisation of nitro group (NO2) to

nitrite (ONO), c) reactions of nonenergetic substituent of the ring (CH3 in TNT) as described in Figure 1.4. (Cohen et al., 2007)

1.8.3 Fenton Oxidation of TNT

Fenton oxidation utilises a solution of hydrogen peroxide in the presence of an iron catalyst to oxidise contaminants, explosives, heavy compounds, leachates and waste water. Fenton oxidation generates hydroxyl radicals from the molecules of hydrogen peroxide, which aids in oxidising explosives into less toxic forms. Fenton oxidation has been described by Barreto-Rodrigues et al., 2009; Liou et al., 2003; Oh et al., 2003; Qi-zhao, 1982; Wang & Lemley, 2002; Zoh & Stenstrom, in 2002. Their studies demonstrated that Fenton oxidation was a good method to degrade TNT since 100% of TNT (156 ml/L) was removed from the media after the solution was subjected to Fenton oxidation at a temperature of 20◦C being agitated at 100 rpm.

1.8.4 Photocatalysis

Contaminants such as TNT are oxidised using magnetic particles that are

coated with TiO2 and exposed to ultraviolet light. During the studies performed by the following order of reactivity was observed: nitrotoluenes > nitrobenzene > dinitrotoluenes > dinitrobenzenes > 2,4,6-trinitrotoluene > 1,3,5-trinitrobenzene, which reflects the known influence of nitro groups on the attack of electrophilic reagents towards the aromatic molecule (Dillert et al., 1995).

1.8.5 Biodegradation of TNT

The earliest study carried out for the microbial degradation of TNT by Fernando et al., (1990) and Spiker et al., (1992) proposed the use of the white rot fungus Phanerochaete chrysosposium. P. chrysosposium is a wood-rotting fungus possessing impressive biodegradative properties. P. chrysosposium has been the organism of choice since it harbors non-specific extracellular peroxidises capable of degrading lignin. These non-specific enzymes facilitate the degradation of other recalcitrant compounds such as explosives.

Figure 1.2: Microbial transformation of nitrate esters. Nitrate ester reductase enzymes catalyse the removal of nitrite (NO2-) from nitrate esters. (A) GTN can be transformed to glycerol in three steps (Meng et al., 1995). Each step requires the oxidation of NAD(P)H and releases nitrite. (Rosser et al., 2001) (B) PETN has two of its nitro groups reduced (Binks et al., 1996).

Figure 1.3: Microbial transformation of TNT. Path a: TNT can undergo reduction of the nitro groups through nitroso, hydroxylamino and amino derivatives (Spain, 1995). Each of the nitro groups may undergo reduction, although triaminotoluene only forms under anaerobic conditions. Azo and azoxy dimers can form from nitroso and hydroxylamino dimerization (Lewis et al., 1992). Path b: TNT can also undergo hydride addition to form hydride- and dihydride- Meisenheimer complexes. Nitrite is produced from this reaction, but other products are unidentified (French et al., 1998).

Figure 1.4 Thermolysis of TNT following three steps: a) C-NO2 homolysis, b) isomerisation of nitro group (NO2) to nitrite (ONO), c) reactions of nonenergetic substituent of the ring (CH3 in TNT) as described in Figure 1.4. Reproduced from Cohen et al., 2007.

Radiolabelled TNT (14 C) was used in the studies carried out to determine the rate at which degradation was carried out by P. chrysosporium. As reported by Fernando et al., (1990), P. chrysosporium is able to cause extensive degradation of [14C] TNT in a reasonably short period of time. It was reported that 35% of [14 C] TNT was mineralised in 12 days of incubation. It has been demonstrated that the extent of mineralisation of [14C] TNT in soil cultures can be extended by increasing the incubation period. However, in liquid cultures addition of supplemental glucose did not stimulate mineralisation after 12 days of incubation. The reasons supplied for this phenomenon was not supported with evidence. However, it was suggested that the cause could be depletion in glucose or the chemical being degraded from the culture medium.

Contradictory theories were reported by Spiker et al., (1992), wherein P. chrysosporium was unable to tolerate the levels of TNT found in contaminated soils. The studies illustrated that the fungus was completely inhibited even by small amounts of TNT present in the contaminated soil. Bumpus et al., (1985) cited evidence that P. chrysosporium is capable of complete degradation of highly chlorinated PCB’s (polychlorinated biphenyls) but mineralisation was observed at very low concentrations (0.05 ppm). Fernando et al., (1990) suggested that P. chrysosporium is a good candidate for TNT bioremediation whereas Spiker et al., (1992), summarised that P. chrysosporium is not a good choice for the bioremediation of TNT- contaminated sites containing very high concentrations of explosives due to the high sensitivity of the fungus to the contaminants. Additionally, the relatively slow growth rate of this fungus means that it would soon be overtaken by bacteria under mixed-culture conditions.

Duque et al., (1993) isolated a Pseudomonas strain from the soil found around an explosives factory and this strain was able to use TNT as a nitrogen source. In the study reported by Duque et al., (1993), a Pseudomonas hybrid strain was able to grow faster on TNT than its parental strain, at both high and low concentrations of TNT. This hybrid strain also

25 did not accumulate nitrite. The explanation provided by Duque et al for this activity was that the hybrid strain exhibited a greater nitrite reductase activity than the parental strain.

Both the parental Pseudomonas sp. strain C1S1 and its hybrid, Pseudomonas sp. Clone A were able to utilise TNT as a nitrogen source by the elimination of the nitro groups. This led to the production of 2,4- and 2,6-dinitrotoulene, 2-nitrotoulene and toluene. The removal of the three nitro groups was thoroughly investigated by Duque et al. Since toluene was detected in the culture supernatants of the TNT-grown cells, the third nitro group could also be eliminated; however the mechanism could not be demonstrated.

A drawback faced by Duque et al., (1993) in their investigations was the reduction of nitrotoulenes to dead-end aminotoulenes, which caused the mineralisation to stop. An explanation for this was not forwarded. A possible reason could be the difference in the pathway for the mineralisation of TNT or a change in an existing one. Even at high concentrations, the Pseudomonas strain is resistant to TNT and consequently the mineralisation was not inhibited by the higher concentrations.

Previous studies relating to the biotransformation of TNT have shown that a number of bacterial consortia can transform TNT by co-metabolism. Co- metabolism can be defined as the transformation of an organic compound by a microorganism incapable of utilising the substrate as a sole source of energy or one of its constituent elements. Boopathy et al., (1994) investigated the use of a number of co-substrates such as succinate, citrate and malic acid with an aim of finding an inexpensive carbon source for large- scale bio-treatment of TNT. Boopathy et al., (1994) confirmed that a soil bacterium isolated from TNT- contaminated soil could degrade TNT (100 ppm) extensively in 12 h when 0.3% of molasses were added to the culture. This transformation was accomplished by co-metabolism wherein among all the substrates screened, molasses was found to produce the best results. These results are in terms of bacterial growth and transformation of TNT. A large

26 scale, soil-slurry reactor for the large-scale treatment of TNT contaminated soil was investigated. The studies conducted by Boopathy et al (4) were useful in identifying the optimal substrate concentration and the suitable substrate for the highest rate of TNT transformation. It was observed that the cultures that received molasses at a concentration of 0·3% transformed 100 ppm of TNT within 12 h of incubation at ambient temperature, whereas the cultures with other carbon sources took more than 100 h to transform 100 ppm of TNT.

The degradation capacity of Pseudomonas sp. clone A was described by Haidour et al., (1996). Pseudomonas sp clone A was able to utilise TNT, 2,4- dinitrotoluene and 2,6-dinitrotoluene as a N-source after the enzymatic removal of nitro groups from the aromatic ring. Haidour et al., (1996) proposed that Pseudomonas sp clone A performs two different types of reduction processes with TNT. The first process leads to the removal of a nitro group from the aromatic ring and allows its utilisation as a N-source, while the second process leads to the formation of unproductive compounds that accumulate in the culture medium.

An important discovery in the degradation of TNT was made by Montpas et al., (1997); the addition of a surfactant, Tween 80 played a key role in facilitating rapid degradation. A strain of Serratia marcescens isolated from a TNT contaminated site was able to degrade TNT by using it as a sole carbon and nitrogen source. A number of microorganisms isolated from the contaminated sites were screened for TNT-degradation activity, but only two bacteria were identified as degraders. These were Serratia marcescens and Alcaligens sp. Degradation experiments were carried out with both bacteria, which demonstrated that S. marcescens was the more effective TNT- degrader. Montpas et al., (1997) conducted experiments with two additives to the working medium, benzyl alcohol and Tween 80. Benzyl alcohol is an enzyme inducer and can increase the rate of oxido-reductase production. Tween 80 is a surfactant, which is used to promote the release of enzymes from micororganisms as well as the dispersion of TNT in aqueous solution. Tween 80, also known as Polysorbate 80 is a non-ionic detergent and

27 emulsifier derived from polyoxylated sorbitol and oleic acid. Nitroaromatics pollutants such as TNT are apolar, having both a low solubility and a high tendency for adsorption onto organic matter. Montpas et al., (1998) surmised that S. marcenscens is able to degrade TNT rapidly in the presence of a surfactant. The study emphasises the versatility of using S. marcescens in the biological treatment of TNT contaminated soil.

A standardized assessment of the ability of different bacterial genera to transform TNT was carried out by Fuller et al., (1997). The studies conducted demonstrated with evidence that aerobic Gram-positive bacteria are much more sensitive to TNT than Gram-negative bacteria. Several possible explanations for the differential sensitivity of aerobic Gram-positive and Gram-negative bacteria to TNT were proposed by Fuller et al., (1997). It was suggested that the reason for the resistance of Gram-negative bacteria towards TNT is because of the structure and composition of the cell wall and the presence of the outer membrane. Since Gram-positive bacteria lack an outer membrane, they are more permeable to TNT, thereby allowing more TNT to enter the cells and disrupt normal function. Another interesting fact pointed out by these studies carried out by Fuller et al, (1997) was that TNT affected only actively growing cells. Two explanations were provided for the resistance of Gram-negative bacteria, 1) they may possess an active transport system that readily removes TNT from the cell, and 2) they may contain enzymes that detoxify TNT.

Fuller et al., (1997) recommended that bioremediation of highly contaminated soil should focus on methods that are favourable to the growth of Gram-negative organisms. The best approach for bioremediation that is both ecologically safe and economical would be the in-situ treatment of soil. However, as pointed out by Fuller et al., only if the microbial ecology of the treated soil is similar to that of the surrounding uncontaminated soil will in- situ bioremediation be helpful. This is because plant growth, biogeochemical cycles and other ecosystems processes are dependent on the microbial community.

A feasibility study was explored to find out whether the bacterium M91-3 could be used for the degradation of TNT (Oh et al., 1998). This bacterium was isolated from agricultural soil containing atrazine. It was concluded from the studies that the soil bacterium M91-3 later identified as Stenotrophomonas maltophilia was capable of aerobically degrading TNT. The most effective degradation of TNT was achieved by optimising the environmental factors such as pH, substrate concentration and additives.

TNT degradation yields a number of different products depending on the microorganism that is used for the degradation. Esteve-Nunez et al., (2001) have shown in their studies that except for a reaction catalysed by pentaerythritol tetranitrate reductase on TNT, very little is known about denitrase activity. Esteve-Nunez et al cloned a denitrase gene cluster of Pseudomonas sp. strain JLR11 in Burkholderia cepacia R34, in order to see if it grows in TNT, which it did. As suggested by Esteve-Nunez, an in depth analysis of this gene cluster will help gain insight of the mechanism underlying the denitrase reactions.

There are a number of bacteria capable of degrading TNT and these bacteria are widely distributed at the contaminated sites, in spite of which the contamination still persists for a very long time.

An observation made by Duque et al., (1993) was the presence of di- and mononitrotoulenes in the culture supernatants of Pseudomonas strain C1S1 growing on TNT as a sole nitrogen source. It was understood from this occurrence that the sequential removal of nitro groups was taking place. Toluene is a good carbon and energy source for a number of bacterial species and hence should result in the complete mineralisation of TNT as discussed by Lewis et al., (2004). Through genetic engineering, this was carried out and complete mineralisation of TNT did take place compared to other bacterial studies.

The mineralisation of TNT is generally accompanied with the production of metabolic dead-ends, that account for most of the TNT removed from the

29 cultures. The microbial activity to remove nitro groups after the genetic manipulation of the microbes was not sufficient to produce efficient mineralisation of TNT (Lenke et al., 1998).

The final product of TNT nitro group reduction is triaminotoulene (TAT), but as investigated by Lewis et al., (2004), TAT has been observed only in anaerobic bacterial cultures.

A study conducted by Claus et al., (2007) investigated the degradation capacity of Raoultella terrigena, a bacterium isolated from soil and water sites contaminated with TNT. R. terrigena strain HB was able to remove TNT from culture supernatants under optimum aerobic conditions within several hours. Nutrient supplements were required in very low quantities for the co-metabolic transformation processes. An important finding was made by Claus et al wherein the main fraction of TNT metabolites was cell- associated. This report deviated from other studies performed where the main metabolites of TNT mineralisation remains in the supernatant in the form of ADNT’s (amino dinitrotoluenes). Since R. terrigena grows rapidly at low temperatures and different redox conditions, it is a promising candidate for the degradation of TNT from the TNT-contaminated water under in-situ conditions (Claus et al., 2007).

Claus et al concluded that the accumulation of TNT metabolites within the bacterial cell allows the opportunity to clean up contaminated water and soil after separation of the biomass. The strain isolated by Claus et al., removed TNT under in-situ conditions, as well as in media containing water and soil that has been contaminated with nitroorganic mixtures. R. terrigena strain HB was not able to grow only in the presence of TNT, an additional nutrient source was required as a carbon or energy source to allow TNT transformation. However, even at low nutrient concentrations TNT transformation did occur and also resting cells were capable of co-metabolic transformation of TNT within a few hours of incubation.

1.8.5.1 Microbial metabolism of TNT

1.8.5.1.2 Aerobic Metabolism of TNT by Bacteria

Studies conducted on aerobic metabolism of TNT by bacteria by Duque et al., (1993) suggested that since TNT is not a substrate for organisms or their oxygenase enzyme, an organism capable of degrading TNT was difficult to isolate. However, Pseudomonas sp C1S1 was isolated from a TNT-contaminated site was able to utilise TNT as a sole nitrogen source (Duque et al., 1993). This capability was thought to be due to the formation of the complex described earlier, called the Meisenheimer complex. The Meisenheimer complex is formed by the nucleophilic substitution of a carbon atom by a hydride ion. But this was later questioned when strain C1S1 was shown to be unable to produce the hydride Meisenheimer complex during incubations with TNT (Vorbeck et al., 1994). Enterobacter cloacae PB2 does use TNT as a sole nitrogen source producing hydride Meisenheimer complex in the process and consecutively releases nitrite from the aromatic ring.

Some other transformation products of TNT reduction include acetylation at the 4 amino position of 2,4-diamino 6-nitrotoluene (2,4-DANT) by Pseudomonas fluorescens.

Studies were conducted by Bradley et al., 1995l to identify the parameters that regulated TNT mineralisation in contaminated soils to explain the long- term persistence of TNT in the contaminated sites. The parameters studied were soil moisture content, drying history, oxygen content, TNT concentration and carbon sources present. The studies demonstrated that the addition of complex carbon sources did not enhance the mineralisation of TNT, it was in fact reduced. Bradley et al., (1995) also observed that the soil moisture content was an important parameter that affected TNT mineralisation. It was observed that the dryness of the soil negatively

affected the TNT mineralisation and could be one of the reasons for the persistence of TNT at the site which was under study. Another important factor demonstrated from the studies was that TNT mineralisation was inhibited in the presence of higher concentrations of TNT. The final parameter to be studied was the effect of oxygen content on TNT mineralisation; it was observed that TNT mineralisation was inhibited in tanks with headspace that resulted in oxygen augmentation as compared to tanks without headspace. This demonstrated that microaerophilic growth of organisms was preferred for TNT mineralisation.

In the mechanism of TNT degradation, the removal of N from the TNT ring via the formation of Meisenheimer complex explains the aerobic degradation of TNT by bacteria. The non-specific reduction of TNT into amino derivatives that accumulates in the supernatant is the most described pathway among aerobic bacteria. Very few studies have been reported regarding the aerobic metabolism of amino aromatic compounds by microorganisms.

1.8.5.1.3 Anaerobic Metabolism of TNT by Bacteria

Anaerobic processes have certain advantages such as rapid reduction at low redox potential. It is therefore advantageous to study anaerobic systems for TNT degradation to obtain more efficient rates of removal, since azoxy- nitrotoluene products are not formed. The final product of TNT nitro group reduction is triaminotoluene (TAT). However, TAT has been observed only in anaerobic bacterial cultures; a reason provided by Lewis et al. (2004) is the presence of low-midpoint-potential redox mediators that are able to achieve the reduction of the third nitro group, in those organisms that have evolved to live in low redox environments.

Two genera of bacteria have been investigated in great detail because of their anaerobic metabolism of TNT, and these include Clostridium and Desulfovibrio. Clostridium species when used to degrade nitro groups in

TNT leads to the production of TAT and products yet to be identified. Hughes et al. (1998) reported the formation of 2-amino-4-hydroxylamin-5- hydroxyl-6-nitrotoluene and 2-hydroxylamino-4-amino-5-hydroxyl-6- nitrotoluene while working with cell extracts of Clostridium. This may have arisen from Bambeger rearrangement of dihydroxydinitrotoluene as described by Hughes et al. (1998).

Desulfovibrio strains have also been investigated because of their capacity to metabolize TNT anaerobically. Preuss et al., (1993) isolated a pure culture of Desulfovibrio which was selected by using TNT as the sole nitrogen source and pyruvate as the carbon and energy source. This strain of Desulfovibrio was able to catalyse the complete reduction of TNT to TAT. It was suggested by Esteve-Nunez et al., (2001) that if TNT degradation by Clostridium and Desulfovibrio sp. was to proceed via TAT, the mineralisation of TNT under anaerobic conditions should involve the elimination of the amino groups from TNT

1.8.5.2 Fungal Metabolism of TNT

The interest in fungi comes from their ability to degrade a diverse group of environmentally persistent and toxic chemicals. The white-rot fungus Phanerochaete chrysosporium has been investigated widely for its ability to transform TNT. The initial steps of fungal degradation of TNT involve the reduction of nitro groups. P. chrysosporium mycelia reduces TNT to a mixture of 4-ADNT, 2-ADNT, 4-hydroxylamino-2,6-dinitrotoluene and azoxytertranitrotoluenes. The aromatic compounds and azoxytertranitrotoluenes disappear under ligninolytic conditions and mineralisation can be quite extensive. This initial attack is due to the extracellular enzymes of the lignolytic system.

Bayman et al., (1997) reported several fungal species that are able to tolerate high TNT concentrations. They proposed a two-step process in which TNT is reduced to ADNT’s by a soil fungus called C. resinae and then the mineralisation is carried out by P. chrysosporium. This two-step

method is important since the rate of mineralisation can be increased, and P. chrysosporium can be protected from the toxicity of TNT that is caused by hydroxylaminodinitrotoluene. Previous studies show that P. chrysosporium is affected by very high concentration of TNT- contamination and is inhibited by it.

A number of other fungal strains have also been investigated for their use in the degradation and mineralisation of TNT. These include wood and litter- decaying basidomycetes. However, a disadvantage of using wood fungi is that since they are native to wood and not soil, this may lead to competitive growth with the soil fungi in the remediation situations.

1.9 Degradation of PETN

Methods currently in use for the degradation of PETN are similar to those used for other explosives. Certain studies utilising different methods are described below.

1.9.1 Degradation of PETN on metal surfaces

A study carried out by Mileham et al., 2008 investigated the chemical

stability of PETN when it was placed with metal oxides MnO2, CuO, MoO3,

and Fe2O3. The study showed that when PETN was placed in physical

contact with MoO3, there was rapid formation of a gas, which on analysis

indicated the presence of N2O4, N2O and CO2. This demonstrated that

PETN is degraded when in the presence of MoO3.

1.9.2 Degradation of PETN by Granular Iron

A study performed by Zhuang et al., (2008) looked at PETN transformation using flow-through iron columns. It was observed that when both a 100% iron column and a mixed column were utilized PETN was sequentially

denitrated. The denitration was as follows, with PETN being reduced to pentaerythritol along with the formation of pentaerythritol trinitrate (PETriN) and pentaerythritol dinitrate (PEDN); the formation of pentaerythritol mononitrate was not detected. On testing for nitrogen mass recovery, 100% recovery was obtained indicating that all nitro groups were removed from PETN.

The study also went on to demonstrate that nitrite was released in each + denitration step and was subsequently reduced to NH4 by iron as well as suggesting that since nitrate was not detected that hydrolysis was not involved in PETN degradation.

1.9.3 Microbial metabolism of PETN

A number of microorganisms have been characterised as being able to degrade nitrate esters such as PETN and GTN. Further studies of these microorganisms have led to the identification of a number of similar enzymes capable of producing nitrite and alcohol from the reductive cleavage of a nitrate ester. Rosser et al., (2001) went on to suggest that these enzymes are dependent on a nicotinamide cofactor, which aids in nitrate ester cleavage. This group of enzymes are labelled as “oxidoreductases” and can be found in a number of microorganisms including Agrobacterium radiobacter, Enterobacter cloacae, Escherchia coli, Pseudomonas flurescens, Pseudomonas putida, and Saccharomyces cerevisiae.

Oxidoreductases are related to the Old Yellow Enzyme (OYE) flavoprotein family. They may differ in substrate and cofactor preferences and can be either constitutively expressed or induced as observed by Rosser et al. The studies performed by Rosser et al., (2001) and Blehert et al., 1997 suggested that these enzymes help in detoxification. Old Yellow Enzyme is an NADPH oxidoreductase that contains flavin mononucleotide (FMN) as

35 its prosthetic group (Brown et al., 1998). OYE was first isolated from brewer’s bottom yeast (Warburg and Christian, 1933).

Genetic similarities have been observed between OYE and a number of genes from different organisms. These include the gene nemA found in Escherichia coli which encodes for N-ethylmaleimide (NEM) reductase; this gene is involved in the degradation of the toxic compound NEM, a cysteine-alkylator and an inhibitor of transvascular protein passage (Carlsson et al., 2001). It was found that NEM reductase was highly similar with morphinone reductase from Pseudomonas putida M10 that participates in the metabolism of codeine and morphine (French and Bruce, 1994). Another homologue of OYE is 12-oxophytodienoate reductase from Arabidopsis thaliana, which has the physiological function of reducing the olefinic bond of an α, β unsaturated ketone (Brown et al., 1998).

