Pyrene Degradation by Mycobacterium sp. KMS: Biochemical Pathway, Enzymatic Mechanisms, and Humic Acid Effect

Yanna Liang

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Pyrene Degradation by Mycobacterium sp. KMS: Biochemical Pathway, Enzymatic Mechanisms, and Humic Acid Effect

Copyright © 2006 Yanna Liang All rights reserved. No part of this book may be reproduced or transmitted in any form or by any

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ISBN-10: 1-59942-717-6

ISBN-13: 978-1-59942-717-1

ii

ABSTRACT

Pyrene Degradation by Mycobacterium sp. KMS: Biochemical Pathway,

Enzymatic Mechanisms, and Humic Acid Effect

by

Yanna Liang, Doctor of Philosophy

Utah State University, 2006

Major Professor: Dr. Ronald C. Sims Department: Civil and Environmental Engineering

Pyrene, a four-ring polycyclic aromatic hydrocarbon (PAH), was identified as the

chemical that requires the largest land area for soil bioremediation due to the slow rate of

biodegradation at the Libby, Montana, Superfund site. Prepared bed land treatment is the

specific bioremediation technology that is currently employed at this site. Although

bioremediation has been widely accepted for treatment of contaminated soil due to its

low cost, the effective application of bioremediation is often hindered by the lack of

information related to: 1) biochemical pathways, 2) enzymatic mechanisms, and 3)

effects of amendments.

Mycobacterium sp. KMS is a new strain isolated from the land treatment units of

the Libby site and has been found to utilize pyrene as a carbon and energy source. The

genome of Mycobacterium sp. KMS was sequenced by Joint Genome Institute (JGI) and

is publically available in the NCBI database.

iii This dissertation is comprised of seven chapters. Chapter I provides information

concerning PAH characteristics, the Libby Superfund site, accelerated bioremediation

approaches, and the hypotheses for this dissertation. Chapter 2 addresses the pyrene

degradation pathway used by Mycobacterium sp. KMS based on isolating and identifying

pyrene degradation intermediates. Chapter 3 describes the enzymatic mechanism of

pyrene degradation by Mycobacterium sp. KMS.

Chapter 4 presents the effect of Elliott soil HA (ESHA) amendment on pyrene

solubility in soil slurry systems and pyrene mineralization in unsaturated soil microcosms.

Chapter 5 describes the overall effect of ESHA amendment on pyrene distribution in a

soil slurry system.

Chapter 6 addresses the engineering significance of this study and future research

recommendations. Chapter 7 summarizes the dissertation. The main theme of this

dissertation is to provide the basic scientific information and knowledge for better

understanding, better control, and improvement of the bioremediation process at PAH-

contaminated sites.

(182 pages)

iv

ACKNOWLEDGMENTS

Sincere appreciation is extended to my major advisor, Dr. Ronald C. Sims. Thank

you for your support, patience, understanding, and inspiration through my Ph. D studies.

You have touched my life in many areas and you will continue to do so.

I am very appreciative of the help given to me by my committee members, Dr. Dale

R. Gardner, Dr. Charles D. Miller, Dr. Bart C. Weimer, Dr. Darwin L. Sorensen, and Prof.

Joan McLean. Thank you for being available, providing direction, technical expertise,

and support.

I am also indebted to Dr. Anne J. Anderson. Thank you for providing insight,

technical guidance, encouragement, and inspiration to me. I will always remember the

long talks that we had.

I would like to thank others involved in this research, including Dr. Karl Nieman,

Brian Pettee, Dr. Dong Chen, Dr. David W. Britt, Mark Greenwood, Greg Sullivan,

Robert Child, Dr. Michael Peterson, Frank Olsen, and Dr. Piotr Dobrowolski. Thank you

for giving me your advice, support, and help. Help and support from CEE, BIE

department offices and the business office at UWRL are greatly acknowledged.

Finally, I would like to thank my husband, Jianming. Thank you for the

understanding, support, and encouragement on everything that I want to do. Thank you

for always standing by me. Thanks are also extended to my wonderful daughter, Judy.

Thank you for being such an understanding, kind, and nice girl. Thank you for coming to

the lab with me on weekends and holidays.

Yanna Liang

vCONTENTS

Page

ABSTRACT ................................................................................................................ ii ACKNOWLEDGMENTS ......................................................................................... iv LIST OF TABLES ...................................................................................................... ix LIST OF FIGURES ................................................................................................... xi

CHAPTER

1. INTRODUCTION .................................................................................1

Sources of PAHs ........................................................................1 Toxicity of PAHs .......................................................................1 Biodegradation...........................................................................2 Accelerated biodegradation .......................................................4 Hypotheses.................................................................................5 References..................................................................................6

2. IDENTIFICATION OF A NOVEL PYRENE DEGRADATION METABOLITE: PYRENE-4,5-DIONE BY MYCOBACTERIUM SP. KMS ....................................................8

Abstract ....................................................................................8 Introduction ...............................................................................8 Materials and Methods.............................................................10

Chemicals.....................................................................10 Bacteria and growth condition .....................................11 Pyrene mineralization in liquid culture........................12 Isolation of pyrene metabolites in small culture ..........12 Isolation of pyrene and pyrene-4,5-dione intermediates in large culture....................................13 Identification of pyrene and pyrene-4,5-dione metabolites....................................14

Results......................................................................................16 Pyrene mineralization in liquid culture..........................16

vi Identification of pyrene degradation intermediates.......17 Identification of pyrene-4,5-dione culture metabolites .....................................................19 Discussion................................................................................20 References................................................................................24

3. PROTEOMIC PROFILE OF MYCOBACTERIUM SP. KMS INDUCED BY PAHS USING 2-D SDS PAGE .................................35

Abstract ...................................................................................35 Introduction .............................................................................36 Materials and Methods ............................................................39

Chemicals ....................................................................39 Pseudo gel creation ......................................................40 Culture setup ...............................................................40 Cell pellet preparation .................................................41 Cell lysis and proteome extraction...............................41 Isoelectric focusing (IEF).............................................42 2-D PAGE....................................................................43 Gel image analysis............... ........................................44 Protein identification by mass spectrometry................45

Results......................................................................................46 Discussion................................................................................51 References................................................................................58

4. HA EFFECT ON PYRENE DEGRADATION: FINDING OF AN OPTIMAL RANGE FOR PYRENE SOLUBILLITY AND MINERALIZATION ENHANCEMENT..................................73

Abstract ....................................................................................73 Introduction..............................................................................74 Materials and Methods ............................................................76

Chemicals.....................................................................76 Bacteria and growth condition .....................................77 Soil ..............................................................................77 Surfactant effect ...........................................................78 ESHA effect on pyrene aqueous solubility..................78 ESHA effect on pyrene mineralization in soil microcosms.....................................................79 ESHA effect on pyrene mineralization in microcosms

vii inoculated with Mycobacterium sp. KMS.................80 Statistical analysis........................................................80

Results .....................................................................................81 Surfactant effect ...........................................................81 ESHA effect on pyrene aqueous solubility..................81 ESHA effect on pyrene mineralization by indigenous microorganisms in soil microcosms .......82 Effect on pyrene mineralization with inoculation of Mycobacterium sp. KMS to soil..........................83 Discussion................................................................................83 Conclusion ...............................................................................88 References ...............................................................................89

5. PYRENE FATE AFFECTED BY HUMIC ACID AMENDMENT IN SOIL SLURRY SYSTEMS .................................98 Abstract ...................................................................................98 Introduction..............................................................................98 Materials and Methods.......................................................... 101

Chemicals...................................................................101 Soil .............................................................................101 HA effect on pyrene mass balance.............................102 Statistical analysis......................................................104

Results................................................................................... 104 Discussion............................................................................. .107 References............................................................................. 111 6. ENGINEERING SIGNIFICANCE AND FUTURE RESEARCH RECOMMENDATIONS ........................... 123

Pyrene degradation pathway study ........................................123 Enzymatic mechanism of pyrene degradation .......................125 HA effects on PAH biodegradation .......................................127 Recommendation for Libby site.............................................131 References .............................................................................133

7. CONCLUSION...................................................................................137 APPENDICES ...............................................................................................140 Appendix A: Example of protein identification (P7) by LC-MS-MS

viii analysis and by Mascot search.....................................141 Appendix B: Supporting data for Chapter 4.................................. ....149 Appendix C: Supporting data for Chapter 5...................................... 163 CURRICULUM VITAE................................................................................171