Binks et al., (1996) performed one of the most significant studies on the biodegradation of PETN, whereby a strain of Enterobacter, designated as E. cloacae PB2 was isolated from explosive contaminated soil samples and was found to aerobically metabolise PETN under nitrogen limiting conditions. The study showed that when E. cloacae was grown in culture containing PETN/NH4Cl as nitrogen sources, even though NH4Cl was more readily assimilated the growth yield was higher when PETN was the nitrogen source. They also concluded that E. cloacae PB2 was unable to utilise PETN as a carbon source, which leads to the assumption that E. cloacae PB2 was utilising nitrogen atoms from PETN (two to three of four). Further purification and kinetic analysis of E. cloacae PB2 showed the production of PETN reductase. Binks et al., (1996) analysed PETN reductase and found it to have properties of an oxidised flavoprotein and further sequencing studies showed that it belonged to the α/β barrel family of flavoproteins. Similar observations were also made by Barna et al, (2001); PETN reductase on analysis by ion-exchange and affinity chromatography was found to be covalently bound to FMN. On testing the activity of PETN reductase, it was found that it was dependent on cofactor NADPH. It was suggested by Binks et al., (1996) that 1 mol of nitrite is

36 released for each mol of NADPH oxidised. The function of NADPH in the activity of the enzyme was explained as such NADPH reduces the enzyme in a reductive half-reaction to give rise to the dihydroquinone from of the enzyme-bound FMN (FMNH2). The enzyme is then oxidised in the oxidative half reaction by the nitro-containing explosive (Barna et al., 2001; Khan et al.¸ 2004; Khan et al, 2005). PETN reductase has been shown to catalyse the liberation of nitrite from GTN, PETN and ethylene glycol dinitrate (EGDN) and it has also shown degrading activity with nitroaromatics such as TNT and cyclic triazine compunds such as RDX, hence having a significant importance in the remediation of land contaminated with explosives.

The deduced amino acid sequence of PETN reductase shares similarities with OYE, morphinone reductase, and oestrogen-binding protein in Candida albicans, however PETN reductase differs from OYE from the fact that it is a monomer, and PETN reductase is also able to reduce nitroaromoatics such as TNT and picric acid (Barna et al., 2001; Binks et al., 1996; French et al., 1994; Khan et al., 2004). French et al., (1994) first cloned the PETN reductase gene and it was designated onr (organic nitrate reductase). It was over-expressed in E. coli where a specific activity of 6.1 U/mg was obtained against GTN for crude enzyme extract with around 30- 50% of cellular protein being PETN reductase.

Agrobacterium radiobacter isolated from sewerage sludge was found to be able to produce GTN reductase and was able to metabolise both GTN and PETN in aerobic, nitrogen-limiting conditions as described by White et al., (1996). This study also reported that nitrite was produced in stoichiometric quantities from GTN, with an increased degradation rate in the presence of NADH. This suggests a cofactor-dependent reductive cleavage of the nitrate ester bond. When nucleic acid and protein databases were searched and compared a high level of similarity was found between GTN reductase and OYE, PETN reductase, NEM reductase, and morphinone reductase as observed by Snape et al., (1997).

It was observed that cofactor preference was different in E. cloacae PB2 and A. radiobacter where GTN reductase preferred NADH and PETN reductase preferred NADPH (Barna et al., 2001; French et al., 1994; Snape et al., 1997; White et al.¸1996). This perhaps may be explained by the differences in the pathways in which these cofactors are involved. Snape et al., (1997) went on to explain that the NADH/NAD+ pair is involved in the catabolic pathway resulting in ATP production, which contrasts with the NADPH/NADP+ pair that is used primarily in biosynthetic pathways.

Pseudomonas species have also been isolated from explosive sites contaminated with the nitrate ester GTN. Blehert et al., (1997) described two isolates of Pseudomonas, which were able to tolerate concentrations of GTN (1.76 mM and higher) inhibitory to other soil microbes, and one which was able to utilize GTN as a sole nitrogen source.

A number of other organisms have been shown to degrade PETN and GTN including Arthrobacter sp., Klebsiella sp. and Rhodococcus sp. (Marshall & White, 2001; Meng et al., 1995; Ye et al., 2004). There have also been a number of other organisms that possess similar oxidoreductase flavoproteins to those that are able to degrade GTN and PETN (Williams et al., 1992). These oxidoreductases are known to generally act in detoxification systems (Blehert et al., 1997).

1.9.4 Fungal metabolism of PETN and GTN

Microorganisms capable of degrading nitrate ester GTN have been isolated from contaminated sites. These include Geotrichum candidum, P. chryosporium and Pencillium corylophilum (Ducrocq et al., 1989; Ducrocq et al., 1990; Zhang et al., 1997). Ducrocq et al., (1989) demonstrated that certain yeasts and mycelial fungi can convert GTN to a mixture of glyceryl dinitrates (GDNs) and glyceryl mononitrates (GMNs). The studies performed by Zhang et al., (1997) demonstrated that complete removal of GTN was observed in the presence of Penicillium corylophilum Dierckx, was able to

38 completely degrade glyceryl trinitrate (GTN) in a buffered medium (pH 7.0) containing glucose and ammonium nitrate.

AIMS

The objectives of this study were to:

1. Isolate TNT, PETN and Pentolite degrading microorganisms from

contaminated soil and water.

2. Characterise these organisms and their enzyme activity as well as

determine optimum conditions for removal of the explosives.

3. Identify genes similar to previously described genes responsible for

degradation.

CHAPTER 2

MATERIALS AND METHODS

2.1 General Procedures

All chemicals were of analytical and molecular reagent grade obtained from Sigma Aldrich or Merck unless specified otherwise. Solutions were prepared in

deionised water (dH20) delivered from a Millipore Milli-Q® System, unless specified otherwise. Solutions were dispensed using Finnpipette micropipettes (Pathtech Pty Ltd., Australia) with the following volume ranges: 200 to 1000 μL, 20 to 200 μL, 5 to 50 μL, and 0.5 to 10 μL. Micropipettes were calibrated according to manufacturers’ instructions.

All glassware was washed using Pyroneg detergent, rinsed in tap water and

rinsed finally in dH20. All glassware, micropipette tips, plasticware, media and solutions used for bacterial, DNA and molecular work was sterilized by autoclaving at 121ºC for 20 min, unless stated otherwise.

General chemicals and equipment used throughout this study is listed in Appendix I.

2.2 Bacteriological Methods

2.2.1 Antibiotics, Media and Chemicals An account of the antibiotics media and chemical used are described below.

2.2.1.1 Antibiotics

Ampicillin was dissolved in sterile dH20 at a concentration of 100 mg/ml. All antibiotic stock solutions were sterilized by filtration through a 0.2 μm membrane filter and stored at -20ºC.

2.2.1.2 Luria Bertani Agar (LBA)

Tryptone (1.0% w/v), yeast extract (0.5% w/v), NaCl (0.5% w/v) and

bacteriological agar (1.0% w/v) was dissolved in dH2O and autoclaved under standard conditions. After cooling to 50ºC, the agar was poured into Petri dishes and allowed to set. LBA plates were stored at 4 ºC.

2.2.1.3 LBA Containing Ampicillin

LBA was prepared as stated above. After cooling agar to 50 ºC, ampicillin was added to a final concentration of 100 µg/mL before pouring into Petri dishes. Plates were stored at 4 ºC.

2.2.1.4 Luria Bertani Broth (LBB)

Tryptone (1.0% w/v), yeast extract (0.5% w/v) and NaCl (0.5% w/v) was

dissolved in dH2O and autoclaved under standard conditions.

2.2.1.5 LBB Containing Ampicillin

LB broth was prepared as stated above. Before use, ampicillin was added at a final concentration of 100 µg/mL.

2.2.1.6 M9 Minimal Nitrogen-free Medium

5X M9 salts (N-free)

Na2HPO4.7H2O 64 g/L

KH2PO4 15 g/L NaCl 2.5 g/L

All the salts were dissolved in 1L of dH2O, autoclaved and stored at room temperature.

M9 salts 5X

MgSO4 1M

CaCl2 1M

After cooling to room temperature, the required amount of filter-sterilised glucose (0.2% w/v) was added to the solution. 1X solutions were prepared by diluting the stock solutions with sterile Milli Q water.

2.2.1.7 Plate Count Agar

Tryptone (5g/L), yeast extract (2.5g/L), glucose (1g/L), Agar (15g/L) was

dissolved in dH2O and autoclaved under standard conditions. After cooling to 500C, the agar was poured into petri dishes and allowed to set. The plates were stored at 4 ºC.

2.2.1.8 X-gal Agar

Using LBA plates supplemented with the appropriate antibiotics, 40 µL of both 24 mg/mL IPTG and 40 mg/mL X-gal was spread evenly onto the surface of the agar just before use. These plates were incubated wrapped in foil, as X-gal is light sensitive.

2.2.1.9 2YT broth

Tryptone (16 g/L), yeast extract (10 g/L),NaCl (5g/L), and glucose (2g/L),

was dissolved in dH2O and autoclaved under standard conditions.

2.2.1.10 2YT Agar

Tryptone (16 g/L), yeast extract (10 g/L),NaCl (5g/L), glucose (2g/L), and

bacteriological agar (5 g/L), was dissolved in dH2O and autoclaved under standard conditions. After cooling to 50 ºC, the agar was poured into petri dishes and allowed to set. The plates were stored at 4 ºC

2.2.1.11 Alkaline lysis solution I

Glucose (BDH) 50 mM EDTA (Merck) 10 mM Tris-base (Roche) 25 mM

All ingredients were dissolved in dH2O, pH adjusted to 8.0 with HCL; stored at 4ºC.

2.2.1.12 Alkaline lysis solution II

NaOH (BDH) 0.2 M SDS (Merck) 1% (w/v)

Dissolved in sterile dH2O. This solution is prepared fresh before every use.

2.2.1.13 Alkaline lysis solution III

Potassium acetate (Sigma) 5M Glacial acetic acid (BDH) 2M º Dissolved in 28.5 mL of dH2O, autoclaved and stored at 4 C.

2.2.1.14 Cetyltrimethylammonium bromide (CTAB)/NaCl

CTAB (Sigma) 10% (w/v) NaCl (Chem Supply) 0.7M

Heated to 65 ºC and stored at room temperature.

2.2.1.15 Chloroform/isoamyl alcohol (C:I 24:1)

Chloroform was dissolved with Isoamyl alcohol in the ratio of 24:1

2.2.1.16 Deoxynucleotide triphosphates (dNTPs) 25 mM of each dATP ,dTTP, dGTP, and dCTP (Bioline); stored at -20ºC.

2.2.1.17 EDTA

EDTA (0.5M) was dissolved in sterile dH2O adjusted to the appropriate pH with NaOH and autoclaved.

2.2.1.18 Ethanol

70%(v/v) ethanol prepared from absolute ethanol (96%) commercial grade (Merck)

2.2.1.19 Ethidium Bromide

0.5 μg/mL Ethidium Bromide (Fluka); 1 mg per 2 L MQ water bath

2.2.1.20 10x Gel loading dye

Ficoll- 400 (BDH) 10% (w/v) Glycerol (Ajax) 50% (v/v) Orange G (BDH) 0.5% (w/v) SDS 1% EDTA 10 mM Tris-base 50 mM pH to 8.0 with HCl; stored at 4ºC.

2.2.1.21 Glycerol

10-80% (v/v) glycerol, autoclaved and then stored at room temperature.

2.2.1.22 GTN (Merck)

1% solution in alcohol. Stored at 4ºC. (Merck, Cat.#107753, Melbourne,Australia)

2.2.1.23 KOAC :60 ml

5 M KOAC,11.5 mL acetic acid dissolved in 28.5 ml water.

2.2.1.24 Lambda ( ) PstI DNA ladder

100 µg DNA (500 µg/mL, Promega), 100 U PstI (10 U/µL, Promega), 90 µL 10 x restriction enzyme Buffer H (Promega), MQ water to 900 µL; incubated overnight at 37ºC, then added 100 µL 10 x gel loading dye; stored at -20ºC.

2.2.1.245 Lysis Buffer

Tris (pH 8.) 50 mM NaCl 100 mM Triton X-100 0.5% The required volume is prepared and then stored at 4 ºC.

2.2.1.26 Milli Q water

Sterile MQ water (Millipore) or deionised water was used to prepare reagents.

2.2.1.27 Molecular grade water

Distilled water, DNAse-free and RNAse-free (Invitrogen) for PCR reactions and genomic DNA.

2.2.1.28 NaCl

5M NaCl

2.2.1.29 NAD(P)H

NAD(P)H (20 mM) dissolved in NaOH (0.01M). Stored at -20 ºC.

2.2.1.30 Nitrite Reagent (Sigma)

10 mg/mL of Nitrite reagent dissolved in Milli-Q water. Nitrite Reagent from Sigma Cat. #37410

2.2.1.30 Pentolite (Orica Mining Services)

1% solution in acetone. Stored at 4ºC.

2.2.1.31 PETN (Orica Mining Services)

1% solution in acetone. Stored at 4ºC.

2.2.1.32 Phenol/Chloroform/Isoamyl Alcohol (25:24:1)

Phenol and Chloroform/Isoamy Alochol was mixed in a ration of 1:1 in a sterile bottle or Falcon tube (wrapped in aluminum foil to keep out the light and stored at 4ºC. IPTG: 2g IPTG (DCL) in 10 mL water, filter sterilized, dispensed into 1 ml aliquots and stored at -20ºC , used at concentration of 100 mM.

2.2.1.33 Potassium phosphate buffer (KPi)

KPi (pH 7.2) 50 mM

100 mL of 1M KPi (pH 7.2) prepared by mixing 71.1 mL of 1 M K2HPO4

with 28.3 mL of 1 M KH2PO4; diluted 1 in 20 with water.

2.2.1.34 Propan-2-ol (Isopropanol)

100% (v/v) Isopropanol (BDH)

2.2.1.35 Sodium acetate (NaOAc)

3M NaOAc (BDH), pH 4.6 with HCl, autoclaved

2.2.1.36 Sodium dodecyl sulphate (SDS)

10% (w/v) SDS

2.2.1.37 Stop mix

Prepared fresh on the day by mixing equal volumes of 20 mM phenazine

methosulfate (PMS) and 50 mM K3Fe(CN6).

2.2.1.38 TAE buffer

40 mM Tris-base, 20 mM glacial acetic acid, 2 mM EDTA

2.2.1.39 TE buffer, pH 8.0

10 mM Tris-base, 1 mM EDTA, pH to 8.0 with HCl; for alkaline lysis method, add 20 µg/mL RNAse A fresh on day; stored at 4ºC.

2.2.1.40 TNT

1 % solution in acetone. Tubes were wrapped in foil, kept away from light and stored at 4ºC.

2.2.1.41 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal)

40 mg/mL X-gal (Progen) in dimethylformamide (BDH); stored at -20ºC protected from light.

2.2.2 Enzyme stocks 2.2.2.1 DNase

10 mg/mL (BM) stock solution prepared in molecular grade water and stored at -20ºC.

2.2.2.2 T4 DNA ligase

3 U/µL T4 DNA ligase (Promega); stored at -20ºC

2.2.2.3 Proteinase K

20 mg/mL Proteinase K (Sigma) stored at -20ºC

2.2.2.4 Ribonuclease A (RNAse)

10 mg/mL RNAse (Sigma); stored at -20ºC.

2.2.2.5 10 x Multi-Core buffer

10 x Multi-Core Buffer (Promega); stored at -20ºC.

2.2.2.6 10 x Ligation buffer

10 x Ligation Buffer (Invitrogen); stored at -20ºC.

2.2.2.7 ATP

10mM ATP (Sigma); stored at -20ºC.

2.2.2.8 DNase (RNase-free)

RQ1 RNase –free DNase with 10xbuffer(Promega)

2.2.2.9 Pfu DNA polymerase:

3 U/µL (Promega)

2.2.2.10 Taq DNA polymerase:

5 U/µL Ampli Taq(Perkin Elmer)

2.2.2.11 Restriction enzymes:

All restriction enzymes used were purchased from Promega (Sydney, Australia) and were used as per manufacturer’s instructions.

2.2.3 Microbiological Methods

2.2.3.1Bacterial Growth Conditions

Plate counts of sample volumes (0.1 mL) from microcosms were performed and plates incubated at 30ºC for 24-36 h under aerobic conditions. Colony forming units/gram of soil was determined for each of the soil samples. Single colonies of variable morphology were streaked inoculated from the original PCA plates and streaked onto fresh PCA plates. These were incubated for 24 hours at 30ºC. The plates were sub-cultured until pure colonies were obtained. When pure colonies were obtained, colonies were inoculated into LB broth, incubated at 30ºC overnight shaking on the orbital shaker at 180 rpm until the required OD was obtained. E. coli strains were grown in LB broth or LB Agar at 37ºC overnight, shaking at 180 rpm on an orbital shaker. Appropriate antibiotics were added to the agar plates as per the requirements of various concentrations such as ampicillin 100 µg/mL.

2.2.3.2 Storage of bacterial strains

All isolated strains were stored at -80ºC in 80% glycerol. All the strains were inoculated in LB broth and once the required OD was obtained, the strains were prepared for long term storage. The bacterial culture was mixed with an equal volume of 80% glycerol and dispensed into a sterile cryogenic vial for long-term storage. For short-term storage of these bacterial strains, strains were stored at 4ºC on appropriate media, and sub- cultured whenever required. E. coli DH5 alpha strains were stored at -80ºC in glycerol freezing medium.

Bacterial stocks were reconstituted by scraping the surface of the frozen culture with a sterilized loop and streaking onto a PCA plate followed by 24-hour incubation at 30ºC.

2.2.3.3 Estimation of Bacterial Cell Concentration

The cell culture densities of all the strains and E. coli were estimated by measuring the absorbance or optical density (OD) at 600 nm. Uninoculated LB broth was used as a blank for absorbance readings.

2.2.3.4 Bacterial profiles

Biochemical testing of the isolated strains was carried out to identify the strains. API test kits were used to perform the biochemical tests. Additional oxidase, catalase, urease and indole biochemical tests were also performed using standard methods. PETN and TNT degrading isolates were Gram- stained by heat-fixing on to a glass slide, stained for 30 seconds with Crystal Violet, 30 seconds with iodine decolorized with acetone, and stained for 10 seconds with Carbol Fuschia. The slides were rinsed briefly under running tap water between chemicals. Isolates were viewed under oil immersion and all the details like Gram-reaction, colony morphology and arrangement were recorded.

2.3 DNA Molecular Techniques 2.3.1 Genomic DNA extraction

Genomic DNA extraction was performed using the CTAB extraction method (Ausubel et al., 1994). Cultures were grown overnight in 5 to 10 mL of LB broth at 30ºC. Bacterial cells were obtained by centrifugation at 4000 x g for 10 min. Cell pellets were resuspended in 565 µL of TE (pH 8.0) in a sterile microcentrifuge tube. Lysis of cells was achieved by the addition of SDS and proteinase K to a final concentration of 0.5% and 100 µg/mL, respectively. Tubes were mixed by inversion and incubated at 37ºC for 1h. 100 µL of 5M NaCl and 80 µL of CTAB was added and after thorough mixing, the tubes were incubated at 65ºC for 15 mins.

An approximately equal volume of chloroform/isoamyl alcohol was added to the mixture and the tube was centrifuged. The aqueous phase was recovered and extracted with an equal volume of phenol/chloroform/isoamyl alcohol (PCI). Phase separation in both cases was achieved by centrifugation at 10,000 x g for 10 mins. DNA was precipitated by the addition of 0.6 volumes of isopropanol. Precipated DNA was collected by centrifugation at 10,000 x g for 10 min. DNA pellets were washed with 70% ethanol and then

resuspended in 30-50 µL of TE or sterile dH2O. The isolated DNA was stored at -20ºC.

2.3.2 Quantification of DNA concentration

Concentration of the extracted DNA was determined using a spectrophotometer. DNA concentrations of chromosomal DNA were estimated by measuring the optical density at wavelengths 260nm and 280 nm. The DNA concentration was calculated by using the ratio of 1 OD to 50 µg/mL of dsDNA and taking the dilution factor in account. The purity of the sample was determined by the ratio of ODs obtained at 260 nm and 280 nm (>1.8 for clean DNA) respectively on an Eppendorf Biophotometer. Alternatively, DNA concentration was estimated by running the sample on an agarose gel and comparing band intensity with the intensity of DNA markers of known concentration.

2.3.3 Polymerase Chain Reaction (PCR)

PCR was performed on the PCR-express Thermal cycler in sterile PCR tubes. Bacterial cells and the isolated DNA samples were used to perform PCR reaction. Colony PCR was performed by using isolated colonies that were picked up using sterile toothpicks. The colony was resuspended in 50 µL sterile molecular grade water. The resuspended mix was used as the template for the reaction. PCR Master Kit supplied by Roche and Taq polymerase (Qiagen) was used to perform PCR. PCR was performed using the general protocol described in the instruction manual. The details of the PCR cycling conditions are described below.

2.3.3.1 Primer Design

Primers were designed using the Clone Manager suite of analysis tools (Sci Ed Central website) using normal criteria for primer design. When RE sites

were incorporated, an additional 4 bp were added to the 5’- end of the primer sequence to allow binding of the RE.

2.3.3.2 General PCR Amplification

PCR was carried out according to protocol below, unless specified otherwise PCR using Roche Master mix Roche Master mix 25 µL Forward primer 0.25 µM Reverse primer 0.25 µM DNA template 100 ng Sterile Mol. Grade water to a volume of 50 µL PCR using Taq polymerase 10 x PCR buffer 1 X

25 mM MgCl2 2.5 mM dNTP mix 200 µM Forward primer 0.25 µM Reverse primer 0.25 µM DNA template 100 ng Taq polymerase 0.25 U Sterile Mol. Grade water to a volume of 25 µL

The reaction mixture was subjected to the following cycling conditions unless stated otherwise: 1 cycle of 95ºC for 4 min followed by 35 cycles of 94ºC for 30 s, annealing temperature for 30 s, and 72ºC for 1 min, with a final extension time between 5-10 min. All PCR products were stored either for 4ºC (short term) or -20ºC (long term).

2.3.3.3 Gradient PCR

The reaction mixture was subjected to the following cycling conditions: 1 cycle of 95ºC for 4 min followed by 35 cycles of 94ºC for 30 sec, annealing

temperature was set at a gradient such that at each step of the cycle the annealing temperature would be increased. The remaining conditions remain the same. Gradient PCR were performed to determine the most optimum annealing temperature for the primers used as well as when degenerate primers were used in the experiments.

2.3.3.4 Touchdown PCR

Touchdown PCR’s are a modification on the conventional PCR that were performed to reduce non-specific amplification. It involves the use of an annealing temperature higher than the target optimum in PCR cycles. The annealing temperature is decreased by 1ºC every cycle until a specific temperature is reached. The touchdown temperature is used for the remaining cycles. Using a touchdown PCR allows the enrichment of correct products over any non-specific products.

The reaction mixture was subjected to the following conditions: 1 cycle of 94ºC for 10 min followed by the first stage of touchdown which was 15 cycles of 94ºC for 40 s, 60ºC for 30 s (with a temperature decrease of 1ºC), 72ºC for 40 s. This was followed by the second stage of touchdown, which was 20 cycles of 94ºC for 40 s, 55ºC for 30 s and 72ºC for 40 s. The final extension at 72ºC for 7 min after which the PCR products were stored at 4ºC (short term) or -20ºC (long term) storage.