ixLIST OF TABLES

Table Page 1.1 Processes and sources producing or containing PAHs ....................................1 2.1 Bacterial pyrene degradation intermediates identified to date.......................29 3.1 Spot and protein number comparison between two lysis buffers ..................63 3.2 Mascot search results of individual spot with only significant hits reported .........................................................................64 3.3 Identification of proteins up-regulated or newly synthesized due to PAH treatment ......................................................72 A1 Protein summary ..........................................................................................142 A2 Protein description .......................................................................................143 A3 Peptide identification ...................................................................................144 A4 Mascot search results ...................................................................................146 A5 Peptide summary report ...............................................................................147 B1 Surface tension measurements of ESHA at pH 7.0......................................150 B2 Surface tension measurements of ESHA at pH 11.8....................................151 B3 Supporting data for pyrene solubility study .................................................152 B4 Supporting data for pyrene mineralization by indigenous soil microorganisms ..................................................................154 B5 Supporting data for pyrene mineralization with the inoculation of Mycobacterium sp. KMS.........................................159 C1 Measurements aqueous phase pyrene fraction in treatments and control over time .............................................................164 C2 Measurements of mineralized pyrene as 14CO2 in treatments and control over time .............................................................166

x C3 Measurements of solvent extractable pyrene fraction in treatment s and control over time ............................................................167 C4 Measurements of bound pyrene fraction in treatments and control over time..................................................................169

xiLIST OF FIGURES

Figure Page 1.1 Mechanisms of PAH carcinogenesis..................................................................3 2.1 Percentage of 14C pyrene mineralization with time in BSM+ and in DDW by Mycobacterium sp. KMS.......................................................30 2.2 HPLC chromatogram showing UV absorbance at 254 nm of solid-phase extractable pyrene residue in Mycobacterium sp. KMS culture after one week exposure to pyrene...................................... .31 2.3 Identification of peak II in pyrene culture. ......................................................32 2.4 HPLC chromatograms of culture with or without pyrene-4,5-dione addition ................................................................................33

2.5 Proposed pyrene degradation pathway by Mycobacterium KMS ...................34

3.1 Proposed metabolic pathway for the biodegradation of pyrene and phenanthrene in Mycobacterium species.......................................65 3.2 Pseudo gels created by calculated pI and Mr based on amino acid sequences of the enzymes listed above .........................................66 3.3 Graphic demonstration of sample analysis and gel image analysis strategy... 67 3.4 2-D PAGE images of samples treated by lysis buffer 1 with a pI range of 4-7.......................................................................................68 3.5 2-D PAGE images of samples treated by lysis buffer 2 at pI range of 4-7 .............................................................................................69 4.1 Average (n = 3) surface tension measurements for ESHA concentrations at a solution pH 7.0. ..........................................................................................93 4.2 Average (n = 3) surface tension decreases with increasing ESHA concentration at pH11.8.........................................................................94 4.3 14C pyrene fraction in aqueous phase at different concentrations of ESHA and at different time..................................................95

xii4.4 Cumulative percentages of 14C pyrene mineralization by indigenous microorganisms at different ESHA concentrations over time .........................96 4.5 Cumulative percentages of 14C pyrene mineralization with the inoculation of Mycobacterium sp. KMS at different ESHA concentrations over time...........97 5.1 The distribution of 14C pyrene in soil slurry phase with time for treatment with standard Elliott soil humic acid (ESHA) amendment of 1875 µg ESHA/g soil slurry...................................................116 5.2 14C pyrene in aqueous phase changing with time for the three treatments amended with standard Elliott soil humic acid (ESHA) and the control with no ESHA addition................................................................117 5.3 During the first 24 hours, percentages of 14C pyrene in aqueous phase changing with time for the three treatments amended with standard Elliott soil humic acid (ESHA) and the control with no ESHA addition .................118 5.4 Percentages of 14C pyrene as mineralized CO2 changing with time for the three treatments amended with standard Elliott soil humic acid (ESHA) and the control with no ESHA addition............................................119 5.5 Percentages of 14C pyrene as solvent extractable (SER) in the soil matrix changing with time for the three treatments amended with standard Elliott

soil humic acid (ESHA) and the control with no ESHA addition .................120 5.6 Percentages of 14C pyrene as bound residue (BR) changing with time for the three treatments amended with standard Elliott soil humic acid (ESHA) and the control with no ESHA addition............................................121 5.7 Percentages of 14C pyrene as bound residue (BR) changing with time for the three treatments amended with standard Elliott soil humic acid (ESHA) and the control with no ESHA addition by day 1 ............................122 A1 Peptide fragmentation ....................................................................................145 B1 Instrument record for surface tension measurements of ESHA at pH 11.8 ............................................................................................151

1CHAPTER 1

INTRODUCTION

SOURCES OF PAHs Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds

composed of two or more fused benzene rings in linear, angular, and cluster

arrangements. PAHs are major constituents of crude oil, creosote and coal tar. They have

been detected in air, water, soil, and food and are ubiquitous contaminants in nature.