2.3.3.5 DNA Sequencing Reactions

DNA sequencing was carried out according to the protocol below: Sequencing mix 2 µL 5 x Dilution buffer 3 µL Forward primer (3 µM) 1 µL Reverse primer (3 µM) 1 µL Purified DNA template 100-1000ng Sterile Mol. Grade water to a total volume of 20 µL

The reaction mixture was subjected to the following cycling conditions: 1 cycle of 96ºC for 1 min followed by 25 cycles of 96ºC for 10 s, 50ºC for 5 s and 60ºC for 4 min. 2.3.3.5.1 Sodium-Acetate Ethanol Clean –up

The clean-up of the extension products were performed as below:

3M Sodium Acetate pH 5.2 3 μl Ethanol (78% (v/v) solution) 77 μl Sequencing reaction 20 μl

Twenty microlitres of the sequencing reaction was added to the acetate/ethanol solution in the 1.5ml tube. The sample was vortexed and allowed to incubate at room temperature for 15 minutes. The sample was centrifuged at 16,100 x g for 5 minutes. The solution was aspirated using a pipette. The supernatant was completely removed and the pellet washed with 200 μl of 70% ethanol. The tube was subjected to centrifugation at 16,100 x g for 5 minutes. The washing step was repeated again after aspirating the solution. The tube was placed on a heating block with the temperature set at 80ºC for 3 minutes. Once cooled the samples were sent for analysis.

2.3.4 Agarose Gel Electrophoresis

Agarose gel electrophoresis was used to separate DNA fragments by length and to estimate the size of DNA.

2.3.4.1 Agarose Gel Preparation

Agarose gels for electrophoresis of DNA samples were prepared by boiling required amount of agarose in 1X TAE (50 mL for mini gels and 100 mL for midi gels) until completely dissolved. After cooling to 50ºC, the mixture was poured into a casting tray and allowed to set. Well’s were created by inserting combs into the mixture before allowing the agarose to set.

2.3.4.2 Sample preparation

The sample prior to running on the gel was mixed with 1/10 volume of 10X loading dye.

2.3.4.3 Electrophoresis

Samples were mixed with loading dye and then loaded into well of the gel. Power supply units or power packs were used to perform electrophoresis. These were set at the required voltage for the appropriate amount of time. Generally for mini gels, the voltage was set at 75V for 1h and 100V for 1.5h, respectively.

2.3.4.4 Staining of Agarose Gels

All DNA gels were stained after electrophoresis in 1 mg/mL ethidium bromide solution for 5 -10 min followed by destaining in running tap water for 30 min.

2.3.4.5 Visualisation of DNA

DNA gels were visualized after staining and destaining with UV transilluminator and photographed using the Gel-Doc System from Biorad.

2.3.4.6 Purification of DNA from PCR amplification

Amplified PCR products were purified using Qiagen QIAquick® PCR purification kit. 5 volumes of buffer PB1 was added to one volume of PCR product. The DNA was bound by applying the sample along with buffer PB1 to QIAquick® spin column, which was centrifuged at maximum speed for 60 s. The flow through was discarded and 750 µL of buffer PE was dispensed to wash the DNA and the column which was centrifuged for another 60 s. The flow through was discarded and the column was centrifuged for an additional minute. The column was placed in a sterile microcentrifuge tube into which the DNA was eluted by dispending 15 µL of Elution buffer directly to the centre of the column. The set up was allowed to stand for 5 min and then was

centrifuged at maximum speed for 60 s. The eluted DNA was stored at -20ºC. This purified DNA could then be used as the template for DNA sequencing.

2.3.4.7 Purification of DNA fragments from Agarose Gel (Gel extraction)

The DNA bands of interest obtained as a result of agarose gel electrophoresis were excised from the agarose gel using a sterile scalpel blade. The excised bands with the agarose was put into a microcentrifuge tube, weighed, and purified using the QIAquick® Gel Extraction Kit (QIAgen) as per the manufacturer’s instructions. Three volumes of QIAgen buffer were added to 1 volume of the excised DNA bands. The agarose was melted by placing the tubes in the waterbath at 50ºC for 10 minutes. Every 2 min the tubes were inverted to allow the particles to remain in suspension. The sample was then centrifuged and the pellet was washed with the washing buffer supplied with the kit. At the completion of the gel extraction, the purified samples were eluted in 30 µL Elution Buffer, and stored at -20ºC. It is understood that there is a ~30% loss in DNA quantity (ng) during the gel extraction procedure. It was tested by loading the samples on a 1.5% agarose gel and confirmed by calculating the quantity of the extracted DNA fragments.

2.3.4.8 Purification of PCR products for sequencing

Dye-labelled DNA was precipitated by the addition of 0.1 volumes of sodium acetate (pH 4.6) and 2.5 volumes of 100% ethanol. Tubes were vortexed, allowed to stand at room temperature for 10 min and centrifuged at 16,100 x g for 20 min. The supernatant was discarded and the pellets rinsed twice with 250 µL of 70% ethanol and air dried before being sent for sequencing analysis to the Micromon facility at Monash University, Melbourne, Australia. ABI Prism 377 DNA sequencer was used to perform the reaction, which was performed by staff at Monash University

2.3.4.9 Electroporation

2.3.4.9.1 Preparation of electro competent cells

A single colony of E. coli DH5α was inoculated into 5 mL of LB broth, which was grown overnight at 37ºC on a shaker. 2 mL of the overnight culture was transferred to 200 mL of pre-warmed LB broth and the cells were º grown at 37 C with vigorous shaking to an OD600 of 0.6 (~2-3 hrs). The cells were chilled on ice for 30 min to slow down the cell growth. The cells were harvested by centrifugation at 4700 rpm for 15 minutes at 4ºC and the cell pellet was resuspended in 200 mL chilled sterile Milli-Q water. The supernatanent was completely drained off after centrifugation and then resuspended in 100 mL of 10 % glycerol in chilled sterile water and centrifuged as above. The cells were pelleted and resuspended again in a final volume of 2 ml of ice cold 10% glycerol. Centrifugation was repeated and resuspended in 250 µL of 10% glycerol and aliquoted out in 50 μL volumes and stored immediately at -80ºC freezer.

2.4. Protein assays

Protein concentrations was determined by mixing 100 μL of sample with 200 mL of 20% v/v Bradford reagent (Bio-Rad) and were loaded into flat bottomed 96 well microtitre plates incubated at room temperature for 5 min, and read at 595 nm. Blanks were 0.15 M NaCl, which was also used to dilute samples to give valid absorbance readings within the range of 0.05-0.6. Bovine Serum Albumin was used as standard to establish the relationship between OD595 and protein concentration. Standard curves were generated for each batch of protein assay reagent used.

Further methods pertaining to specific analyses will the described in the corresponding chapters.

CHAPTER 3

Isolation, Identification and characterisation of bacteria capable of degrading TNT and PETN

3.1 Introduction

Bacteria with specific degradative abilities may be isolated using selective enrichments with inocula from contaminated soil and water. Soil enrichments are usually nutrient limited, and bacteria found in such contaminated soil develop the ability to scavenge nutrients from the soil. Contaminants usually remain in the soil for a number of years and bacteria often mutate rapidly in such a way that they are able to utilise these contaminants as energy sources. This is the rationale behind isolating bacteria from contaminated soil and water sources. Selective enrichment is a process in which bacteria are inoculated into a medium with a defined composition; usually the inocula are the contaminated soil or water. Media with defined compositions encourage the growth of specific types of bacteria such as those capable of breaking down the contaminant (which is also incorporated into the defined media). This will selectively encourage the growth of bacteria with desired degradative ability.

A number of studies show that bacterial isolation using selective enrichment is a feasible method. Arthrobacter globiformis CECT 4500 was isolated from contaminated soil around a munitions factory and was capable of tolerating high concentrations of nitrate (Pinar et al., 1998). Arthrobacter aurescens TC1 was isolated from a site contaminated with atrazine and was capable of metabolizing s-triazine ring compounds. (Strong et al. 2002). Many

chlorobenzene degraders have been isolated from contaminated groundwater and identified as gram-positive strains or Pseudomonas strains (Nishano et al. 1992). Other chlorobenzene degraders have been isolated from a chorobenzene contaminated well, while uncontaminated wells did not yield bacteria with such abilities. To isolate bacteria able to degrade TNT or PETN, soil and water contaminated with the TNT/PETN or other nitroaromatics is the most logical source of microorganisms

Studies have shown that genes encoding proteins capable of degrading xenobiotic compounds are often located in plasmids or transposons. Often more than one protein is necessary for the complete degradation of xenobiotic compounds especially when a degradation pathway is followed; these genes are often found in clusters. Some examples of gene clusters include the nah cluster for naphthalene degradation (Grund et al. 1983; Yen et al. 1982), and the xyl cluster for xylene/toluene degradation. Often, genes for the degradation of other heavy compounds are also found on the same plasmid including genes for degradation of alkane, isopropylbenzene and polyaromatic hydrocarbons (PAH) in strains of Rhodococcus and Pseudomonas (Dabrock et al., 1994; Foght et al., 1996; van Beilen et al. 2001).

TNT and PETN are environmental pollutants for which biodegradation may be a good method of remediation. The majority of the strains isolated from contaminated soil have been shown to degrade TNT, however few strains capable of degrading PETN have been isolated; hence it is desirable for this study to isolate and identify further strains of TNT and /or PETN degrading bacteria. This will allow us to better understand the range of degrading bacteria and give us an insight into the mechanisms of degradation.

3.1.1 Co-metabolism

Co-metabolism may be described as the process by which simultaneous degradation of two compounds occur. It follows the principle in which the

degradation of the secondary compound depends on the presence of the primary compound. Jensen et al. (1963) described the term “co-metabolism” to explain the process of dehalogenation reactions frequently carried about by microbial consortia. Co-metabolism has also been described as the oxidation of a non-growth substrate alongside the growth of microorganisms on carbon and other energy sources. It was suggested that soil organic matter became a better source of nutrients for the microbes present in the soil when decomposable matter was added to the soil, this additional matter aided in the breakdown of pollutants via co-metabolism (Horvath, 1972). Some microbes that exhibit co- metabolsim are described in Table 1.

Table 1 Microbial strains exhibiting the phenomenon of co-metabolism

Microorganism Reference

Achromobacter sp. Horvath, 1970; Horvath et al., 1970 Horvath, 1972 Arthrobacter sp. Horvath et al., 1970 Bacillus sp. Matsumura et al., 1967 Pseudomonas sp. Chambers et al., 1964; Gibson et al., 1967 Serratia marcescens Montpas et al., 1997 Vibrio sp. Ali et al., 1962, Callely et al., 1965

3.1.2 Co-substrates

Studies performed recently have indicated that co-metabolism can lead to the complete mineralization of xenobiotics such as chlorinated aromatic compounds, polycyclic aromatic compounds and polychlorinated biphenyls by mixed bacterial cultures, if a carbon and energy source were supplied to the microorganisms in the form of a biodegradable compound. Table 2 describes the products formed when certain compounds under-go co-metabolism in the presence of co-substrates.

There are a number of inexpensive carbon and energy sources which have been tested for the treatment of heavy compounds. These include succinate, citrate, malic acid, acetate, glucose, sucrose, and molasses as well surfactants such as Tween-80 and Triton X-100.

Table 2 Compounds subjected to co-metabolism and products formed Reproduced from Horvath, 1972 and Montpas et al., 1997

Primary Co-substrate Product substrate

Ethane NT Acetic acid Propane NT Propionic acid, acetone Butane NT Butanoic acid Ethylbenzene NT Phenlyacetic acid 2,4,6-Trinitrotoluene Tween-80 2-amino-4,6-dinitrotoluene 4-amino-2,6-dinitrotoluene 2,4,6-Trinitrotoluene Molasses 2-amino-4,6-dinitrotoluene 4-amino-2,6-dinitrotoluene Methyl tert-butyl ether Pentane Tert butyl alcohol

NT: Not tested

3.1.2.1 Surfactants

Surfactants consist of a hydrophilic headgroup and one or more hydrophobic tails. It is the hydrophobic tail, usually a hydrocarbon or fluorocarbon chain, that reduces the solubility of the surfactant in water. The polar head on the other hand has the opposite effect. These properties of surfactants which can either reduce or increase the solubility contributes to their usefulness in a numerous applications including co-metabolism.

Studies have shown that surfactants can enhance the solubility and dissolution of heavy compounds from contaminated soils (Li et al. 2009). Table 3 demonstrates the effects some surfactants have on the biodegradation of aromatic compounds.

3.1.2.1.1 Trition X-100

Triton X-100 is a water-soluble, nonionic surfactant, which has a hydrophilic polyethylene oxide group and a hydrophobic group. It is a commonly used detergent in laboratories during DNA extraction as part of the lysis buffer. It is also used as a co-substrate for the degradation of heavy compounds as shown in Table 3.

3.1.2.1.2 Tween-80

Tween-80 is a nonionic, viscous, water-soluble, yellow, liquid surfactant also known as polysorbate 80. It is a surfactant that can be used to promote the release of enzymes from microorganisms as studies by Montpas et al., (1997) have shown. Surfactants such as Tween-80 were observed to disperse aromatic pollutants such as TNT in aqueous solution. Since TNT and other aromatic pollutants are apolar having low solubility in aqueous media, adding a surfactant enhances the dispersion of TNT and reduces sorption to soil. Tween- 80 is also biodegradable and can act as a carbon source. Co-metabolism experiments have shown that using Tween-80 along with TNT have led to

higher rates of transformation of TNT than in the absence of Tween-80. Sabatini et al., (1995) demonstrated that the bioavailability enhancement of apolar pollutants by added surfactants or by surfactants produced by microorganisms (biosurfactants) greatly improve the rate of biodegradation.

The isolation and comparison of bacterial strains that can utilize TNT and/or PETN as a sole nitrogen source are described in this chapter. Studying the mechanism of TNT and PETN degradation by these isolates can provide further information as to whether the explosives are being mineralized, degraded or broken down into other compounds that maybe recalcitrant.

Table 3. Effects of surfactants on biodegradation of aromatic compounds

Compound Microorganism Surfactant Effects Explanation Reference

TNT Serratia Tween 80 + Promotes Montpas et marcescens release of enzymes al., 1997 from microbes. TNT dispersion in aqueous solution Naphthalene Pseudomonas Triton X-100 + Surfactant Mulder et strain 8909N enhances dissolution al., 1998

Pyrene Pencillium sp. Tween 80 + Surfactant Pinto et enhanced desorption al., 2000

Phenanthrene Cultures isolated Triton X-100 + Enhanced Kim et al., from wastewater solubilisation of PAH’s 2001

3.2 Materials and Methods

Microcosm studies were performed with 4 soil and 2 groundwater samples

contaminated with TNT and/or PETN.

3.2.1 Microcosms

All samples were collected from Australian locations that have been exposed to explosives. Contaminated soil samples, designated SS1 and SS2, and a contaminated water sample, W1, were assessed for TNT, PETN and Pentolite degraders. The second set of contaminated soil designated SI and SII and contaminated groundwater samples designated 201-S and 204-D was set up to isolate only PETN degraders. Soil samples were weighed out (40 g) and mixed with an equal quantity (by mass) of contaminated water (approximately 40 mL) and mixed thoroughly to from a representative sample for microbial analysis. The soil microcosms were set up in 250 ml Schott bottles equipped with hole- caps and silica septa to enable oxygen augmentation. Minimal M9 Nitrogen- free media was dispensed into the bottles along with the explosive of interest as the sole nitrogen source. The redox indicator resazurin (1 mL) was also dispensed in the Schott bottle.

Resazurin is a non-toxic, water soluble, redox sensitive dye that changes from its blue/non-fluorescent state to pink/highly fluorescent state upon reduction at a redox potential around 70 mV. Further reduction to the colourless resofurin then can occur at a redox potential around -51 mV (Guerin et al. 2001). Resazurin is added to viable cells and can become reduced via an unknown mechanism presumed to be either enzymatic or chemical reactions in the cell. It is often used for measuring cell proliferation, viability and cytotoxicity since it’s a simple and quantitative method.

Oxygen augmentation was performed daily by removing 25 cc of air from the headspace of the bottles and then 25 cc of pure oxygen was dispensed using a needle and syringe. This was performed for all the aerobic samples. The anaerobic samples were left undisturbed in a cupboard at room temperature (~22ºC). Samples were taken from all bottles on Day 1 including controls to

determine the microbial load of soil samples. Before sampling, each bottle was inverted by hand to encourage dislodgement of microorganisms from the soil.

Slurry dilutions were made in 0.1 % peptone water (neat to 10-6 for samples SS1 and SS2; neat to 10-3 for all other samples) and inoculated onto PCA in duplicate. The plates were incubated at 30ºC for approximately 48 h, and colony forming units (cfu)/g soil determined. Microcosms were set up to aid in the isolation of possible TNT and PETN degraders. Soil and water parameters were

3.2.2 Cell Passages

Cell passages (sub-culturing) were performed at regular intervals of 3 days to avoid senescence associated with high cell density and nutrient depletion as well as a facilitating the gradual removal of any external nitrogen sources (from contaminated soil or water) via dilution. Four passages were performed to ensure complete removal/dilution of exogenous nitrogen traces. Slurry dilutions were made in 0.1% peptone water (neat to 10-4) and inoculated on PCA in duplicates. Plates were incubated for approximately 36 h, and cfu/ml was determined.

3.2.3 Isolation of TNT and PETN degraders

Single colonies of variable morphology were streak- inoculated from PCA plates onto fresh PCA plates and incubated at 30ºCfor approximately 36 h. Thirty-six hours was used as some of the isolates were slow-growers that required more than the standard 24 h for growth. Colonies were sub-cultured until pure colonies were obtained. Six strains were isolated and designated TNT-1, TNT-2, Pa-3, Pa-4, Pb-5 and Pb-6 (isolated from Site A and B). Thirty strains were isolated from Site C and D and designated ST1 to ST30.

3.2.3.1 TNT and PETN overlays

TNT and PETN overlay plates were prepared so as to provide an additional

selective media. The overlay plates were prepared after PCA was poured, cooled and allowed to set. 100 μL of TNT or PETN (0.5 mM) was spread on a PCA plate and allowed to dry. TNT overlay plates were wrapped in foil to prevent disintegration because of light. All the isolated strains were streaked onto both TNT and PETN overlays. Due to the toxic nature of TNT, only the strains capable of utilizing TNT as a nitrogen source would be able to grow on the overlay. However, PETN overlays could not be used to assess growth since PETN crystallized out of solution.

3.2.4 Utilization of TNT/PETN as a nitrogen source

The strains isolated from the microcosms were then subjected to further cell passages to determine whether TNT/PETN is removed from the media over time.

3.2.4.1 TNT Preliminary passages

Cell passages were performed using Minimal M9 Nitrogen free media in which TNT (100 ppm) was the sole nitrogen source. A pure colony of either TNT-1 or TNT-2 was inoculated into the media. The experiment was performed in duplicate to ensure the reproducibility of the results. The inoculated media was incubated in a shaker-incubator at 30ºCand shaken at 130 rpm. One percent of the original inoculum was used for the consecutive passages. The passages were carried out between 2 and 4 weeks or until the required OD600 of between 1 and 2 was obtained

TNT (100ppm) was dispensed into the media. After passage 4 was performed and the reaction had proceeded for the appropriate number of days (15-21 days), an equal volume of solvent, acetonitrile was added to stop the reaction/degradation of TNT/PETN and solubilise the analytes. The solvent when added to the solution containing explosives solubilizes the explosives and stops the reaction from proceeding. Subcultures of the passages were taken before the solvent was added and purity streak plates were performed to ensure purity of the passage. The reactions were analyzed by HPLC by external

contractor Analytical Consulting Services Australia (Kensington, VIC).

3.2.4.2 PETN Preliminary passages

3.2.4.2.1 Passages of strains from Site A and B

PETN passages were performed as described as for the TNT passages with a PETN concentration of 50 ppm. PETN being highly insoluble often required that the cultures be incubated for longer periods of time so that the desired optical density could be obtained. Due to the insoluble nature of PETN, often the OD readings were not due to the growth of the isolates but just the turbidity arising from insolubility. Therefore plate counts were performed during the passages to determine if the isolates were growing in the presence of PETN. PETN passages were performed for an average of 4 weeks; with the passage being performed once every week for 4 weeks. Subcultures of the passages were taken before adding solvent. Purity streak plates were also performed. The addition of equal volumes of acetonitrile to the tubes before being sent for analysis to ACS stopped the reactions as well as solubilized PETN; which would otherwise result in incorrect readings due to the insoluble nature of PETN.

3.2.4.2.2 Passages from strains from Site C and D

Passages were performed with the strains isolated from the microcosm studies. All the strains were provided only with PETN as a nitrogen source, so as to isolate more PETN degraders. Thirty isolates were identified after sub-culturing and spreading on PCA plates. Care was taken to ensure that all other sources of nitrogen were removed by repetitive passaging. Pure colonies were inoculated into Minimal M9 Nitrogen free media with 50 ppm of PETN as a nitrogen source. The reaction was allowed to proceed for 6 weeks; the first 2 weeks were to ensure removal of any nitrogen carried over from the growth on PCA plates, and then a further 4 weeks until it was stopped by the addition of solvent.

3.3 Identification of bacterial isolates

Soil and water samples from the contaminated sites were evaluated for microorganisms able to degrade TNT and PETN. Once the strains able to grow on minimal media with either TNT or PETN were isolated; these strains were further identified and characterized as described below:

3.3.1 Phenotypic characteristics

A preliminary characterization of the isolates was performed, based on the colony characteristics after growth on PCA. The colonies were grown on PCA plates to allow easy counting of the colonies. The strains were also grown on 2YT plates, so as to promote better growth for genomic DNA extraction. Phenotypic characterization was performed to enable preliminary grouping of the bacteria.

3.3.2 Gram reaction determination

A preliminary identification of the isolated bacteria was performed to determine if the strains were gram positive or negative. The Gram reaction method is based on the differences in cell wall composition of gram positive and negative strains. The cell wall of gram-negative strains is lysed by using 3% KOH, whereas gram-positive strains are not. Controls were performed with known gram-negative bacterium (E. coli) and gram-positive bacterium (S. aureus). Of the 30 strains isolated from Site C and D, gram reactions were performed on the strains showing best growth as well as those microorganisms which when identified by 16S ribosomal RNA genes (rDNA) were different from those previously described to degrade TNT or PETN.

3.3.3 Biochemical tests

Gram staining provides valuable information about different bacteria

including the presence of structures such as endospores and capsules. There is a limit to how much information microscopic analysis can provide. Therefore, to identify bacteria a series of biochemical tests were performed. The bacteria can be identified by the biochemical reactions each organism undergoes. Different bacteria due to the presence of unique enzymes are capable of different biochemical reactions. Some common biochemical tests that help distinguish the strains were performed

3.3.3.1 API® 20 NE Test

API® strips (20 NE) were used to identify isolates TNT 1 and 2 as well as the PETN isolates to species level. API® strips were used according to manufacturer’s instructions. API® strips are very useful since they give accurate identifications based on API® database. The kit contains strips that include 21 miniature biochemical tests.

3.4 Co-Metabolic Degradation studies of TNT 3.4.1 Co-metabolic Degradation Experiments

The strains tested for the co-metabolism experiments were TNT 1, TNT 2, Pa-3 and Pb-5. These strains were selected based on their ability to grow on TNT overlay plates as well as from the biochemical tests performed. Since many of the colonies were identified as being the same species, representative colonies were chosen and used for the experiments. The strains were streaked onto PCA plates and allowed to grow at 30ºCuntil single colonies were obtained (approximately 36 h). A single colony from each of the 4 strains was inoculated into Minimal M9 media with either TNT or PETN as the sole nitrogen source. Tween-80 (0.1 %) or 0.1 % of Triton X-100 was added as the co-substrate depending on the experiment being performed. The set up was left in a shaker incubator for 3 days and plate counts performed every 2 days. Passages were prepared and performed every 7 days.

3.5 Results 3.5.1 Details of soil and water samples from the contaminated sites A, B, C and D.

The soil and water samples from all the contaminated sites were observed and the results of the observation are described in Table 4.

Table 4 Details of soil/water samples used for microbial analysis Sample Sampling Sample Sampling Explosive origin date number observations tested

Site A 5.9.07 SS1 Red/brown, soil TNT Black/grey, PETN gravel/soil Pentolite pH 6.0 Site B 5.9.07 SS2 Brown soil TNT Black/grey PETN gravel/soil Pentolite pH 5.5 Site A 5.9.07 W1 Cloudy TNT PETN Pentolite pH 6.3 Site C 13.12.08 SI Red/brown,soil PETN Black/grey gravel/soi pH 5.5 Site D 13.12.08 SII Red/brown,soil PETN Black/grey gravel/soil pH 6. Site C 13.12.08 201-S Brown cloudy PETN water

71 pH 6.5 Site D 13.12.08 204-D Brown/grey PETN cloudy water pH 6.2

3.5.2 Microbial load from Sites A, B, C and D

Slurry dilutions were prepared and plated onto PCA plates. Table 5 describes the microbial load of the contaminated soil and water samples evaluated on Day 1. The Schott bottles containing TNT as the explosive from Site A (SS1) shows the highest load of microbes. Contaminated water from Site D (204-D) containing PETN as the explosive shows the lowest microbial load.

Table 5. Microbial load of contaminated soil and water samples

Sample Sample Explosive Microbial load Origin number tested (cfu)/g

Site A SS1 PETN 3.41x109 TNT TNTC Pentolite 2.42x109 Site B SS2 PETN > 5x109 TNT > 5x109 Pentolite > 5x109 Site C SI PETN 2.8x106 Site D SII PETN 2.3x106 Site C 201-S PETN 9.0x107 Site D 204-D PETN 2.0x105

TNTC: Too numerous to count

3.5.3 Microbial content of soil samples from Site C and D

Slurry dilutions were prepared and plated onto PCA plates. Table 6 describes the microbial content of the contaminated water and soil samples on Day 1 as well as after passages were performed.

Table 6 Microbial content of samples obtained from Site C and Site D

Sample Sample Passage Microbial Origin number number Load (cfu)/ml

Site C SI PI-10% transfer from original microcosm > 5x103 Site D SII I > 2.6x103 Site C 201-S I 1.00x102 Site D 204-D I No growth Site C SI PII- 5% transfer from PI 5x103 Site D SII II 2.8x105 Site C 201-S II 5x103 Site D 204-D II 4.5x104 Site C SI PIII- 1% transfer from PII > 6x103 Site D SII III > 5x103 Site C 201-S III > 5x103 Site D 204-D III 1.6x103 Site C SI PIV- 1% transfer from PIII 1.93x104 Site D SII IV 3.37x104 Site C 201-S IV 5.0x103 Site D 204-D IV 2.12x104

3.5.4 TNT Preliminary passages

HPLC-UV results obtained from the analysis of TNT-degradation passages are described in Table 4. The data shows that the four strains were able to partially degrade TNT from the initial concentration of 100 ppm down to 32- 46 ppm after 4 weeks with one passage carried out every week. By-products of degradation were also detected: 2-amino-DNT and 4-amino-DNT, providing further evidence that biodegradation was occurring.

Table 7 Results of HPLC-UV analysis of TNT passages from Site A and B The test was carried out for 4 weeks with the Falcon tubes being shaken at 150 rpm and temperature set at 30°C. Passage 4 was sent to ACS for HPLC anlaysis.

Sample ID TNT (ppm) 2-amino-DNT 4-amino-DNT (ppm) (ppm)

Sterile control 50 <0.1 <0.1 Negative control < 0.2 < 0.2 <0.2 TNT 1 (1A) 32 0.1 0.2 TNT 1 (1B) 40 0.1 0.5 TNT 2 (2A) 46 0.1 0.2 TNT 2 (2B) 38 0.2 0.4

3.5.5 PETN Preliminary passages

Samples of passages of strains from all the sites were sent to ACS for HPLC analysis and the results are indicated in Tables 8 and 9. The results show that PETN was degraded by the isolates from Site A and B with isolate Pb-5 showing the maximum degradation from 50 ppm of PETN to 23 ppm. The presence of PETN by-products was not tested. The strains isolated from Site

C and D showed better degradation of PETN.

Table 8. Results of HPLC-UV analysis of PETN passages of Samples from Site A and B. The test was carried out for 6 weeks at with the Falcon tubes being shaken at 150 rpm and temperature set at 30°C. Passage 4 was sent to ACS for HPLC anlaysis.

Sample ID PETN (ppm)

Sterile control 50 Negative control <0.2 Pa-3 47 Pa-4 47 Pb-5 23 Pb-6 47

Table 9. Result of HPLC-UV analysis of PETN passages of Samples from Site C and D. The test was carried out for 6 weeks at with the Falcon tubes being shaken at 150 rpm and temperature set at 30°C. Passage 4 was sent to ACS for HPLC anlaysis.

Sample ID PETN (ppm)

1- SC <0.1 Control 25 2- ST 2 11.4 3-ST 3 0.2 4-ST 7 12.8 5-ST 15 12.4 6-ST 27 14.9

3.5.6 Phenotypic characteristics

Colony differentiation was noticed when strains were grown on PCA and particularly 2YT agar, which is a richer medium than PCA, so stronger pigmentation was produced. All strains formed smooth colonies on PCA and 2YT agar. The major variations in the strains were the differences in colony size and the extent to which the colonies were mucoid. A few of the strains were very mucoid; ST3 and ST4, which swarmed the agar to the extent that there were no single colonies to measure diameter. Colony size of the isolates varied between 0.5 – 1.6 mm in diameter after 2 days of growth on PCA at 30°C. The phenotypic results of the growth on PCA are further shown in Table 10.

Table 10 Phenotypic characterization of colonies of bacterial isolates grown on PCA after 48 h at 30°C

Source Bacterial Colour Morphology Mucoid Colony Isolate size (mm)

SS1 TNT 1 Yellow Smooth - 0.8 SS2 TNT 2 Yellow Smooth - 0.8 SS1 Pa-3 White Smooth ++ 1.3 SS1 Pa-4 White Smooth ++ 1.4 SS2 Pb-5 Beige Smooth ++ 1.3 SS2 Pb-6 Beige Smooth ++ 1.3 SI ST 1 Yellow Smooth - 0.5 SI ST 2 Yellow Smooth - 0.5 SI ST 3 Beige Smooth +++ * SI ST 4 Beige Smooth +++ * SII ST 5 Beige Smooth - 0.6 SII ST 6 Beige Smooth - 0.5 SII ST 7 Yellow Smooth - 0.6 SII ST 8 Beige Smooth - 0.5

Source Bacterial Colour Morphology Mucoid Colony Isolate size (mm)

SII ST 9 Yellow Smooth - 0.5 201-S ST 10 Beige Smooth - < 0.5 201-S ST 11 Beige Smooth - < 0.5 201-S ST 12 White Smooth ++ 1 201-S ST 13 White Smooth ++ 1 204-D ST 14 White Smooth ++ 1 204-D ST 15 Yellow Smooth - 0.6 204-D ST 16 Beige Smooth - 0.5 204-D ST 17 White Smooth + 1 SI ST 18 Yellow Smooth - 0.6 SI ST 19 Yellow Smooth + 0.5 SI ST 20 Beige Smooth - 0.7 SI ST 21 Beige Smooth - 0.8 SI ST 22 Yellow Smooth - 0.6 SI ST 23 White Smooth + 1 SI ST 24 Yellow Smooth - 0.5 SII ST 25 Yellow Smooth - 0.5 SII ST 26 White Smooth - < 0.5 SII ST 27 Yellow Smooth - 0.5 SII ST 28 Beige Smooth - 0.6 SII ST 29 Beige Smooth - 1 SII ST 30 Yellow Smooth - 1

*Swarming growth- no single colonies were present to measure the colony size.

3.5.7 Gram reaction

Gram reactions were performed on all isolates from Site A, B C and D of which the results are described in Table 11.

Table 11 Gram reactions of isolated strains

Isolates Gram reaction

TNT-1 Gram-negative TNT-2 Gram-negative Pa-3 Gram-positive Pa-4 Gram-positive Pb-5 Gram-positive Pb-6 Gram-positive ST 1 Gram -negative ST 2 Gram-negative ST 3 Gram-negative ST 7 Gram-positive ST 12 Gram-positive ST 14 Gram-positive ST 15 Gram-positive ST 24 Gram-negative ST 27 Gram-positive ST 30 Gram-positive

3.5.8 Biochemical tests

Six strains were chosen for further biochemical testing, they included: TNT-1, Pa-3, ST1, ST3, ST4 and ST7. A number of biochemical tests were performed and the results are described in Table 12.

Table 12. Biochemical test with Isolates

Tests TNT-1 Pa-3 ST1 ST3 ST4 ST7

CTA sugars * Glucose + + + + + + * Maltose + + + + + + * Sucrose + + - + + - * Lactose + + - + + - ONPG + + + + + - TSI Media No Glucose No Glucose and NT NT change change gas Catalse + + + + + + Urea - + - - - NT Indole - - - - - NT Oxidase - - - - - + Phenylalamine - - NT - - - Citrate - - - + + - Bile aesculin ------Nitrate + V - + + V Methyl Red - - - + - NT

V=Variable NT=Not tested

Both TNT 1 and TNT 2 were identified as Achromobacter xylosoxidans according to certainty thresholds (> 80%). Isolates TNT 1 and TNT 2 were identified as A. xylosoxidans (99.4% certainty) according to the API™ database. Table 13 shows the tests included in API® test kit (20 NE). The six strains were tested using this kit. However, only strains TNT-1 and TNT-2 demonstrated results on analysis within the stipulated time of 24 hours. All other strains failed to give results within 24 hours and hence the results obtained after that could not be considered valid. The biochemical attributes

of isolate TNT 1 and TNT 2 from analysis with API® test kit (20 NE) are listed in indicating that they are Achromobacter xylosoxidans.

Table 13 Biochemical attributes of Achromobacter xylosoxidans from analysis with API® test kit (20 NE)

Tests Active ingredients Enzymes/Reactions Result

NO3 Potassium nitrate Reduction Negative TRP L-tryptophane Indole production Negative GLU D-glucose Fermentation Negative ADH L-arginine Arginine dihydrolase Negative URE Urea Urease Negative ESC Esculin,ferric citrate Hydrolysis by β-glucosidase Negative GEL Gelatin Hydrolysis by protease Negative PNG 4-nitrophenyl-βD-galactopyranoside β-galactosidase Negative GLU D-glucose Assimiliation Positive ARA L-arabinose Assimiliation Negative MNE D-mannose Assimiliation Positive MAN D-mannitol Assimiliation Negative NAG N-acetyl-glucosamine Assimiliation Negative MAL D-maltose Assimiliation Negative GNT Potassium gluconate Assimiliation Positive CAP Capric acid Assimiliation Positive

ADI Adipic acid Assimiliation Positive MLT Malic acid Assimiliation Positive CIT Trisodium citrate Assimiliation Positive PAC Phenylacetic acid Assimiliation Positive OX Oxidase Cytochrome oxidase Positive

3.5.9 Co-metabolism experimental results

It was observed that the Falcon tubes containing TNT and Tween-80 as the co- substrate were turning brown which is an indication of the formation of the hydride-Meisenheimer complex which occurs during TNT degradation. The culture media developed a reddish-brown colour which disappeared after 24 hours of incubation. Samples were taken and send for HPLC analysis (results not shown). Plate counts were performed to observe the rate of growth of the strains and to study whether the growth rate (cfu/ml) increased after the solutions turned brown. It was hypothesized that since TNT degradation was occurring, the number of colony forming units would increase. Tables 14, 15, 16 and 17 show the results of growth curve experiments. The experiments were performed with either Tween-80 or Triton X-100 as the co-substrate. During TNT metabolism, the tubes containing TNT and Tween-80 were turning brown, the colour change due to the formation of azo compounds formed during TNT metabolism. Similar experiments were performed with the PETN strains, however color changes in the media were not observed as with TNT co-

metabolism experiments suggesting that azo compounds are not formed durin PETN metabolism. However, only strain Pa-3 was able to utilize Tween -80 and Triton X-100. As observed with TNT strains Tween-80 was more readily utilized as a co-substrate when compared to Triton X-100.

Table 14. Results of co-metabolism experiment using Tween-80 as co-substrate with TNT

Day Strain Passage 1 Passage 2 Passage 3

0 TNT 1 2x105 9x105 1.8x106 4 TNT1 8x105 1.4x106 2.6x106 0 TNT 2 3x105 8x105 1.2x106 4 TNT2 7x105 1.0x106 2.6x106

Table 15. Results of co-metabolism experiment using Triton X-100 as co- substrate with TNT

Day Strain Passage 1 Passage 2 Passage 3

0 TNT 1 7.4x106 2.4x106 2.1x106 7 TNT1 4.1x106 2.1x106 1.1x106 0 TNT 2 4x105 2.5x105 2.2x105 7 TNT 2 1.8x106 3x105 9x105

Table 16. Results of co-metabolism experiment using Tween-80 as co-substrate with PETN

Day Strain Passage 1 Passage 2 Passage 3

0 Pa-3 1x104 2x104 3x104 4 Pa-3 2x104 6x104 8x104 0 Pb-5 NG NG NG 4 Pb-5 NG NG NG

NG=No growth

Table 17. Results of co-metabolism experiment using Triton X-100 as co- substrate with PETN

Day Strain Passage 1 Passage 2 Passage 3

0 Pa-3 3x104 3x104 5x105 4 Pa-3 5x104 6x104 6x105 0 Pb-5 NG NG NG 4 Pb-5 NG NG NG

NG=No growth

3.6 Discussion

Thirty-six bacterial strains were isolated from explosive contaminated soil and water. Two of these were isolated from TNT microcosms and the others from PETN microcosms. The strains were capable of utilizing either TNT or PETN as a sole nitrogen source. The number of bacteria isolated in this study shows that there are several environmental bacteria that can degrade TNT and PETN and use it as a sole source of nitrogen and that these microorganisms may be isolated by selective enrichment.

Previous studies performed on other xenobiotics show that degrading strains are often species of Pseudomonas and Rhodococcus. Studies have also shown that species closely related to Pseudomonas and Rhodococcus are able to degrade herbicides, phenols, halogenated phenols, chlorinated herbicides, polyaromatic hydrocarbons etc. (Behki et al., 1993; Finnerty et al., 1992; Häggblom et al., 1992; Warhusrt et al., 1994). The initial testing to evaluate the microbial load of the different samples from the various sites showed that Site B had the highest number of bacterial strains for each of the explosives. This may have been due the presence of contaminating nitrogen sources from the soil itself that promoted growth of these microorganisms.

Cell sub-culturing greatly reduced the number of the colony forming units, with the last passage being the most accurate number of colony forming units able to survive on the explosive of choice. As explained previously, cell passages ensured the removal of all other contaminating nitrogen sources. Thereby, promotes the growth of bacteria that were able to degrade the explosive and utilize it as a nutrient source. The high number of colony forming units shows that the strains were capable of utilizing the explosive as a nitrogen source and thus are able to degrade and/or break down the explosive and utilize the nitrogen released from the break-down.

The initial phenotypic analysis showed that most of the strains were beige and had formed smooth colonies, while several of the strains had a mucoid phenotype. Studies have observed changes in colony morphology when the same strain is grown on different media. The same trait was observed here as some of the strains seemed to be more mucoid on 2YT media as compared to PCA media. A reason for this occurrence could be that in the presence of richer media, energy is perhaps diverted towards biosynethetic pathways such as polysaccharide production that makes the colonies more mucoid. This can be a biosynthetic pathway which is followed only in the presence of high energy components such as those present in rich media.

Gram reaction determination showed that most of the isolates were gram negative and a few were gram positive. This coincides with studies previously described that report the majority of degrading strains to be gram negative, such as Enterobacter sp., along with a few gram positive bacteria such as Arthrobacter sp. able to degrade heavy compounds.

TNT and PETN degraders were isolated and identified by subjecting the strains to degradation passages wherein a strain was inoculated in minimal media with only TNT or PETN as the sole nitrogen source. The results show that the TNT degraders were able to degrade TNT via pathways leading to ADNT metabolites. The primary bacterial metabolites of TNT transformation are aminodinitrotoluenes (ADNTs), diaminomono-nitrotoluenes (DANTs), and

84 tetranitroazooxytoluenes (AZTs) via the intermediate hydroxylamino- dinitrotoluenes (HADNTs). This is consistent with the ADNTs formed during the degradation passages, although further analysis of other degradation products was not conducted during this study. This further suggests that strains TNT-1 and TNT-2 are capable of degrading TNT and utilizing the nitrogen from the degradation of TNT as a nitrogen source. The complete reduction of TNT to triaminotoluene (TAT) has been shown to require very strict anaerobic conditions as studied by Ederer et al., (1997) and has not been reported in aerobic soils. Studies were not performed under anaerobic conditions with the TNT strains isolated so complete reduction of TNT to TAT was unlikely to have occurred under the experimental conditions used in this study. This would be the subject of future studies.

The initial degradation experiments were conducted with a controlled amount of TNT and PETN, and degradation was observed in the multiple passages carried out. However, a set of passages performed with higher concentration of TNT (200ppm) and PETN (100 ppm) in the media did result in the lower cell density was described by Martin et al. (1997). The decline in the growth of the strain could be attributed to the accumulation of toxic metabolites in the media.

Biochemical tests were performed with the strains and the results of the tests are shown in Table 9. The API 20 NE test was performed for the strains as well, but was only able to identify only strains TNT 1 and 2 as Achromobacter xylosoxidans. The same tests for the other strains were also performed, but since some of the isolates took more than 36 h for the tests to yield a result (which was beyond the time limit of the kit); the results obtained could not be considered valid.

Co-metabolism experiments showed that using Tween-80 as a co-substrate promoted the growth of the strain in minimal media. As studied by Montpas et al, (1997) using Tween-80 along with TNT aided in the transformation of TNT. This was further observed by the color change of the media suggesting that TNT was being broken down by the strains in the presence of Tween-80.

However, it was observed that co-metabolism experiments using Triton X-100 as the co-substrate did not show any improvement in growth. Control passages in which Tween-80 was not added took 2 weeks longer to reach an OD of 1 when compared to the passages in which Tween-80 were added. Possible explanations could be the toxicity of the media once Triton X-100 was added or membrane disruption of the cell in the presence of the surfactant. In contrast, the results showed that colony forming units/ml increased 10 fold when Tween-80 was used as the co-substrate. A possible explanation was that the process of co-metabolism was occurring and hence promoted the growth of the strain in the minimal media. It is probable that Tween-80 was being used as an additional source of carbon by the isolates thereby increasing the bioavailability of TNT and PETN which improved the growth of the strains

Similar results were observed when PETN strains were subjected to co- metabolism experiments. The PETN strains were able to grow better in the presence of Tween-80 as compared to Triton X-100 which proved detrimental to growth of the strains. Since most of the plate counts were performed between day 0 and day 4, perhaps the strains required longer incubation times when trying to transform PETN in the presence of Triton X-100 or the cell counts had already peaked. Control strains which did not contain Tween-80 or Triton X-100 took longer to reach an OD of 0.5. Further study into the action of the co-substrate Triton X-100 on the degradation of strains containing TNT or PETN need to be performed. The study performed indicated that explosive mineralization progresses at a faster when a co-substrate is provided. The study also demonstrates that Triton X-100 is not a co-substrate that aids in co- metabolism.

The bioremediation of soils is a very useful process mainly because it occurs in situ. Also, the transformation of toxic compounds into biomass that is not a threat to the environment is an additional advantage since the cost of excavating the soil can be avoided. The biotransformation processes ultimately depends on enzyme action. Identification of the enzymes present in the microbes and strains capable of transforming explosives is the first step in the process of bioremediation or biotransformation.

The next chapter describes the processes involved in identifying and characterizing the strains at the molecular level

The results presented in this chapter demonstrate the isolation of TNT and PETN degrading bacteria, as well as demonstrating the prominence of certain gram-negative bacteria known to be able to degrade explosives. Further analysis and comparison of these strains may provide information pertaining to whether the genetic basis of the ability to degrade TNT or PETN is common to all the isolates, and also if it may be transferred between the strains.

CHAPTER 4

Molecular identification and characterisation of isolates on TNT and PETN

4.1 Background

Thirty-six isolates were compared in the last chapter and six representative strains were chosen for further characterisation based on the TNT and PETN degradation passages. . These strains were able to degrade either TNT or PETN as a sole nitrogen source. This study aimed to characterise these strains further again using molecular techniques, as these isolates may be useful in bioremediation.

Bacterial identification is the main activity of many clinical microbiological laboratories. Conventional tests such as the biochemical tests and phenotypic tests for species differentiation are often tedious and time-consuming. Often it also required that specialized testing be performed that is beyond the capabilities of clinical laboratories. Combining molecular biology and bioinformatics to yield processes that are more universal and less time- consuming have become very popular. Molecular identification methods using one or more genes have become important since they yield quick results (Devulder et al, 2003).

There has been an exponential increase in the use of rapid template purification, PCR and automated DNA sequencing over recent years. These rapid processes have dramatically reduced the time required to yield high- quality sequences. 16S rRNA gene sequencing is a well-established method to analyse the relatedness of prokaryotic species. Since all bacteria contain 16S

rRNA, it is a good method to identify bacteria isolated from soil and water. Databases such as GenBank that contain 16S rDNA sequences and information have also increased over the years leading to easier access to information. The sequence differences in the 16S gene have been used to discriminate between species as well as subtyping and identifying hypervirulent bacterial clones (Gee et al., 2003).

Studies performed previously on Enterobacter cloacea PB2 (French et al, 1996) led to the discovery of pentaerythritol tetranitrate reductase (onr gene), as well as the discovery of ner gene from Agrobacterium radiobacter (White et al, 1996). Since most of the isolates belonged to Enterobacter species, the isolates were tested for the presence of these published genes known to aid in the degradation of TNT, PETN and GTN.

4.2 Materials and Methods

Universal primers were selected for PCR amplification of the 1.5 kb 16S rDNA sequence for each isolate. Universal primers 616V (Juretschlo et al., 1998) and 1492R (Loy et al., 2002) were ordered and used to amplify the 16S rDNA from the isolates.

Table 18 Universal primers 616V and 1492R

Primer Sequence Tm(°C) 616V 5’-AGA GTT TGA TYM TGG CTC-3 50

1492R 5’-GGY TAC CTT GTT ACG ACT T-3’ 50

Mixed base codes: M=A/C, Y= C/T

Four methods of preparing template DNA were evaluated including genomic DNA extraction (2.3.1), boiling method, colony PCR’s and the use of commercially available DNA extraction kits obtained from Qiagen. The best method was determined and used in the remainder of the study.

4.2.1 Genomic DNA extraction

Phenol-chloroform extraction is a method widely used in laboratories around the world to extract DNA. It is a liquid-liquid extraction method that is used in partitioning DNA, RNA and proteins. This method involves the separation of phases by the centrifugation of a solution containing an aqueous phase and a saturated solution of phenol and chloroform. This method ensures the removal of all proteinaceous materials from the cell suspension; however the method is unable to remove non-biological substances such as chemicals, which could inhibit PCR

4.2.2 Boiling method

The boiling method is another method used to extract DNA from the cells; it is a crude method of extracting DNA, however a number of studies show that a good yield of DNA can be obtained when the boiling method is performed. The advantages of the boiling methods include the simplicity of the protocol. The boiling method does not require any additional reagents which is an added advantage. A disadvantage of the method would be the purity of the gDNA extracted and the proteinaceous matter may not be completely removed after centrifuging the samples.

Fresh colonies grown on PCA plates were suspended in Milli-Q water and boiled for 10 min to lyse the cells. The solution was then immediately placed on ice for 1 minute to quench the cell solution. The solution was centrifuged at 16,100 x g for 5 minutes at room temperature and the supernatant was used as the template in PCR.

4.2.3 Colony PCR

Colony PCR’s were performed to ascertain the amount of DNA that can be obtained when a PCR is performed without DNA extraction first. Previously performed studies show that colony PCR is best suited when screening for successful recombinant clones as well as screening bacterial colonies. It is often the fastest method when screening bacterial colonies, as it is simple and does not have multiple steps. Colony PCR was performed by preparing a microbial suspension (half of one fresh PCA colony suspended in 50 μL sterile water), an aliquot of this microbial suspension (2 µL) was used directly as the DNA template without any further manipulation.

4.2.4 DNA extraction using QIAamp® DNA mini kit

QIAamp® DNA mini kit was the commercial kit used for the extraction of DNA. The extraction was performedaccording to the manufacturer’s instructions. Isolates from which DNA was to be extracted were spread plated on PCA (50 μL from glycerol stocks of the isolates) plates and incubated and allowed to grow overnight. The fresh colonies from the plates were then removed from the plates with an inoculation loop and stirred into 180 μL of Buffer ATL. Twenty microlitres of Proteinase K was added and mixed by vortexing. The microcentrifuge tube was then centrifuged to remove drops from the lid. To the microcentrifuge tube was added 4 μL of RNase A. The tube was vortexed before adding 200 μL of Buffer AL to the sample. It was once again pulse vortexed and then incubated at 70ºCfor 10 min in a waterbath. After centrifuging for a few seconds, 200 μL of ethanol was added and mixed by vortexing.

The mixture was then applied to the QIAamp® spin column and centrifuged at 10,000 x g for 1 min. The column was placed in a clean collection tube and the filtrate was discarded. A volume of 500 μL of Buffer AW1 was added and the

microcentrifuge tube centrifuged for 1 minute. The spin column was placed in a clean collection tube.

A volume of 500 μL of Buffer AW2 was added and the tube centrifuged at maximum speed for 3 minutes. The spin column was placed in a new collection tube and centrifuged at maximum speed for 1 minute so as to collect the filtrate, which was discarded. The spin column was placed into a sterile microcentrifuge tube and 200 μL of Buffer AE or distilled water was added. The tube was incubated at room temperature for 5 minutes and then centrifuged at 10,000 x g for 1 minute. The final step was repeated and once the genomic DNA was eluted it was stored at -20ºCuntil required.

4.3 Results

4.3.1 Genomic DNA extraction

The phenol-chloroform method of extracting genomic DNA (gDNA) was evaluated. The extracted gDNA was then used as the template in PCR. This was the method used to extract samples from Site A and the resultant gel picture depicting the PCR product is shown in Figure 4.1. The gel picture shows bands of moderate signal intensity for all isolates except strain Pb 6 which gave a weak signal. The size of the product obtained was a 1.6 kbp which was the expected size based on the primers used. The concentration of gDNA was estimated using a Eppendorf Photometer (Biorad) and between 300 and 600 ng of DNA was extracted from the isolates. The purity of the DNA extracted was within the acceptable range (OD260:280 of 1.8-2). Approximately 10-40 ng of PCR product was used for sequence analysis.

4.3.2 Boiling method

Genomic DNA of isolates from Site A was extracted using the boiling method. This gDNA extract was used as the template in a PCR. The resulting products were run on an agarose gel which is shown in Figure 4.2. All the samples showed moderate signal intensity except for strain Pa 3. The size of

the product obtained was a 1.6 kbp which was the expected size. The concentration of DNA extracted by the boiling method was between 100 and 200 ng. The gDNA extracted using boiling method gave a purity reading of 1.4 which was lower than the purity range required for PCR. However a considerable amount of contamination was expected since this is a crude method of extraction.

4.3.3 Colony PCR

A colony PCR of samples from Site A was performed and the PCR products were visualised on an agarose gel as shown in Figure 4.3. All the samples gave very weak signals which may be explained since most of the gDNA did not travel down the gel. As can be seen in the gel, there appears to be considerable DNA present in the wells. The reason for this may be explained by the genomic DNA being heavily contaminated. On testing the purity of the samples, colony PCR’s gave the lowest readings between 0.5 and 1.2 and the concentration of DNA was high

4.3.4 DNA extraction using QIAamp® DNA mini kit

The last method to be evaluated was DNA extraction using QIAamp® DNA mini kit and the resultant agarose gels are shown in Figures 4.4, 4.5a, 4.5b, 4.6, and 4.7. It was demonstrated from the agarose gels that extracting gDNA using a commercial was the best method of extraction since the bands give high intensity signals in all the samples. Therefore, this method was chosen for the remainder of the study whenever gDNA needed to be extracted. The gDNA extracted using the kit had the highest purity of OD 260:280 =1.9 and highest concentration of approximately 400 and 800 ng per µL. After the PCR, some primer dimmer formation was observed on the gels along with some smearing. The primer dimers could have been generated when the annealing temperature that the PCR was performed at was too low. Smearing is a result of too much DNA being loaded into the agarose gels. Figures 4.5a and 4.5b show the results from the strains that are of importance for this study.

PCR cycles were performed as using an annealing temperature between 50°- 60ºCunless specified otherwise. The PCR products obtained were subjected to clean-up and extension before being sent to the Micromon DNA sequencing facility to be analysed. The sequence data were aligned into a single contiguous stretch of DNA (contig) by aligning the forward and reverse sequences with only the good stretch of data with the use of Clone Manager™ in the SECentral™ bioinformatic program. Clone Manager™, a software consisting of a number of tools used for enzyme operations, cloning simulation, graphic map drawing, primer design and analysis, global and local sequence alignments, similarity searches, and laboratory-sized sequence assembly projects (http://www.scied.com/pr_cmpro.htm). The contigs obtained were subjected to BLAST searches. The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program works by comparing nucleotide or protein sequences to existing databases and calculates the statistical significance of matches. BLAST is a program which can also be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families (http://blast.ncbi.nlm.nih.gov/Blast.cgi).The contig sequences were usually between 1.3 Kbp and bacterial identification was based on the highly similar 16S rDNA sequences returned which are shown in Table 18.

A phylogram was generated using Jalview which is a multiple sequence alignment editor. All the sequences were first aligned using the multiple sequence alignment tool, Clustal W and then a phylogram indicating the distance- which is the percentage differences in sequence between isolates was generated. Figure 4.7 shows the phylogram generated. Clustal W is a multiple alignment program for DNA and protein sequences (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Table 19 shows the results of 16S rDNA sequencing. It shows the top 5 matches for TNT and PETN degrading isolates.

M 1 2 3 4 5 6

11501

1700 1600

805

Figure 4.1: Visualised gel picture of PCR using template extracted by phenol- chloroform method Key: Well M: Lambda/Pst1 DNA ladder, 1= Achromobacter xylosoxidans TNT 1, 2= Achromobacter xylosoxidans TNT 2, 3= Arthrobacter sp Pa 3, 4= Microbacterium sp. Pa 4 , 5= Arthrobacter sp. Pb 5, 6= Microbacterium sp. Pb 7

M 1 2 3 4 5 M 6 7 8 9

11501

1700 1600

805

Figure 4.2: Visualised gel picture of PCR using template extracted by boiling method Key: Well M: Lambda/Pst1 DNA ladder, 2= Achromobacter xylosoxidans TNT 1, 4= Achromobacter xylosoxidans TNT 2, 5=Negative Control, 6= Arthrobacter sp. Pa 3, 7= Microbacterium sp. Pa 4, 8= Arthrobacter sp. Pb 5, 9= Microbacterium sp. Pb 6

M 1 2 3 4 5 6 7

11501

1700 1600

805

Figure 4.3: Visualised gel picture of Colony PCR Key: Well M: Lambda/Pst1 DNA ladder, 1=Negative Control, 2= Achromobacter xylosoxidans TNT 1, 3= Achromobacter xylosoxidans TNT 2, 4= Arthrobacter sp. Pa 3, 5= Microbacterium sp. Pa 4, 6= Arthrobacter sp. Pb 5, 7= Microbacterium sp. Pb 6

M 1 2 3 4 5 6 7 8 M 9 10 11 12

11501

1700 1600

805

Figure 4.4: Visualised gel picture of DNA extracted using QIAamp® DNA mini kit Key: Well M: Lambda/Pst1 DNA ladder, 3= Achromobacter xylosoxidans TNT 1, 4= Achromobacter xylosoxidans TNT 2, 5=Negative Control 7= Arthrobacter sp.Pa 3, 8= Microbacterium sp Pa 4, 9= Arthrobacter sp. Pb 5,10= Pb 5, ,12= Microbacterium sp Pb 6.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 M 14 15 16 17 18

11501

1700 1600

805

Figure 4.5a: Visualised gel picture of a 16s rDNA PCR for PETN degrading isolates from microcosm 2 extracted with the QIAamp® DNA mini kit Key: Well M: Lambda/Pst1 DNA ladder, 1=Negative control, 2= Pseudomonas sp. ST1, 3=ST2, 4= Enterobacter sp., ST3, 5= Klebsiella sp., ST4, 6=ST5, 7=ST6, 8= Microbacterium sp., ST7, 8=ST8, 10=ST9, 11=ST10, 12=ST11, 13=ST12, 14=ST13, 15=ST14, 16=ST15, 117=ST16, 18=ST17.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

11501

1700 1600 805

Figure 4.5b: Visualised gel picture of a 16s rDNA PCR for PETN degrading isolates from microcosm 2 extracted with the QIAamp® DNA mini kit Key: Well M: Lambda/Pst1 DNA ladder, 1=ST18, 2=ST19, 3=ST20, 4=ST21, 5=ST22, 6=ST23, 7=ST24, 8=ST25, 9=ST26, 10=ST27, 11=ST28, 12=ST29, 13=ST30, 14=TNT1, 15=TNT2, 16=Pa 3, 17=Pb 5, 18=Negative Control.

M 1 2 3 4 5 6 7

100

Table 19. 16S rDNA BLAST results showing the top 5 matches for the TNT and PETN degrading isolates

Iso. Accession Description Max Total score Query Max Identity

score coverage

TNT1 HQ185400.1 Stenotrophomonas 2764 2764 100% 99% maltophilia strain 5633 16S ribosomal RNA gene, partial sequence AM743169.1 Stenotrophomonas 2764 2764 99% 99% maltophilia K279a complete genome strain K279a NR_041577.1 otrophomonas 2764 2764 99% 99% ophilia strain 5517 ribosomal RNA gene , partial sequence Stenotrophomonas 2764 2764 99% 99% HQ185398.1 maltophilia strain 5517 16S ribosomal RNA gene partial sequence AB294557.1 Stenotrophomonas 2764 2764 99% 99% maltophilia gene for 16S ribosomal RNA, partial sequence, strain: NCB0306-284 Pa-3 FJ267577.1 Arthrobacter sp. 2493 2493 100% 99% I_GA_A_3_4 16S ribosomal RNA gene partial sequence HQ331132.1 Arthrobacter sp. 2419 2419 100% 99% LKSO216S ribosomal RNA gene partial sequence HQ331126 Arthrobacter sp. 2419 2419 100% 99% LKRO3 16S ribosomal RNA gene, partial sequence HQ331125.1 Arthrobacter oxydans 2419 2419 100% 99% strain LKRO2 16S ribosomal RNA gene EF550164.1 Arthrobacter sp. 2419 2419 100% 99% GW1416S ribosomal RNA gene, partial sequence

101

Iso. Accession Description Max Total score Query Max Identity

score coverage

Pb-5 EU699610.1 Actinomycetales 1514 1514 100% 99% bacterium

TLI146 16S ribosomal RNA gene

JF496482.1 Arthrobacter oxydans 1508 1508 100% 99% strain

WA4-316S ribosomal RNA gene Arthrobacter oxydans 1508 1508 100% 99% strain JF496431.1 EA6-1016S ribosomal RNA gene, partial sequence HQ331132.1 Arthrobacter sp. 1508 1508 100% 99% LKSO2 16S ribosomal RNA gene, partial sequence ST 1 JF700472.1 Pseudomonas sp. 2486 2486 100% 100% HR99 16S ribosomal RNA gene partial sequence JF700467.1 Pseudomonas sp. 2486 2486 100% 100% HR94 16S ribosomal RNA gene partial sequence JF792088.1 Pseudomonas 2486 2486 100% 100% azotoformans strain 22A 16S ribosomal RNA GQ417893.1 Uncultured 2486 2486 100% 100% Pseudomonas sp clone F3Boct.37 16S ribosomal

RNA gene, partial sequence GQ417892.1 Uncultured 2486 2486 100% 100% Pseudomonas sp. clone . F3Boct.36 16S ribosomal RNA gene,

partial sequence

102

Iso Accession Description Max Total score Query Max Identity

score coverage

ST 3 AB641889.1 Enterobacter sp. 2547 2547 98% 99% NCCP-280 gene for 16S rRNA, partial sequence

AB641900.1 Enterobacter sp. 2545 2545 98% 99% NCCP-291 gene for 16S rRNA, partial sequence JF894166.1 Enterobacter cloacae 2533 2533 98% 99% strain FR 16S rRNA, partial sequence HQ336043.1 Enterobacter 2527 2527 98% 99% dissolvens strain AGYP1 16S ribosomal RNA gene,

partial sequence HQ154578.1 Enterobacter cloacae 2527 2527 98% 99% strain R10-1A 16S ribosomal RNA gene, partial sequence

ST 4 FJ907195.1 Klebsiella 1575 1575 96% 94% e isolate, 12 16S NA gene AM232720.1 Klebsiella sp. OPB2 1575 1575 91% 95% 6S rRNA gene, isolate

Klebsiella sp. SZH11 GU384262.1 al RNA gene, 1575 1564 97% 93%

nce

HQ591433.1 Klebsiella sp. SOR89 1575 1530 97% 93% 16S ribosomal RNA gene, partial sequence GU384263.1 Klebsiella 1575 1530 96% 93% pneumoniae strain SZH12 16S

ribosomal RNA gene partial sequence

103

Iso Accession Description Max Total score Query Max Identity

score coverage

ST 7 FJ444664.1 Uncultured 2259 2259 100% 100% Microbacterium sp.

clone 2y-10 16S ribosomal GQ329713.1 Microbacterium sp. 2259 2259 100% 100%

GIMN1.002 16S ribosomal RNA gene,

partial sequence

EU741114.1 Microbacterium 2259 2259 100% 100% arborescens strain13635B 16S ribosomal RNA gene partial sequence AB641893.1 Microbacterium sp. 2259 2259 100% 100% NCCP-284 gene for 16S rRNA, partial sequence

104

Figure 4.6: Phylogram indicating the distance between strains isolated from contaminated sites.

105

4.3.5 Experiments evaluating the presence of onr and ner genes in strains

4.3.5.1 Attempted Enterobacter sp onr gene identification

Binks et al. (1996) have previously described a strain of Enterobacter isolated from explosive contaminated soil, designated as Enterobacter cloacae PB2, that aerobically metobolises PETN under nitrogen-limiting conditions. E. cloacae PB2 when grown in a culture containing only PETN as a nitrogen source, was found to give a growth yield 1.64 times the growth observed when

the culture contained NH4Cl suggesting that E. cloacae was able to utilise

PETN much more readily as compared than NH4Cl.

Analysis of the strain PB2 showed that it was able to produce PETN reductase encoded by the onr (organic nitrate reductase) gene. The onr gene is a 1.5 Kbp gene which was first cloned by French et al., (1996) and was over-expressed in E. coli where a specific activity of 6.1 U/mg was obtained against GTN for crude extracts of enzyme, with 30-50% of cellular protein being accounted for by PETN reductase.

Primers were designed from the onr gene to determine if any of the isolates Arthrobacter sp., Pa-3, Achromobacter xylosoxidans TNT1, Pseudomonas sp. ST1, Microbacterium sp., ST7, Enterobacter sp., ST3 or Klebsiella sp., ST4 contain the onr gene. Since Enterobacter and Klebsiella are evolutionarily similar, it was anticipated that these isolates may contain a gene similar to onr.

Table 20. Primers designed to amplify the onr gene

Primer Sequence Tm(°C) onr F1 5’ATTACTGCGCCTGGCGTGTAG-3’ 56.8 onr F2 5’CAGCATTACTGCGCCTGGCGTGTGG- 3’ 60 onr R1 5’-ATACAGCGCGTCCGCTTCTTC-3’ 56.8 onr R2 5’-CAGATACAGCGCGTCCGCTTCTTCG-3’ 60

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Gradient PCR’s were performed to determine the optimal annealing temperature as well as to optimise the PCR’s as shown in Figure 4.8. Once the optimum temperature was obtained, which was 56. 8° the PCR was repeated using the same temperature and the products purified as shown in Figure 4.9. Although a PCR product was obtained it was shown not to be the onr gene. This was the annealing temperature that was made use of in all the remaining experiments where onr gene was to be amplified. Based on band intensity, the amount of DNA concentration is approximately 60ng.

4.3.5.2 Attempted Agrobacterium sp. nerA gene identification

Agrobactierum radiobacter isolated from sewage sludge by Snape et al. (1997) was found to be able to produce the enzyme GTN reductase and to metabolise both GTN and PETN under nitrogen-limiting, aerobic conditions (White et al., 1996). Analysis of GTN reductase by Snape et al. (1997) showed that it was a 39 kDa monomeric protein encoded by the nerA gene (nitrate ester reductase).

A strain of Agrobacterium was obtained from Microbiology department of RMIT University was used as the positive control. Primers were ordered according to the literature as per Snape et al (1997) study for the amplification of the nerA gene. According to the study conducted by Snape et al., (1997) a part of the gene approximately 560 bp in length was amplified using primers ner AD- AE. The positive control was used as the template for PCR which was extracted using the previously mentioned kit and the product was visualised using agarose gel electrophoresis as shown in Figure 4.10. The primers were able to amplify a product 560 bp in length, which was the expected size, however some non-specific binding was also observed. This could be explained since the species is not Agrobacterium radiobacter. This result indicated the presence of the nerA gene in this type strain of Agrobacterium sp.

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M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

11501

1700 1159

800

Figure 4.7 Gradient PCR with strain Enterobacter sp., ST3 using onr primers onr R1 and F1 The same Enterobacter sp. ST3 sample is used at different temperatures to determine the best annealing temperature. Well M= Lamda/Pst 1 ladder, 5=56.8°. The arrow indicates the band demonstrating the highest intensity.

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7 M 1 2 3 4 5 6

11501

1700 1159 800

Figure 4.8 Purified PCR products amplified using onr R1 and F1. Well M= Lambda/Pst 1 ladder, 1=,Arthrobacter sp., Pa-3, 2= Enterobacter sp., ST3, 3= Klebsiella sp., ST4, 4= Klebsiella sp., ST4.

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The ner primers were then used with gDNA extracted from samples of Klebsiella sp. ST4, Arthrobacter sp. Pa-3, Enterobacter sp. ST3, and Achromobacter xylosoxidans TNT1 to amplify the nerA gene. The PCR products were visualised by agarose gel and are shown in Figure 4.11. Positive results were not obtained since the size of the amplified products did not match the positive control and multiple bands were also observed.

Table 21. Primers designed to amplify nerA from Agrobacterium sp. (Snape et al.,

1997)

Primer Sequence Tm(°C) ner A-D 5’-GCIAAYCGIATYGTIATGGC-3’ 47 ner A-E 5’-ATISWICCICCRTAYTCRTC 3’ 46 ner A-H 5’-ACRTCIGGRTTIGCDATRAA -3’ 47 where R=A/G, W=A/ T, S=C/G, D= A/G/T, Y= C/T, and I= A/ C/ G/ T.)

4.3.6 Experiments using Degenerate primers

Degenerate PCR is a method using degenerate primers that are used to amplify unknown DNA sequences that are related to a known DNA sequence. It may be also used to amplify a mixture of related sequences in a single PCR reaction. PCR’s using degenerate primers are useful to identify new members in a gene family or to identify orthologous genes from different organisms. However, the more distantly related the organisms are, the more difficult it is to design the primers, due to the fact that the sequences are less similar.

Degenerate primers are designed by first gathering sequences from a large range of organisms, translating them to amino acid sequences and performing an alignment. The aligned sequences are then analysed for highly conserved regions. These conserved regions become the targets of the degenerate primers.

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M 1 2 3 4 5 6 7

11501

1700 1159 900 560

Figure 4.9 Visualised gel picture of PCR using ner primers on Agrobacterium sp. (positive control). Well M=Lambda/Pst 1 DNA ladder, 2= ner A-D to A- E, 3= ner A-D to A-E, 4= ner A-D to A-H, 5= ner A-D to A-H, 6=Negative Control .

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M 1 2 3 4 5 6 7

11501

1700 1159 900 560

Figure 4.10. Visualised gel picture of PCR using ner primers Well M=Lambda/Pst 1 DNA ladder, 1= Klebsiella sp., ST4, 2= Achromobacter xylosoxidans TNT1, 3== ner A-D to A-E, 4= Arthrobacter sp., Pa-3, 5= Enterobacter sp., ST3, 6=Negative Control, M=Lambda/Pst 1 DNA Ladder .

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In the experiments described here, degenerate primers were designed based on the amino acid alignment of sequences homologous to the onr gene. The amino acid sequences were taken from published sequences for Enterobacter sp., E. - Pseudomonas putida (xenA) and Pseudomonas fluorescens (xenB). Details are provided in Appendix II. The sequences were aligned using ClustalW and the most conserved regions are selected as primers.

PCR was performed with two sets of primers (Table 22) using gDNA as the template extracted using the kit. The PCR products were visualised by performing agarose gel electrophoresis, as shown in Figure 4.11. Some products were amplified using the degenerate primers, however the sizes of the bands amplified were between 200 and 700 bp. The products thus obtained were far too small to be the entire nitrate reductase gene. This could be explained due to non-specific binding since the primers are not specific for a particular gene. A number of larger bands were also observed on the agarose gels, which is again probably due to non-specific binding.

Table 22. Degenerate Primers

Primer Sequence Tm(°C) DF 108 5’-ATGGGCNCCRCTGACSCG -3’ 57.5

DR 348 5’-TGNGAYATHCGNCCVRYRTGCC- 3’ 57.5

DF 588 5’-ACGGHTAYYTNHTVSAYCARTT-3’ 57

DR 1001 5’-CCAAAVGCBACAACYTC-3’ 57

(where mixed base codes are R=A/G, Y=C/T, M=A/C, K=G/T, S=G/C, W=A/T, H=A/C/T, B=G/C/T, V=A/G/C, D=A/G/T, N=A/G/C/T)

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M 1 2 3 4 5 6 M 7 8 9 10 11 12 M

11501

1700 1159 805 700

250

Figure 4.11. Visualised gel picture of PCR using degenerate primers. Well M=Lambda/Pst 1 DNA ladder, 1=Arthrobacter sp., Pa-3., 2= Achromobacter xylosoxidans TNT1, 3= Klebsiella sp. ST4, 4= Enterobacter sp., ST3, 5= Microbacterium sp., ST7, 6= Achromobacter xylosoxidans TNT 2 (where wells 1-6 were amplified with degenerate primers DF108 and DR 348) Well M=Lambda/Pst 1 DNA ladder, 7= Arthrobacter sp., Pa-3, 8= Achromobacter xylosoxidans TNT1, 9= Enterobacter sp., ST3, 10= Microbacterium sp., ST7, 11= Achromobacter xylosoxidans TNT 2 12= Negative control (where wells 7-11 were amplified with degenerate primers DF588 and DR 1001)

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4.4 Discussion

Thirty-six microbial strains were isolated from contaminated soil and water samples and 6 strains were further identified and characterised. These included Enterobacter sp. ST3, Pseudomonas sp. ST1, Klebsiella sp. ST4, Microbacterium sp. ST7, Arthrobacter sp. Pa-3, and Achromobacter xylosoxidans TNT1. To the authors knowledge, this is the first report of Arthrobacter sp. and Achromobacter xylosoxidans stains with the ability to degrade PETN and TNT, respectively.

All the bacteria isolated in this study have been previously described to be able to degrade a number of other soil contaminants. For example, Arthrobacter globiformis CECT 4500 is a Gram-positive microbe isolated from the grounds surrounding a munitions factory that was described to be tolerant of up 1 M nitrate (Pinar et al., 1998). These studies demonstrated that under strict conditions, A. globiformis used a wide variety of xenobiotic carbon sources. The bacterium was able to use nitrate when the concentration was below 150 mM along with other carbon and energy sources. Achromobacter xylosoxidans (also known as Alcaligenes xylosoxidans) has been described to contain a gene, nirA, encoding the blue dissimilatory nitrite reductase (Prudencio et al., 1999). Dissimilatory nitrite reductase is an important enzyme in the denitrification process, in which nitrate undergoes step-wise reduction to the gaseous products nitrous oxide and dinitrogen.

Four methods of genomic DNA extraction were employed to obtain template for PCR. Since different methods can yield different results depending upon the the strain of bacteria, the most efficient method of extracting genomic DNA from soil bacteria was determined. Phenol-chloroform extraction is one of the most common methods of extracting genomic DNA and was the first method to be studied and performed followed by the boiling method, colony PCR and lastly DNA extraction using a QIAamp® DNA mini kit.

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All methods yielded gDNA that could be used as a template for PCR. It can be inferred from the gel pictures that the using QIAamp® DNA mini kit was the best method for extracting genomic DNA. The concentration and purity of gDNA extracted using the kit was far higher than any of the other methods. Although the other methods of DNA extraction also gave rise to the template DNA for PCR (of varying intensity) each method had its own limitations. Phenol-chloroform method of extracting gDNA was the next best method of the four methods evaluated. The concentration of DNA extracted was slightly lower than using the kit. Colony PCR in spite of being the least time- consuming did not give rise to high purity gDNA. This is to be expected, as no purification steps are taken before use of colony suspensions as template. Nonetheless, colony PCR remains a useful method for certain applications where DNA purity is of less importance, especially for high-throughput applications. The boiling method required no additional reagents, but could still be used to successfully extract gDNA of high concentration but not very high purity. When performing the extraction, care also needed to be taken that no PCR inhibitors were added to the reaction. Only molecular grade water was used when performing the boiling method so as to prevent the inhibition of the PCR from chemicals or other agents in sterile tap water.

The strains were identified to genus level using BLAST with matches of approximately 99% identity. Achromobacter xylosoxidans TNT1 was identified using the API biochemical kit within acceptable certainty thresholds. The partial 16S rRNA gene was sequenced initially for the identification of the Gram-negative bacilli (TNT 1 and TNT 2) isolated from contaminated Site A, and showed maximum similarity to Stenotrophomonas maltophilia. However, testing with the API 20 NE kit showed that the bacilli were Achromobacter xylosoxidans.

Achromobacter xylosoxidans and Stenotrophomonas maltophilia are aerobic, nonfermentative, Gram-negative bacilii that are found in a wide variety of aquatic, soil and rhizosphere environments. They have been known to cause infections in patients suffering from Cystic Fibrosis (Krzewinski et al., 2001),

116 but are otherwise non-pathogenic to healthy individuals. Stenotrophomonas maltophilia was previously grouped in the genus Xanthomonas, to which Achromobacter xylosoxidans also belonged before the two were reclassified as type species Stenotrophomonas and Achromobacter. The 16S results showed more similarity to Stenotrophomonas maltophilia; however, previous studies have shown that the two organisms are commonly misidentified. The correct nomenclature of A. xylosoxidans is an ongoing discussion. The species has been names Achromobacter xylosoxidans, Alcaligenes denitrificans subsp. xylosoxidans, and Alcaligenes xylosoxidans. The name Achromobacter xylosoxidans was also proposed, but this has not been accepted by all. Correct discrimination of the organism from others is difficult using biochemical tests. Therefore, the proper species identification for strains TNT-1 and TNT-2 is debatable, but for the purposes of this study, Achromobacter xylosoxidans TNT-1 and TNT-2 has been proposed.

Microbacterium sp. was found at both sites Pa-3 and Pb-5, suggesting that it is a common soil bacterium. However, there have not been many studies on the degradative abilities of this strain on explosives. Heavy compounds such as quinoline-4-carboxylic acid (Roger et al., 1989), and polycyclic aromatic hydrocarbons (Sheng et al., 2009) have been described to be degraded by Microbacterium sp.

Klebsiella sp. and Enterobacter sp. are known soil microorganisms having been described as capable of degrading a range of products as well as known to contain genes encoding proteins for the degradation of explosives such as PETN and GTN. Some Pseudomonas sp. are capable of degradation of xenobiotics as studied previously and also contain degradative genes such as xenA, xenB, xenC and xen D( Fuller et al.,2009; Fernandez et al., 2009).

Arthrobacter sp. is another organism that has been previously described to be able to degrade heavy compounds. However, studies showing TNT and PETN degradative capabilities have not been previously described to the authors’ knowledge. Since some the organisms isolated from the contaminated soil have

117 been previously described to be able to degrade other pollutants, further studies into their degradation activity was performed, as will be described in the following chapter.

The next series of experiments performed were to determine the presence of the onr gene from Enterobacter cloacae PB2 in any of the six isolates. Primers were designed using the onr gene and tested against the genomic DNA extracts.

Since the optimum Tm had to be used in the PCR, a gradient PCR testing different temperatures was set up. The optimum temperature was determined from the gel picture by observing the band of the highest intensity (Fig 4.8). The remaining PCR’s were performed at 56.8ºCfor the remaining isolates to determine if any of them contained the onr gene. PCR products were obtained for Arthrobacter sp., Pa-3, Enterobacter sp., ST3 and Klebsiella sp., ST4. The expected size of the onr gene was 1.5K bp, but the products amplified from the isolates were 800 bp (Fig 4.9), which indicated that the product was not the onr gene, perhaps due to non-specific binding. Since the onr gene was first isolated from Enterobacter cloacae PB2, it was presumed that a similar gene would be present in strains Enterobacter sp. ST3 and Klebsiella sp. ST4. The amplified product was sequenced and analysed, however the sequence did not match the onr gene (data not shown).

Another gene encoding for a GTN degrading enzyme is the nerA gene from Agrobacterium radiobacter. Primers to amplify this were designed according to the published paper by Snape et al., (1997). A positive control, Agrobacterium sp. was provided by the Microbiology Department at RMIT, Bundoora. The positive control was first tested with the ner primers and a product of approximately 560 bp was obtained which was the expected size.. The ner primers were then used against three of the isolates: Klebsiella sp., ST4, Arthrobacter sp., Pa-3 and Achromobacter xylosoxidans TNT1. From Fig 10 it can be observed that amplification occurred; however, since it is not the estimated size (from the previous experiment), it cannot be considered as the actual product. The amplified product was sequenced and analysed which confirmed that it was not the nerA gene (data not shown).

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Since there was difficulty in obtaining a PCR product using onr and ner primers, degenerate primers were designed and used with the genomic DNA of the six isolates as the template DNA. The degenerate primers were designed based on nitroreductase genes from a number of organisms. These include E. cloacae, E. coli, A. radiobacter, P. fluorescens, and P. aeroginosa. The nucleotide sequences of the genes onr, nerA, xenA, xenB and nemA were aligned using Clustal W and the most conserved region was used to design the primers. Two sets of primers were designed. However, using degenerate primers was not helpful in amplifiying genes that might assist in the degradation of explosives. This may indicate that in the species that have been identified here, the divergence of these genes from published sequence is sufficient to ensure that primers cannot bind in a specific manner. Whole- genome sequence analysis of the isolates would be one way to reveal the enzymes involved in the degradative pathways.

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CHAPTER 5

Nitrate Ester Reductase Activity Assays

5.1 Introduction

The main aim of work described in this chapter was to determine the PETN- degrading activity of the six isolates: Arthrobacter sp. Pa-3., Enterobacter sp. ST3, Klebsiella sp. ST4, Microbacterium sp. ST7, Pseudomonas sp. ST1, and Achromobacter xylosoxidans (TNT-1). Studies performed by Binks et al. (1996); French et al., (1996); Snape et al, (1996); White et al., (1996) have shown nitrite is the major degradation product formed when the nitrate esters GTN and PETN are degraded and the reaction proceeds via a reductase enzyme that uses an NAD(P)H cofactor. Therefore, the change in nitrite concentration was assessed using the Griess assay to determine the nitrate ester reductase activity of the isolates with GTN or PETN as a substrate. The effect of different cofactor addition was also assessed.

5.1.1 Colorimetric Griess Assay

The Griess test is a colorimetric chemical test that detects the presence of nitrite and was developed by the industrial chemist, Peter Griess in 1858. The Griess test is based on a diazotization reaction. The Griess diazotization follows the principle that sulfanilic acid is quantitatively converted to a diazonium salt by reaction with nitrite in acid solution. The diazonium salt is then coupled to N- (1-naphthyl) ethylenediamine, forming an azo dye, which forms a pink colour that can be spectrophotometrically quantitated based on its absorbance at 530

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nm (Griess Reagent kit, 2011). The detection limit for the test is usually 1.0 µM nitrite.

Of the 36 strains isolated from contaminated soil and water, 6 isolates were chosen for further characterisation; Arthrobacter sp. Pa-3, Achromobacter xylosoxidans TNT-1 Klebsiella sp. ST4, Pseudomonas sp. ST1, Enterobacter sp. ST3 and Microbacterium sp. ST7. These six isolates were selected on the basis of their ability to grow in minimal media containing either TNT or PETN as the nitrogen source. The ability of the aforementioned isolates to degrade PETN when supplied as the substrate was investigated as well as their potential for use in bioremediation. Nitrate ester reductase enzyme activity was also assessed for inducibility in the presence of PETN/GTN to evaluate the regulation pathways of PETN and GTN-degrading ability. White et al., (1996) described that the nerA gene was inducible in Agrobactirium radiobacter and Binks et al., (1996) showed in their studies that the onr gene is constitutively expressed in Enterobacter cloacae PB2.

5.2 Materials and Methods

5.2.1 Strains

Arthrobacter sp. Pa-3, Achromobacter xylosoxidans TNT-1, Klebsiella sp. ST4 Pseudomonas sp. ST1, Enterobacter sp. ST3 and Microbacterium sp. ST7 were selected as representative isolates of each genus for assessment of enzyme activity. Stock cultures were stored in a final concentration of 40% glycerol at - 80ºC and grown on PCA when required and stored at 4°C.

5.2.2 Culture conditions

Strains were grown in two types of media: Luria broth (LB) and Minimal media with PETN (mM9+PETN). These media were selected as they represent a rich non-selective medium and a lean selective medium and were used to

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compare the growth rates of the strains in the different media and whether the degradation gene(s) are constitutively expressed or inducible by PETN.

Single colonies of the isolates were inoculated into LB media and incubated overnight at the appropriate temperature (28ºC- 30ºC). Adequate head space was provided when carrying out incubation to allow mixing and oxygen transfer (a maximum of 20% vessel capacity was maintained). Six of the strains were inoculated at 1% v/v into 10 ml of LB in 50 mL Falcon tubes (with lids loosened) and 200 ml of mM9+PETN (in 1 L baffled flasks). LB cultures were

harvested after approximately 24 hours of incubation when an OD600 of approximately 2 was reached. PETN cultures grown in minimal media were

harvested when an OD600 of approximately 0.2 was reached. LB media, being a rich media allowed the strains to proliferate at a faster rate whereas minimal

media did not. Optical density (OD600) was determined every two days to ensure that the strains were growing in minimal medium with PETN as the sole nitrogen source.

5.2.3 Harvesting of culture

Once the required optical density was reached, the cells were harvested. Falcon tubes were centrifuged at 5000 x g for 20 minutes at 4 ºC, the supernatant was discarded and the pellet stored in the -20ºC freezer (as required) or used immediately. Fresh cell pellets were always used for resting cell assays. The same procedure was followed for strains grown on both media.

5.2.4 Nitrite concentration assays

Nitrite concentration was determined by transferring 100 µL of supernatant from suspended reactions (for resting cell assays) or 100 µL of reactions terminated with stop mix (for cell-free lysate assays) to 900 µL of nitrite

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reagent (10 mg/mL, Sigma-Aldrich catalogue # 37410), incubating at room

temperature for 10 min, and measuring the absorbance at 530 nm (A530). Nitrite concentrations were read from a standard curve according to the following equations:

Nitrite concentration of resting cells (mM) = (0.2411 x A530) – 0.0003 Nitrite concentration of cell-free lysate (mM) = (0.2351 x A530) – 0.0026 contains stop mix)

Two sets of experiments were performed with the harvested cells: 5.2.4.1 Whole cell resting cell activity assays 5.2.4.2 Cell-free lysate activity assays that determine cofactor and substrate requirements.

5.2.4.1 Resting Cell Activity Assay

The resting cell activity assay was performed for all the strains and was adapted from White et al (1996). The assay was performed using GTN or PETN as the substrate. When using GTN as a substrate, cell pellets were resuspended in 50

mM potassium phosphate (KPi) pH 7.2 to an OD600 equivalent of 1 unit in a final volume of 1 mL. GTN was added to the suspension to a final concentration of 0.2 mM. The reactions were performed within time intervals of 5, 15, 25, 35 and 45 minutes. Sub samples of 150 μL were taken and immediately centrifuged for 1 minute at 16,100 x g at room temperature. The nitrite concentrations of the sub-samples were determined using the supernatant. When using PETN as substrate, cell pellets were resuspended in

KPi pH 7.2 to an OD600 equivalent of 5 units in 1 mL, as nitrite release was consistently slower when this substrate was used.

Along with the above assays, nitrite utilisation assays were performed in parallel to evaluate whether resting cells were metabolising nitrite that was produced from the degradation of GTN and PETN.

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Experiments were performed using the method described above, but NaNO2 was used as the substrate instead of GTN/PETN, at a concentration of 0.05 mM. This concentration corresponds to roughly the maximum concentration achieved for resting cell assays when GTN/PETN was used as substrate under the assay conditions.

Enzyme activity was determined from the calculated slope, with the detection limit defined as the amount of enzyme resulting in a change in absorbance at 2 least 0.01 A530/h as well as a correlation coefficient (R ) greater than or equal to 0.85. One unit of enzyme activity was defined as the amount of enzyme required to liberate (from 0.2 mM GTN or PETN) or metabolise (from 0.05

mM NaNO3) 1 μmol of nitrite per minute at 22°C and pH 7.2.

5.2.4.2 Cell-Free Lysate Activity Assay

Lysate Activity assays were performed with harvested samples subjected to sonication in the presence of salt and detergent (Triton X-100) and then clarified by centrifugation. Lysates were assayed both with and without the NADP) H cofactor.

5.2.4.2.1 Sonication

Cell pellets (from 10 mL of LB culture or 200 mL of mM9+PETN) stored at - 20ºC were thawed and then resuspended in 1.5 mL of 1X Lysis buffer (50 mM Tris pH 8.0, 100 mM NaCl, 0.5% Triton X-100). The resuspended samples were subjected to sonication at 27% power for 3 x 15 second pulses with 30 second rests between pulses (Branson Digital Sonifier). The sonicated samples were incubated on ice for two hours and then centrifuged at maximum speed for five minutes at room temperature. The pellets were discarded and the supernatant was used for the assays. The supernatant was aliquoted into microcentrifuge tubes and stored at 4ºC.

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5.2.4.2.2 Nitrate ester reductase activity assay

The PETN and GTN degrading activity of the strains was measured by performing nitrate ester reductase assays. The degrading activity of the strains was monitored by measuring the nitrite production in a 1 ml assay reaction that contained 100 µl of crude enzyme lysate, 0.2 mM GTN/PETN and 0.2 mM NAD(P)H in 50 mM KPi buffer (pH 7.2). Reactions of 98 µl were stopped at various time points with 2 µl of stop mix (10 mM phenazine

methosulfate and 25 mM K3Fe (CN6)), which oxidised any remaining cofactor, and the nitrite concentration was determined. One unit of activity was defined as the amount of enzyme required to release 1 µmol of nitrite per minute from GTN/PETN at 22°C. The detection limit was based on a slope of

greater than or equal to 0.01 A530/h and a correlation coefficient greater than or equal to 0.85.

Assays were performed initially with GTN/PETN as the substrate and with or without cofactor (NADH, NADPH or no cofactor). Both GTN and PETN degrading activity were determined using both cofactors in triplicate.

5.2.4.2.3 Nitrite Utilisation Assay

Nitrite utilisation was evaluated by setting up controls analysing the utilisation of nitrite by cell-free lysates in the absence of GTN/PETN. One ml

reactions containing 0.05 mM NaNO2 diluted in 50 mM KPi buffer (pH 7.2) were prepared to measure enzyme activity. 98 µl samples were taken at different time points (e.g. 5, 25 and 45 min) and nitrite concentration determined.

5.2.4.2.4 Protein Assay

The Bradford protein assay was performed as per the standard protocol (2.4.4.4). Protein concentration for unknown samples was estimated by using

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Bovine Serum Albumin as a standard. Protein was quantified by measuring the absorbance at 595 nm and reading against the standard curve.

5.3 Results: Characterisation of Isolates

5.3.1 Resting cell activity

Six isolates were evaluated for resting cell activity. . Studies comparing GTN and PETN degrading activities were performed for cells grown in LB and M9 N-free media with PETN as a nitrogen source. Degradation rates were generally higher when GTN was used as a substrate compared with PETN with the degradation rates being approximately 5-20 folds higher for GTN. However, the activity was not significantly enhanced in minimal media with the addition of GTN/PETN as the substrate as shown in Figure 5.1 and 5.2, when compared to the degradation rates in LB media The studies revealed that all the isolates preferred GTN to PETN when the strains were grown in mM9+PETN media (Figure 5.3) The negative U/OD results could be due to nitrite utilisation. However significant nitrite utilisation was not observed (Appendix IV). PETN degrading activity was highest when the strains were cultured in LB media. Comparison studies were also performed on the isolates to compare the GTN and/or PETN degrading activity. It was demonstrated from the experiments that strains Achromobacter xylosoxidans TNT-1, Klebsiella sp. ST4, Enterobacter sp. ST3, Microbacterium sp. ST7, and Pseudomonas sp. ST1 showed a 5 -20 fold increase in GTN-degrading activity when compared to PETN degrading activity for the cells cultured in mM9+ PETN media. No significant nitrite utilisation was observed with

resting cells in the presence 0.05 mM NaNO2 (data not shown). Units per OD is calculated by taking into consideration the U/mL, sample volume, OD and sonication volume. U/OD= U/mL/(OD*Sample volume)/Sonication volume.

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Of all the isolates, Klebsiella sp. ST4 showed the highest increase (5 fold) in GTN degrading activity when cultured in mM9+PETN media when compared to other isolates cultured in mM9+PETN using either GTN or PETN as the substrate. Isolate Arthrobacter sp. Pa-3 showed a 1.5 fold increase of GTN-degrading activity when cultured in mM9+PETN. It was also observed that the GTN degrading activity in resting cells of all strains cultured in LB was lower than expected (Figure 5.1). Previously performed studies show that GTN-degrading activity in resting cells that have been cultured in LB are higher (up to 1.9 fold increase) (Constantinos, 2010) The reason for the unexpectedly low activity is unknown and further testing needs to be performed to assess this. Since no additional exogenous cofactors are provided to the resting cells, the reduced cofactors need to be supplied from within the cells themselves. Cells that are overgrown or older may have less reduced cofactor available which could be why activity was lower than expected. All the isolates demonstrated expected PETN-degrading activity when cultured in LB media.

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0.008

0.006

0.004

0.002 M9 +GTN

U/OD LB 0 AS AX KS ES MS PS

-0.002

-0.004

-0.006 Isolates

Figure 5.1 GTN-degrading activities of resting cells of isolates cultured on LB and mM9+PETN. GTN was used as the substrate in the assay. Explain U/OD AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1 KS: Klebsiella sp. ST4 ES: Enterobacter sp. ST3 MS: Microbacterium sp. ST7 PS: Pseudomonas sp. ST1

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0.01

0.009

0.008

0.007

0.006 M9+PETN 0.005

U/OD LB 0.004

0.003

0.002

0.001

0 AS AX KS ES MS PS Isolates

Figure 5.2 PETN-degrading activities of resting cells of isolates cultured on LB and mM9+PETN. PETN was used as the substrate in the assay. AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1

KS: Klebsiella sp. ST4

ES: Enterobacter sp. ST3

MS: Microbacterium sp. ST7

PS: Pseudomonas sp. ST1

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0.0009

0.0008

0.0007

0.0006

0.0005 M9 +GTN

U/OD 0.0004 M9+PETN

0.0003

0.0002

0.0001

0 AS AX KS ES MS PS Isolates

Figure 5.3 GTN and PETN-degrading activity of resting cells of isolates cultured on mM9+PETN. GTN or PETN were used as substrates in the respective assays. AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1

KS: Klebsiella sp. ST4

ES: Enterobacter sp. ST3

MS: Microbacterium sp. ST7

PS: Pseudomonas sp. ST1

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5.3.2 Cell-free lysate activity

The GTN/PETN degrading activity was evaluated for all six strains by taking into account the effect of different cofactor conditions when cell-free lysates were used in the assay. All six strains preferred NADPH over NADH, with GTN degradation rates being around 5-20 fold higher in the presence of NADPH when cultured in mM9+PETN (Figure 5.4). Strain Arthrobacter sp. Pa-3 was the exception which showed similar enzyme activities using NADPH or NADH when GTN was used as the substrate (Figure 5.5). Arthrobacter sp. Pa-3 also demonstrated higher PETN-degrading activity when the strain was cultured in LB as compared to when it was cultured in mM9+PETN (Figure 5.4). Arthrobacter sp. Pa-3 showed the lowest GTN and PETN-degrading activity in the presence of NADPH when the strain was cultured in LB and mM9+PETN (Figure 5.6).

The GTN and PETN-degrading activity analysis of cell-free lysates was performed to determine which substrate is preferentially degraded by the isolates. All the isolates preferred GTN over PETN when cultured in mM9+PETN (Figure 5.7). GTN degradation rates were highest in LB media when compared to mM9+PETN for Klebsiella sp. ST4. The isolate Arthrobacter sp. Pa-3 demonstrated almost an equal PETN-degrading activity in LB and mM9+PETN.

All strains exhibited higher GTN-degrading activity when cultured in mM9+PETN when NADPH was used as the co-factor (Figure 5.7)

131

9.00E-03

8.00E-03

7.00E-03

6.00E-03

5.00E-03 g M9 U/mg protein

U/m 4.00E-03 LB U/mg protein

3.00E-03

2.00E-03

1.00E-03

0.00E+00 AS MS AX PS KS ES Isolates

Figure 5.4 PETN-degrading acitvity of cell-free lysates in the presence of NADPH for isolates cultured on LB and mM9+PETN. PETN was used as the substrate in the assay. AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1 KS: Klebsiella sp. ST4 ES: Enterobacter sp. ST3 MS: Microbacterium sp. ST7 PS: Pseudomonas sp. ST1

132

4.50E-03

4.00E-03

3.50E-03

3.00E-03

2.50E-03 M9 U/mg protein

U/mg 2.00E-03 LB U/mg protein

1.50E-03

1.00E-03

5.00E-04

0.00E+00 AS MS AX PS KS ES Isolates

Figure 5.5 GTN-degrading activities of cell-free lysates in the presence of NADH for isolates cultured in LB and mM9+PETN. GTN was used as the substrate in the assay.

AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1 KS: Klebsiella sp. ST4 ES: Enterobacter sp. ST3 MS: Microbacterium sp. ST7 PS: Pseudomonas sp. ST1

133

2.50E-01

2.00E-01

1.50E-01

g M9 U/mg protein

U/m LB U/mg protein 1.00E-01

5.00E-02

0.00E+00 AS MS AX PS KS ES Isolates

Figure 5.6 GTN-degrading activity of cell-free lysates in the presence of NADPH for isolates cultured in LB and mM9+PETN. GTN was used as the substrate in the assay. AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1 KS: Klebsiella sp. ST4 ES: Enterobacter sp. ST3 MS: Microbacterium sp. ST7 PS: Pseudomonas sp. ST1

134

2.50E-01

2.00E-01

1.50E-01 M9+PETN U/mg protein

U/mg M9+GTN U/mg protein 1.00E-01

5.00E-02

0.00E+00 AS MS AX PS KS ES Isolates

Figure 5.7 PETN and GTN-degrading activities of cell-free lysates in the presence of

NADPH cultured in mM9+PETN. GTN or PETN was used as the

substrate in the respective assays.

AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1 KS: Klebsiella sp. ST4 ES: Enterobacter sp. ST3 MS: Microbacterium sp. ST7 PS: Pseudomonas sp. ST1

135

2.50E-01

2.00E-01

1.50E-01 U/mg U/mg protein (NAD 1.00E-01 U/mg protein (NAD

5.00E-02

0.00E+00 AS MS AX PS KS ES Isolates

Figure 5.8 GTN-degrading activities of cell-free lysates in the presence of NADH/ NAD(P)H for isolates cultured in mM9+PETN media. GTN was used as the substrate in the assay. AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1 KS: Klebsiella sp. ST4 ES: Enterobacter sp. ST3 MS: Microbacterium sp. ST7 PS: Pseudomonas sp. ST1

136

5.3.3 Comparison of resting cell and cell-free lysate activities

It was indicated from the experiments performed that GTN was preferred as a substrate over PETN. A study was also performed comparing the activities between resting cell and cell-free lysates. The activities varied between each of the isolates, but GTN degradation activity was higher in cell-free lysates when cultured in mM9+PETN media for strains Enterobacter sp. ST3, Klebsiella sp. ST4, Microbacterium sp. ST7, Achromobacter xylosoxidans TNT-1 and Pseudomonas sp. ST1. PETN-degradation rates were also higher in cell-free lysates cultured in LB and mM9+PETN when compared to resting cells. All the isolates when cultured in LB or M9 showed higher GTN and PETN-degrading activity in cell free lysates than in resting cells.

The differences in PETN degrading activity were found to be more pronounced in LB than in mM9 media containg PETN. The only isolates showing a variation to the aformentioned results were Arthrobacter sp. Pa-3 and Klebsiella sp. ST4, which demonstrated increased PETN-degrading activity in mM9+PETN (Figure 5.9).

GTN degradation activity was lower than expected in resting cell assays, however expected activities were demonstrated in cell free lysates. Strain Enterobacter sp. ST3 showed the highest activity amongst all the strains when cultured in mM9+PETN. Strain Arthrobacter sp. Pa-3 showed the lowest GTN- degrading activity in both LB and mM9+PETN media.

137

0.4

0.35

0.3

0.25 LB Restinc cells M9 Resting cels 0.2

U/OD LB Cell free lysate M9 Cell free lysate 0.15

0.1

0.05

0 AS AX KS ES MS PS Isolates

Figure 5.9a: PETN-degrading activities of resting cells and cell-free lysates of all isolates cultured on LB and mM9+PETN media AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1 KS: Klebsiella sp. ST4 ES: Enterobacter sp. ST3 MS: Microbacterium sp. ST7 PS: Pseudomonas sp. ST1

138

25 LB Resting cells 20 M9 +GTN Resting cells

U/OD LB Cell free lysate 15 M9+GTN Cell free lyste 10

0 AS AX KS ES MS PS -5 Isolates

Figure 5.9b: GTN-degrading activities of resting cells and cell-free lysates of all

isolates cultured on LB and mM9+PETN media.

AS: Arthrobacter sp. Pa-3 AX: Achromobacter xylosoxidans TNT-1 KS: Klebsiella sp. ST4 ES: Enterobacter sp. ST3 MS: Microbacterium sp. ST7 PS: Pseudomonas sp. ST1

139

5.4 Discussion

Six microbial strains were identified that were capable of TNT and PETN degradation. The strains isolated and characterised for degrading activity were Arthrobacter sp. Pa-3, Enterobacter sp. ST3, Klebsiella sp. ST4, Microbacterium sp. ST7, Pseudomonas sp. ST1, and Achromobacter xylosoxidans TNT-1. The degrading activity was measured for GTN along with PETN since GTN is more soluble. It should be noted that nitrite readings for triplicate samples when using GTN as substrate were generally very consistent, while replicates with PETN as substrate occasionally differed by a considerable amount. This likely reflects the poor solubility of PETN that may occasionally form insoluble crystal structures that may be difficult for the enzyme to access. Hence GTN is a more suitable substrate for routine enzyme assays. The characterisation of the six isolates revealed a preference towards GTN as a substrate.

All six isolates were tested for PETN and GTN-degrading activity using NADH or NADPH as the cofoactor. All the isolates preferred NADPH as a cofactor, however degrading activity was observed even when NADH was the cofactor used.

Experiments using NADH as cofactor and PETN as substrate were performed, however very low absorbance readings were observed for all the isolates with activity below the detection limit. Therefore, remaining experiments for PETN-degrading activity were performed only with NADPH as the cofactor.

Resting cell assays performed with PETN as a substrate indicated high degrading activity when strains were cultured in LB media. The resting cell assays performed using GTN as the substrate resulted in variable results when the strains were cultured in LB with nitrite concentration decreasing with time for some samples. Since such results were not observed in cell-free lysate assays, it is difficult to explain. A possible explanation could be the presence of an inhibiting component in LB media which resulted in the lower

140 activity. Another possible reason is that there may have been a rapid release of nitrite in the first few minutes of the assay followed by nitrite utilisation by other enzymes present in the bactieral system. Therfore, the timepoints for the assay might not have been appropriate to detect trends such as these in nitrite concentration.

On comparing the degradation activities of the resting cell assays for GTN and PETN as substrates, it was observed that strains cultured in mM9+PETN showed a higher magnitude of GTN-degrading activity than PETN-degrading activity. This may be explained due to the increased solubility of GTN as compared to the insoluble nature of PETN. GTN as a substrate would be more readily available to the isolates, which would be indicated by a higher magnitude of degradation, which was the result observed. Another possible reason for the higher magnitude of GTN-degrading activity by the isolates could be the symmetrical nature of PETN that could result in the molecule being more stable and hence more difficult to reduce. GTN, however is inherently less stable and so it was not suprising to find that it was more readily degraded by the enzymes isolated from this study.

Cell-free lysate assays were performed on all isolates to determine substrate preference and GTN and PETN-degrading activity. The results obtained were similar to studies previously described (White et al,1996). Arthrobacter sp., Pa-3 was the only strain that showed similar degrading activity in PETN and GTN. However, the cofactor preference on PETN media was NADPH, while in GTN media, similar activities were observed with either cofactor. All other isolates clearly prefered NADPH over NADH as a cofactor when GTN was used as the substrate. Enterobacter sp. ST3 showed the same PETN and GTN degrading activity when cultured in LB. GTN-degrading activity was clearly induced when cells were grown on PETN, but PETN-degrading activity was not.

Microbacterium sp. ST7 showed moderate PETN degrading acitvity when the strain was cultured in LB. This was similar to the GTN degrading activity observed in the presence of NADH as cofactor. Strain Microbacterium sp.

141

ST7 showed a preference for NADPH as a cofactor and higher GTN- degrading activity was observed when the strain was cultured in mM9+PETN. This indicates that GTN-degrading activity may be inducible in this strain. However, the higher PETN-degrading activity observed for PETN-grown cells indicates that constitutively expressed enzymes are responsible for PETN degradation, while possibly other inducible enzymes are involved in GTN degradation, but are unable to degrade PETN. Further purification of the enzymes expressed under different growth conditions may help to address this question.

Klebsiella sp. ST4 showed very low PETN and GTN-degrading activity when cultured in LB, however the GTN-degrading acitivy was higher when Klebsiella sp. ST4 was cultured in mM9+PETN in the presence of NADPH as the cofactor. This clearly demonstrated that GTN-degrading activity is inducible in this strain, but PETN-degrading activity appears to be constitutively expressed.

Achromobacter xylosoxidans TNT-1 showed similar GTN and PETN- degrading activity when cultured in LB, but showed a slightly higher degree of GTN degradation in mM9+PETN media with NADPH being preferred as the cofactor.

Pseudomonas sp., ST1 also showed very similar PETN and GTN-degrading activity when cultured in LB. A higher magnitude of GTN-degrading activity was observed when the strain was cultured in mM9+PETN, with NADPH being prefered over NADH as the cofactor. Similarly to Enterobacter sp. ST3, Microbacterium sp. ST7, Klebsiella sp. ST4 and Achromobacter xylosoxidans TNT-1, GTN-degrading activity appeared to be induced, while PETN degradation was not.

This increase in the NADPH-dependent activity in mM9+PETN media indicates that GTN-degrading activity is inducible. All the strains except Arthrobacter sp. Pa-3 cultured in mM9+PETN media demonstrated higher activity in cell-free lysates. The higher rates could perhaps be explained by

142 the addition of the exogenous cofactor to the reaction mix, which may have been rate-limiting in the resting cell assays since they rely on cofactors that are generated from the intact cells. Another possible explanation could be the diffusion of susbtrate through the cell membrane that may have caused the reduced activities. Another possibility could be that Arthrobacter sp. contains nitroreductase genes that are constitutively expressed.

All strains had higher resting cell activity against PETN when cultured in LB media than in minimal media, suggesting a constitutive expression of the genes that are associated with PETN degradation. The higher activity for the resting cells grown in LB may also have been due to the richness of the media relative the the lean minimal medium. It is possible that cells grown in a richer medium may accumulate a greater reduced cofactor pool within the cells that may improve resting cell activity. Conversely, cells grown in minimal media may down-regulate non-critical metabolic pathways, of which PETN-degrading enzymes may be one.

The strains that were isolated in this study may be of use in biorememdiation, and further studies need to be performed to isolate and characterise the enzymes responsible for degradation. Another area that could be studied further is the recycling of cofactors used such that a reduced cofactor can be supplied cheaply.

This additional information could assist in the further characterization, optimization and production of explosive-degrading enzymes.

143

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PhD Thesis referred:

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163

APPENDIX I

16S rDNA Sequences

Isolate TNT 1

AGAGTTTGAT CCTGGCTCAG AGTGAACGCT GGCGGTAGGC CTAACACATG CAAGTCGAAC GGCAGCACAG GAGAGCTTGC TCTCTGGGTG GCGAGTGGCG GACGGGTGAG GAATACATCG GAATCTACTT TTTCGTGGGG GATAACGTAG GGAAACTTAC GCTAATACCG CATACGACCT ACGGGTGAAA GCAGGGGATC TTCGGACCTT GCGCGATTGA ATGAGCCGAT GTCGGATTAG CTAGTTGGCG GGGTAAAGGC CCACCAAGGC GACGATCCGT AGCTGGTCTG AGAGGATGAT CAGCCACACT GGAACTGAGA CACGGTCCAG ACTCCTACGG GAGGCAGCAG TGGGGAATAT TGGACAATGG GCGCAAGCCT GATCCAGCCA TACCGCGTGG GTGAAGAAGG CCTTCGGGTT GTAAAGCCCT TTTGTTGGGA AAGAAATCCA GCTGGCTAAT ACCCGGTTGG GATGACGGTA CCCAAAGAAT AAGCACCGGC TAACTTCGTG CCAGCAGCCG CGGTAATACG AAGGGTGCAA GCGTTACTCG GAATTACTGG GCGTAAAGCG TGCGTAGGTG GTCGTTTAAG TCCGTTGTGA AAGCCCTGGG CTCAACCTGG GAACTGCAGT GGATACTGGG CGACTAGAAT GTGGTAGAGG GTAGCGGAAT TCCTGGTGTA GCAGTGAAAT GCGTAGAGAT CAGGAGGAAC ATCCATGGCG AAGGCAGCTA CCTGGACCAA CATTGACACT GAGGCACGAA AGCGTGGGGA GCAAACAGGA TTAGATACCC TGGTAGTCCA CGCCCTAAAC GATGCGAACT GGATGTTGGG TGCAATTTGG CACGCAGTAT CGAAGCTAAC GCGTTAAGTT CGCCGCCTGG GGAGTACGGT CGCAAGACTG AAACTCAAAG GAATTGACGG GGGCCCGCAC AAGCGGTGGA GTATGTGGTT TAATTCGATG CAACGCGAAG AACCTTACCT GGCCTTGACA TGTCGAGAAC TTTCCAGAGA TGGATTGGTG CCTTCGGGAA CTCGAACACA GGTGCTGCAT GGCTGTCGTC AGCTCGTGTC GTGAGATGTT GGGTTAAGTC CCGCAACGAG CGCAACCCTT GTCCTTAGTT GCCAGCACGT AATGGTGGGA ACTCTAAGGA GACCGCCGGT GACAAACCGG AGGAAGGTGG GGATGACGTC AAGTCATCAT GGCCCTTACG GCCAGGGCTA CACACGTACT ACAATGGTAG GGACAGAGGG CTGCAAGCCG GCGACGGTAA GCCAATCCCA GAAACCCTAT CTCAGTCCGG ATTGGAGTCT GCAACTCGAC TCCATGAAGT CGGAATCGCT AGTAATCGCA GATCAGCATT GCTGCGGTGA ATACGTTCCC GGGCCTTGTA CACACCGCCC GTCACACCAT GGGAGTTTGT TGCACCAGAA GCAGGTAGCT TAACCTTCGG GAGGGCGCTT GCCACGGTGT GGCCGATGAC TGGGGTGAAG TCGTAACAAG GTAGCCGTAT CGGAAGGTGC GGTTGGATCA CCTCCTTACT

Isolate Pa-3

GATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGTGAAGCCAAGCTTGCTTGGTGGAT CAGTGGCGAACGGGTGAGTAACACGTGAGCAACCTGCCCTGGACTCTGGGATAAGCGCTGGAAAC GGCGTCTAATACTGGATATGAGCTCCTATCGCATGGTGGGGGTTGGAAAGATTTTTCGGTCTGGGAT GGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGTCGACGGGTAGCCGGCC TGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGG GGAATATTGCACAATGGGCGGAAGCCTGATGCAGCAACGCCGCGTGAGGGATGACGGCCTTCGGGT TGTAAACCTCTTTTAGCAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAAAAGCGCCGGCTAACT ACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGC TCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCCGAGGCTCAACCTCGGGCCTGCAGTGGGTACGG GCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGG AGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGG AGCAAACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGACCAT TCCACGGTTTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTA AAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACG CGAAGAACCTTACCAAGGCTTGACATATACGAGAACGGGCCAGAAATGGTCAACTCTTTGGACACT CGTAAACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCGTTCTATGTTGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAA CTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTAC AATGGCCGGTACAAAGGGCTGCAATACCGTGAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTC GGATTGAGGTCTGCAACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTG

164

CGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCAAGTCATGAAAGTCGGTAACACCTGAA GCCGGTGGCCCAACCCTTGTGGAGGGAGCCGTCGAAGGTGGGATCGGTAATTAGGACTAAGTCGTA ACAAGGTAGCCGTACCGGAAGG

Isolate Pb-5

AACGGGTGAGTAACACGTGAGTAACCTGCCCTTAACTCTGGGATAAGCCTGGGAAACTGGGT CTAATACCGGATATGACTCCTCATCGCATGGTGGGGGGTGGAAAGCTTTATTGTGGTTTTGGA TGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTTACCAAGGCGACGACGGGTAGCC GGCCTGAGAGGGTGACCGGCCACACTGGGACTGAAACACGGCCCAAACTCCTACGGGAGGCA GCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGAC GGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAAAAGCGAAAGTGACGGTACCTGCAAAAAA AGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAAT TATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCCGTGAAAGTCCGGGGCTCAACTC CGGATCTGCGGTGGGTACGGGCAGACTAGAGTGATGTAGGGGAGACTGGAATTCCGGTGTAGC GGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCATTAACTG ACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAA ACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGC CCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC GGCGGAGCATGCGGATTAATTCGATGCAACGCGA

Isolate ST1

AAGTCGAGCG GTAGAGAGAA GCTTGCTTCT CTTGAGAGCG GCGGACGGGT GAGTAATGCC TAGGAATCTG CCTGGTAGTG GGGGATAACG TTCGGAAACG GACGCTAATA CCGCATACGT CCTACGGGAG AAAGCAGGGG ACCTTCGGGC CTTGCGCTAT CAGATGAGCC TAGGTCGGAT TAGCTAGTTG GTGGGGTAAT GGCTCACCAA GGCGACGATC CGTAACTGGT CTGAGAGGAT GATCAGTCAC ACTGGAACTG AGACACGGTC CAGACTCCTA CGGGAGGCAG CAGTGGGGAA TATTGGACAA TGGGCGAAAG CCTGATCCAG CCATGCCGCG TGTGTGAAGA AGGTCTTCGG ATTGTAAAGC ACTTTAAGTT GGGAGGAAGG GTTGTAGATT AATACTCTGC AATTTTGACG TTACCGACAG AATAAGCACC GGCTAACTCT GTGCCAGCAG CCGCGGTAAT ACAGAGGGTG CAAGCGTTAA TCGGAATTAC TGGGCGTAAA GCGCGCGTAG GTGGTTTGTT AAGTTGGATG TGAAATCCCC GGGCTCAACC TGGGAACTGC ATTCAAAACT GACTGACTAG AGTATGGTAG AGGGTGGTGG AATTTCCTGT GTAGCGGTGA AATGCGTAGA TATAGGAAGG AACACCAGTG GCGAAGGCGA CCACCTGGAC TAATACTGAC ACTGAGGTGC GAAAGCGTGG GGAGCAAACA GGATTAGATA CCCTGGTAGT CCACGCCGTA AACGATGTCA ACTAGCCGTT GGAAGCCTTG AGCTTTTAGT GGCGCAGCTA ACGCATTAAG TTGACCGCCT GGGGAGTACG GCCGCAAGGT TAAAACTCAA ATGAATTGAC GGGGGCCCGC ACAAGCGGTG GAGCATGTGG TTTAATTCGA AGCAACGCGA AGAACCTTAC CAGGCCTTGA CATCCAATGA ACTTTCCAGA GATGGATTGG TGCCTTCGGG AACATTGAGA CAGGTGCTGC ATGGCTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGTAACG AGCGCAACCC TTGTCCTTAG TTACCAGCAC GTAATGGTGG GCACTCTAAG GAGACTGCCG GTGACAAACC GGAGGAAGGT GGGGATGACG TCAAGTCATC ATGGCCCTTA CGGCCTGGGC TACACACGTG CTACAATGGT CGGTACAGAG GGTTGCCAAG CCGCGAGGTG GAGCTAATCC CATAAAACCG ATCGTAGTCC GGATCGCAGT CTGCAACTCG ACTGCGTGAA GTCGGAATCG CTAGTAATCG CGAATCAGAA TGTCGCGGTG AATACGTTCC CGGGCCTTGT ACACACCGCC CGTCACACCA TGGGAGTGGG TTGCACCAGA AGTAGCTAGT CTAACCTTCG GGAGGAC

Isolate ST3

TGGGGCGCAGCTACACATGCAGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTG ACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAAC TACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTT CGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGG CTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACT

165

GAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGC GCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTAC TTTCAGCGGGGAGGAAGGTGTTGTGGTTAATAACCACAGCAATTGACGTTACCCGCA GAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGC GTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTG AAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTA GAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATAC CGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGG GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAG GTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGA GTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGG AGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACA GAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGC TGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT ATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACT GGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACA CGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCA TAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATC GCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACAC CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGTTTAGCTTAACCTTCGGG AGGGCGCTACCACTTGATTACGC

Isolate ST4

GCAGCCTACC AGCAAGTCGA GCGGTAGCAC AGAGAGCTTG CTCTCGGGTG ACGAGCGGCG GACGGGTGAG TAATGTCTGG GAAACTGCCT GATGGAGGGG GATAACTACT GGAAACGGTA GCTAATACCG CATAATGTCG CAAGACCAAA GTGGGGGACC TTCGGGCCTC ATGCCATCAG ATGTGCCCAG ATGGGATTAG CTAGTAGGTG GGGTAATGGC TCACCTAGGC GACGATCCCT AGCTGGTCTG AGAGGATGAC CAGCCACACT GGAACTGAGA CACGGTCCAG ACTCCTACGG GAGGCAGCAG TGGGGAATAT TGCACAATGG GCGCAAGCCT GATGCAGCCA TGCCGCGTGT GTGAAGAAGG CCTTCGGGTT GTAAAGCACT TTCAGCGGGG AGGAAGGCGA TAAGGTTAAT AACCTTGTCG ATTGACGTTA CCCGCAGAAG AAGCACCGGC TAACTCCGTG CCAGCAGCCG CGGTAATACG GAGGGTGCAA GCGTTAATCG GAATTACTGG GCGTAAAGCG CACGCAGGCG GTCTGTCAAG TCGGATGTGA AATCCCCGGG CTCAACCTGG GAACTGCATT CGAAACTGGC AGGCTAGAGT CTTGTAGAGG GGGGTAGAAT TCCAGGTGTA GCGGTGAAAT GCGTAGAGAT CTGGAGGAAT ACCGGTGGCG AAGGCGGCCC CCTGGACAAA GACTGACGCT CAGGTGCGAA AGCGTGGGGA GCAAACAGGA TTAGATACCC TGGTAGTCCA CGCCGTAAAC GATGTCGATT TGGAGGTTGT GCCCTTGAGG CGTGGCTTCC GGAGCTAACG CGTTAAATCG ACCGCCTGGG GAGTACGGCC GCAAGGGTTA AAACTCAATG AATTTGACGG GGGCCCGCAC AAGCGGTGGG AGCATGTGGT TTAATTCGAT GCAACGCGAG ACCTTTACCT GGTCTGACAT CACAGAACTT CCAGAGATGA TGGTGCTCGG GACTGTGAGA CAGGTGCTGC ATGCTGTCGT CAGCTCGTGT TGTGAATGTG GGTAAGGTCC GCACCGAGCG CATCTATCCT TCTGCCAGCG TCAGTCGGAC TCAAGGAACT GCAGGTAACT GAGAAGTGGG ATGACGTCA

Isolate ST7

GCTGGGTGGATCAGTGGCGAACGGGTGAGTAACACGTGAGCAATCTGCCCCTGACTCTGGGA TAAGCGCTGGAAACGGCGTCTAATACCGGATACGAGCTGCGGCCGCATGGTCGGCAGTTGGA AAGATTTTTCGGTCAGGGATGAGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACC AAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCC CAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGGAAGCCTGATGCAGCA ACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAG TGACGGTACCTGCAGAAAAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGG CGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTCTGTCGCGTCTGCTGTG AAATCCCGAGGCTCAACCTCGGGTCTGCAGTGGGTACGGGCAGACTAGAGTGCGGTAGGGGAG ATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGC AGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGGAGCAAACAGGCTTAGATAC CCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGTCCATTCCACGGATTCCGTGAC GCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAAT TGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAATCCTT ACCAAGGCTTGACATATAGAGGAAACGTCTGGAAACAGTCGCCCCGCAAGGTCTCTATACAGG TGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTCGTTCTATGTTGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGG AGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAA

166

TGGCCGGTACAAAGGGCTGCAATACCGTGAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTT CGGATTGAGGTCTGCAACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAG

167

APPENDIX II

CLUSTAL 2.0.12 multiple sequence alignment

Enterobacter ATGTCAGCTGAAAAACTTTTCACCCCACTGAAAGTGGGCGCCGTCACCGCACCAAACCGC 60 Escherichia ATGTCATCTGAAAAACTGTATTCCCCACTGAAAGTGGGCGCGATCACGGCGGCAAACCGT 60 E.cloacae ATGTCCGCTGAAAAGCTGTTTACCCCACTGAAAGTGGGTGCCGTTACTGCCCCAAACCGC 60 nerA ------ATGACCAAACTCTTCGACCCGACAAAGCTGGGCGATATCGCCATCGCCAACCGC 54 xenB ------ATGACGACTCTCTTTGATCCGATCAAACTCGGCGATCTCGAACTGTCCAACCGC 54 xenA ------ATGTCCGCACTGTTCGAACCCTACACCCTCAAAGACGTCACCCTGCGTAACCGT 54 ** * ** * * * * *****

Enterobacter GTGTTTATGGCCCCACTCACCCGTCTGCGCAGCATAGAGCCGGGTGATATCC-CAACCCC 119 Escherichia ATTTTTATGGCACCGCTGACGCGTCTGCGCAGTATTGAACCGGGTGACATTC-CTACCCC 119 E.cloacae GTGTTTATGGCCCCACTTACCCGTCTGCGCAGCATCGAGCCGGGCGATATCC-CAACGCC 119 nerA ATCGTCATGGCGCCGCTGAC------GCGCAACCGCTCTCCCAACGCTGTTC-CGAACGA 107 xenB ATCATCATGGCGCCGCTGACCCGC---TGCCGCGCCGATGCCGGCCGCGTAC-CCAACGC 110 xenA ATCGCCAT---TCCGCCGATGTGCCAGTACATGGCCGAAGACGGCATGATCAACGACTGG 111 * ** ** * * * * * *

Enterobacter ACTGATGGGTGAATATTATCGTCAGC--GCGCCAGCTCTGGTCTCATCATCACTGAAGCC 177 Escherichia GTTGATGGCGGAATACTATCGCCAAC--GTGCCAGTGCCGGTTTGATTATTAGTGAAGCC 177 E.cloacae ATTGATGGGTGAGTATTACCGCCAGC--GCGCCAGCGCGGGCCTGATTATCTCCGAAGCC 177 nerA TCTGAACGTCAAGTATTATGCCCAGC--GCGCCACGGCGGGCCTGATCATCACCGAAGCC 165 xenB GCTGATGGCCGAATACTACGTACAAC--GGGCTTCCGCCGGCCTGATCCTCAGCGAGGCC 168 xenA CACCACGTTCACCTGGCCGGCCTGGCCCGTGGTGGTGCCGGCTTGCTGGTGGTCGAGGCC 171 * * * * * * ** * * * ** ***

Enterobacter ACGCAGATTTCCGCACAGGCGAAGGGATATGCCGGCGCACCAGGCCTGCACAGCCCGGAG 237 Escherichia ACGCAAATTTCTGCCCAGGCAAAAGGATATGCAGGTGCGCCTGGCATCCATAGTCCGGAG 237 E.cloacae ACGCAGATTTCTGCTCAGGCAAAAGGCTACGCCGGTGCACCGGGTCTGCACAGCCCGGAA 237 nerA ACCGCCATCACCCATCAAGGCCAGGGCTATGCCGACGTGCCCGGTCTCTACACCAAGGAC 225 xenB ACCTCGGTGACCCCCATGGGCGTGGGCTACCCGGACACCCCCGGCATCTGGTCCAACGAC 228 xenA ACTGCTGTGGCGCCGGAAGGGCGTATCACCCCCGGTTGCGCCGGTATCTGGAGCGATGCC 231 ** * * * * * * ** * *

Enterobacter CAGATTGCCGCCTGGAAGAAAATCACCGCTGGCGTTCACGCCGAAGAGGGTCGCATTGCG 297 Escherichia CAAATTGCCGCATGGAAAAAAATCACCGCTGGCGTTCATGCTGAAAATGGTCATATGGCC 297 E.cloacae CAGATCGCCGCGTGGAAAAAAATCACCGCAGGCGTGCATGCTGAAGATGGCCGTATTGCG 297 nerA GCCCTCGACGGCTGGAAGAAGGTGACCGACGCCGTGCATGCCAATGGCGGCAAGATCGTC 285 xenB CAGGTGCGTGGCTGGGCCAATGTGACCAAGGCAGTACACGGCGCCGGTGGCAAGATATTC 288 xenA CACGCTCAGGCGTTCGTACCGGTGGTGCAGGCCATCAAGGCTGCCGGTTCCGTGCCGGGT 291 * * * * * * *

Enterobacter GTTCAGCTGTGGCACACCGGTCGTATCTCTCACAGCAGCATTCAGCCTGGCGGTCAGGCG 357 Escherichia GTGCAGCTGTGGCACACCGGACGCATTTCTCACGCCAGCCTGCAACCTGGCGGTCAGGCA 357 E.cloacae GTTCAGCTGTGGCACACCGGTCGTATCTCACACAGCAGCATCCAGCCTGGCGGTCAGGCG 357 nerA GTGCAGATGTGGCATGTGGGCCGCATCTCGCACACCAGCCTGCAGCCGAACGACGGCAAA 345 xenB CTGCAGCTGTGGCACGTCGGACGCATCTCCCAC-CCGTCCT--ATCTGAACGGCGAAACC 345 xenA ATCCAGATCGCTCACGCCGGGCGCAAGGCCAGCGCCAACCGCCCGTGGGAGGGTGATGAC 351 * *** * ** ** ** * * * * * *

Enterobacter CCGGTTTCCGCGTCGGCCCTGAATGCGAATACCCGCACTTCCCTGCGCGATGAAAACGGT 417 Escherichia CCGGTAGCGCCTTCAGCACTTAGCGCGGGAACACGTACTTCTCTGCGCGATGAAAATGGT 417 E.cloacae CCGGTTTCTGCCTCTGCCCTGAACGCCAATACCCGCACTTCCCTGCGCGATGAAAACGGT 417 nerA CCGGTCTCGTCGACCTCCAGAGCCGCCAAGGCCAAGACCTATCTCGTCGAAAAGGACGGC 405 xenB CCGGTGGCACCCAGCGCCCTGCAGCCCAAGGGGCATGTCAGCCTGGT------392 xenA CACATTGCCGCCGACGATGCGCGCGGCTGGGAGACCATTGCCCCGTCTGCCATTGCCTTT 411 * * * * *

Enterobacter AACGCGATCCGCGTCGATACCTCAACGCCACGCGCCCTTGAGCTGGATGAGATCCCGGGT 477 Escherichia CAGGCGATCCGTGTTGAAACATCCATGCCGCGTGCGCTTGAACTGGAAGAGATTCCAGGT 477 E.cloacae AATGCGATCCGCGTCGACACCACCACGCCACGCGCGCTGGAGCTGGACGAGATCCCGGGT 477 nerA AGCGGCCACTTTGCCGAGACCTCCGAACCACGGGCGCTTGAAACCGCCGAGATCCCGGGG 465 xenB -GCGCCCGCTG-GCCGACTTCCCAACTCCGCGGGCCCTGGAAACCGCTGAAATCGCCGAC 450 xenA GGCGCGCACCT-GCCGAAA-----GTGCCACGCGAAATGACGCTGGACGATATCGCCCGG 465 * * * ** ** ** * * * ** ** *

Enterobacter ATCGTGAACGATTTCCGTCAGGCGGTGGCTAACGCCCGCGAAGCCGGTTTTGATCTGGTT 537 Escherichia ATCGTCAATGATTTCCGTCAGGCCATTGCTAACGCGCGTGAAGCCGGTTTTGATCTGGTA 537 E.cloacae ATCGTGAATGATTTCCGTCAGGCCGTCGCCAACGCCCGGGAAGCGGGCTTCGACCTGGTT 537

168 nerA ATCGTCGAGGACTATCGCAAGGCCGCTCGCGCTGCGATCGATGCCGGGTTCGACGGCGTC 525 xenB ATCGTCGATGCCTACCGGGTCGGCGCGGAAAACGCCAAGGCCGCCGGTTTCGACGGCGTG 510 xenA GTCAAGCAGGACTTCGTCGATGCCGCCCGCCGTGCGCGTGATGCCGGCTTCGAGTGGATA 525 ** * * * * ** * ** ** ** ** *

Enterobacter GAACTGCACTCCGCGCACGGATACCTCCTGCACCAGTTCCTTTCACCGTCGTCCAACCAC 597 Escherichia GAGCTCCACTCTGCTCACGGTTATTTGCTGCATCAGTTCCTTTCTCCTTCTTCAAACCAT 597 E.cloacae GAGCTTCACTCTGCGCACGGTTACCTGCTGCATCAGTTCCTGTCCCCGTCTTCCAACCAG 597 nerA GAGATCCATGGCGCCAACGGCTATCTTCTCGATCAGTTCCTGCGCGCTGACATCAACGAC 585 xenB GAAATCCACGGCGCCAACGGCTACCTGCTGGACCAGTTCCTGCAAAGCAGCACCAACCAG 570 xenA GAACTGCACTTTGCCCATGGCTACCTGGGCCAGAGCTTCTTCTCCGAGCACTCCAACAAG 585 ** * ** ** * ** ** * * *** * *** *

Enterobacter CGCACCGACCAGTACGGCGGTAACGTCGAAAACCGCGCCCGTCTGGTGCTGGAAGTGGTT 657 Escherichia CGTACCGATCAGTACGGCGGCAGCGTGGAAAATCGCGCACGTCTGGTACTGGAAGTGGTC 657 E.cloacae CGTACCGACCAGTACGGCGGCAGCGTTGAAAACCGCGCGCGTCTGGTGCTTGAAGTGGTG 657 nerA CGCACCGACCAGTATGGCGGCTCGATCGAGAACCGCGCTCGCTTCCTCTTCGAGGTAGTC 645 xenB CGTACCGACCAGTACGGCGGCTCCCTGGAAAACCGTGCCCGCCTGCTGCTGGAAGTCACC 630 xenA CGCACCGATGCCTACGGTGGCAGCTTCGACAACCGCAGCCGCTTCCTGCTGGAGACACTG 645 ** ***** ** ** ** * ** ** ** ** * * * **

Enterobacter GATGCCGTTTGTCAGGAGTGGAGCCCGGACCGTATTGGTATTCGCGTCTCCCCAATCGGT 717 Escherichia GATGCCGGGATTGAAGAATGGGGTGCCGATCGCATTGGCATTCGCGTTTCGCCAATCGGT 717 E.cloacae GATGCTGTCTGTAATGAGTGGAGCGCAGACCGCATTGGTATTCGTGTCTCCCCGATCGGT 717 nerA GACGCCGTGACCAAGGAAATCGGCGCCGGCCGCACCGCTATCCGCATCTCGCCGGTA--- 702 xenB GACGCGGCCATCGAGATCTGGGGTGCCGGCCGGGTAGGCGTGCACCTGGCACCACGTGCC 690 xenA GCCGCCGTGCGTGAAGTGTG-GCCGGAGAACCTGCCGCTGACCGCGCGCT-----TTGGT 699 * ** * * * * * *

Enterobacter ACGTTCCAGAACGTCGACAACGGTCCGAACGAAGAAGCGGACGCGC-TGTATCTGATTGA 776 Escherichia ACTTTCCAGAACACGGATAACGGCCCGAATGAAGAAGCCGATGCAC-TGTATCTGATTGA 776 E.cloacae ACTTTCCAGAACGTCGACAACGGTCCGAACGAAGAAGCAGACGCGC-TGTATCTGATTGA 776 nerA ACGCCGGCAAACGATGCCAGTGATCCGCA-GCCGCAGCCGCTTTTCACCTATGTCATCGA 761 xenB GACTCCCATGACATGGGTGATG-CCAACCTGGCGGA---GACCTTCACCTACGTCGCTCG 746 xenA GTGTTGGAGTACGATGGCCGCGATGAGCAGACCCTGGAAGAGTCGA-TCGAACTGGCCCG 758 ** * * * * *

Enterobacter AGAACTGGCGAAACGCGGTATCGCGTATCTGCACATGTCCGAGCCGGACTGGGCCGGTGG 836 Escherichia ACAACTGGGTAAACGCGGCATTGCTTATCTGCATATGTCAGAACCAGATTGGGCGGGGGG 836 E.cloacae AGAGCTGGCGAAACGCGGTATCGCCTATCTGCACATGTCCGAGACGGACTTGGCAGGCGG 836 nerA AGGCCTGGCAAAATACGATCTCGCCTATATCCACGTCATCGAA---GGCGCAACCGGTGG 818 xenB GGAGCTGGGCAAACGCGGTATCGCCTTTATCTGCTCCCGCGAGAAAGAAGGCGCCGACAG 806 xenA CCGCTTCAAGGCCGGTGGGCTCGACCTGCTGAGCGTGAGTGTC---GGCTTCACCATTCC 815 * * * * * * * *

Enterobacter TCAGCCTTACTCTGAGGCTTTCCGCCAGAAAGTGC-GCGAGC-GTTTCCACGG-GGTGAT 893 Escherichia TGAACCGTATACTGATGCGTTCCGCGAAAAAGTAC-GCGCCC-GTTTCCACGG-TCCGAT 893 E.cloacae CAAGCCTTACAGTGAAGCCTTCCGTCAGAAAGTGC-GCGAGC-GCTTCCACGG-CGTGAT 893 nerA CGCGCGCGACTTCCTGCCCTTCGACTATGACGCGCTGCACGCTGCTTACAAGGCTGCCGG 878 xenB CCTG------GGCCCGCAACTCAAAGAAGCCTTCGGCGGCCCCTAC------AT 848 xenA CGACACCAACATC---CCCTGGGGTCCAGCGTTCATGGGGCCGATTGCCGAGCGCGTGCG 872 * *

Enterobacter CATCGGTGC-----GGGGGCCTA------TACGCCTGAGAA--AGCCGAAGATCTGAT 938 Escherichia TATCGGCGC-----AGGTGCATA------CACAGTAGAAAA--AGCTGAAACGCTGAT 938 E.cloacae TATCGGGGC-----GGGTGCGTA------TACGGCAGAAAA--AGCCGAGGATTTGAT 938 nerA CGGCAAGGCCGGCTGGATGCTTAACAACGGCTACGACCGCGAGTTGGCCGAGGAAGCCAT 938 xenB CGCCAACGA------ACGCTT------CACCAAGGACAG--CGCCAACGCCTGGCT 890 xenA CCGCGAAGCGA---AGCTGCCCG----TGACGTCGGCGTGGGGCTTTGGTACGCCGCAGT 925 * * ** * * *

Enterobacter TAATAAAGGGTTGATCG-ACGCCGTGGCCTTTGGTCGTGATTACATTGCCAACCCGGACC 997 Escherichia CGGCAAAGGGTTAATTG-ATGCGGTGGCATTTGGTCGTGACTGGATTGCGAACCCGGATC 997 E.cloacae CGGTAAAGGCCTGATCG-ACGCCGTGGCCTTTGGCCGTGACTACATTGCTAACCCGGATC 997 nerA CGACAGCGGCAGAGCCG-ACGTTGTCGCCTTCGGCAAGCCGTTCATCGGCAATCCAGATC 997 xenB GGCCGCTGGCAAGGCTG-ACGCCGTGGCCTTTGGTGTGCCCTTCATCGCCAACCCGGACC 949 xenA TGGCGGAGGCCGCATTGCAGGCCAACCAGCTGGATCTGGTTTCGGTAGGGCGCGCGCACC 985 ** * * * * * * * * * * *

Enterobacter TGGTTGCCCGTCTGCAGCAGAAAGCAGCGCTGAACCCACAGCGCCCGGAAAGCTTCTACG 1057 Escherichia TGGTCGCCCGCTTGCAGCGCAAAGCTGAGCTTAACCCACAGCGTGCCGAAAGTTTCTACG 1057 E.cloacae TGGTTGCCCGTTTGCAGAAAAAAGCCGAACTGAACCCGCAGCGTCCTGAAAGCTTCTATG 1057 nerA TCGTCCGCCGACTGAAGGAAAACGCCCCGCTCAATGGCCTCGACCAGGCAACGCTCTATG 1057 xenB TGCCAGCCCGCCTGAAGGCCGATGCGCCGTTGAACGAAGCGCACCCGGAAACCTTCTACG 1009 xenA TGGCCGACCCGCACTGGGCTTACTTTGCTGCCAAGGAGCTGGGGGTGGAAAAAGCCTCCT 1045 * ** * * ** * ** **

169

Enterobacter GCGGCGGCGCGGAAGGCTATACCGACTACCCTACGCTGTAA------1098 Escherichia GTGGCGGCGCGGAAGGCTATACCGATTACCCGACGTTGTAA------1098 E.cloacae GCGGCGGCGCGGAAGGTTATACCGACTACCCTTCACTGTAA------1098 nerA GCGGCGGCGGAAAGGGCTACGCAGACTATCCGACGCTGGACGAAGTAGCCTAG 1110 xenB GCAAGGGCCCGGTCGGCTATATCGACTACCCGACTCTGTAA------1050 xenA GGACCTTGCCGGCGCCTTATGCACACTGGCTCGAGCGTTATCGCTGA------1092 * ** * * * *

170

APPENDIX III

Resting cell activity of the six GTN and PETN-degrading isolates

Isolate Media Substrate U/OD

AS M9 GTN 0.000220 AX M9 GTN 0.000540 KS M9 GTN 0.000760

ES M9 GTN 0.000600 MS M9 GTN 0.000640 PS M9 GTN 0.000640 AS M9 PETN 0.000060 AX M9 PETN 0.000080 KS M9 PETN 0.000080 ES M9 PETN 0.000100 MS M9 PETN 0.000080 PS M9 PETN 0.000100

AS LB GTN -0.004000 AX LB GTN 0.006000 KS LB GTN -0.003300 ES LB GTN 0.000400 MS LB GTN -0.000900 PS LB GTN 0.003500 AS LB PETN 0.001700 AX LB PETN 0.002300 KS LB PETN 0.007000 ES LB PETN 0.006700 MS LB PETN 0.008400 PS LB PETN 0.005700

171

APPENDIX IV

Cofactor preference of the Six GTN/PETN-degrading isolates in cell-free lysate using GTN/ PETN as substrate

M9 media

U/mg Substrate Cofactor Isolate U/mL protein

NADH AS 3.00E-04 1.22E-04 PETN NADH MS 3.00E-04 1.82E-04 NADH AX 3.00E-04 1.74E-04 NADH PS 2.00E-04 1.32E-04 NADH KS 6.00E-04 3.76E-04 NADH ES 1.00E-03 5.94E-04

NADPH AS 8.00E-04 3.25E-04 PETN NADPH MS 5.00E-04 3.04E-04

NADPH AX 5.00E-04 2.90E-04

NADPH PS 3.00E-04 1.97E-04

NADPH KS 7.00E-04 4.39E-04

NADPH ES 6.00E-04 3.57E-04

NADH AS 6.00E-03 2.44E-03 GTN NADH MS 6.00E-04 3.65E-04 NADH AX 5.00E-04 2.90E-04 NADH PS 5.00E-04 3.29E-04 NADH KS 7.00E-05 4.39E-05 NADH ES 5.00E-04 2.97E-04

NADPH AS 8.59E-04 4.86E-03 GTN NADPH MS 1.93E-01 6.38E-02 NADPH AX 1.73E-01 6.05E-02 NADPH PS 2.75E-01 1.02E-01 NADPH KS 4.47E-01 2.11E-01 NADPH ES 3.32E-01 1.69E-01

172

LB Media

U/mg Substrate Cofactor Isolate U/mL protein

PETN NADH AS NT NADH MS NT NADH AX NT NADH PS NT NADH KS NT NADH ES NT

PETN NADPH AS 5.00E-03 1.33E-03 NADPH MS 1.00E-02 2.75E-03 NADPH AX 1.50E-02 4.43E-03 NADPH PS 2.00E-02 7.42E-03 NADPH KS 5.00E-03 1.53E-03 NADPH ES 3.00E-02 7.84E-03

GTN NADH AS 1.00E-02 2.18E-03 NADH MS 5.00E-03 1.07E-03 NADH AX 4.00E-04 8.59E-05 NADH PS 8.00E-04 1.71E-04 NADH KS 1.00E-04 2.12E-05 NADH ES 1.70E-02 3.66E-03

GTN NADPH AS 1.00E-02 2.15E-03 NADPH MS 5.00E-03 1.07E-03 NADPH AX 1.00E-02 2.12E-03 NADPH PS 3.00E-03 1.90E-03 NADPH KS 1.00E-01 2.15E-02 NADPH ES 3.50E-02 7.52E-03

173