Processes and sources that can produce or contain PAHs are listed in Table 1.

TOXICITY OF PAHs

Research on PAH carcinogensis began two centuries ago when physician John Hill

reported the link between excessive use of tobacco snuff and nasal cancer in 1761 (2). In

1775, Percival Pott related chimney sweep’s scrotal skin cancer with exposure to soot. In

1915, Yamigiwa and Ickikawa reported that tumors were formed after repeated

application of coal tar on the ears of rabbits. From 1930 to 1955, Kennaway, Hieger,

Cook, and Hewett established that the carcinogenic fraction of coal tar contained PAHs.

In the 1970s, James and Elizabeth Miller (8) showed that many chemicals require

metabolic activation to express toxicity. It is now well known that PAHs must be

metabolically activated first to elicit their latent mutagenic, genotoxic, and carcinogenic

properties (3).

PAH carcinogenesis has recently been reviewed and related to at least four

mechanisms (4) (Fig. 1): 1) the dihydrodiol epoxide mechanism, which involves

2TABLE 1.1. Processes and sources producing or containing PAHs (3)

Natural oil seeps Combustion of fossil fuels Refinery and oil storage waste Tobacco and cigarette smoke Accidental spills from oil tankers and other ships Forest and prairie fires Municipal and urban wastewater discharge runoff Rural and urban sewage sludge River-borne pollution Refuse and waste incineration Atmospheric fallout of fly ash particulates Coal gasification and liquefaction processes Petrochemical industrial effluents Creosote and other wood preservative wastes Coal tar and other coal processing wastes Chronic input associated with boating activitites Automobile engine exhausts

microsomal cytochrome P450 enzymes to activate PAHs to reactive epoxide and diol-

epoxide intermediates that form covalent adducts with DNA, perhaps resulting in

mutations that lead to tumorigenesis; 2) the radical-cation mechanism, which involves

one-electron oxidation to generate radical-cation intermediates that may attack DNA,

resulting in depurination; 3) the quinone mechanism, which involves enzymatic

dehydrogenation of dihydrodiol metabolites to yield quinone intermediates that may

either combine directly with DNA or enter into a redox cycle with oxygen to generate

reactive oxygen species capable of attacking DNA; and 4) the benzylic oxidation

mechanism, which entails formation of benzylic alcohols that are converted by

sulfotransferase enzymes to reactive sulfate esters that may attack DNA.

BIODEGRADATION

The environmental fate of PAHs is of great concern since they are the largest class

of chemical carcinogens known today (1). Engineering of biodegradation, the biological

3

H

HO

1. Diol-epoxide mechanism

H2OH

HOH

OH

P450Epoxidehydrolase

trans-Dihydrodiol

H

HOH

OH

O

PAH

PAH-DNA adducts

DNADihydrodiol epoxide

PAH-DNA adducts

EpoxideDNA

2. Radical-cation mechanism

P450or peroxidase

[O] DNA UnstablePAH-DNAadducts

Depurinationof DNA

3. Quinone mechanism

H

HOH

OH

trans-Dihydrodiol

Dihydrodioldehydrogenase

OHOH

OHO+

OO

DNA

Hydroquinone Semiquinone radical PAH-quinone

PAH quinone-DNA adducts-H2

Reactive oxygen species (HO-)

Oxidized DNA adducts

DNA

4. Benzylic oxidation mechanism

P450

O2

CH2CH3 CHR-OH CHR-OSO3H

Alkyl PAH Hydroxyalkyl PAHR CH3

Alkyl PAH sulfate

PAH-DNA adductsDNASulfotransferase

Cation radical unstable

P450

O2O2

FIG. 1.1. Mechanisms of PAH carcinogenesis (4).

4catalyzed reduction in complexity of chemicals (11), was developed in the 1970’s and

has been widely accepted as a cost effective tool for site decontamination.

One site currently utilizing biodegradation is the Champion International Superfund

Site in Libby, MT. This site experienced heavy contamination of PAHs and

pentachlorophenol (PCP) from wood treatment operations from 1946-1969. Prepared bed

land treatment, a process of stimulating soil microorganisms to degrade contaminants,

was initiated in 1989 in two 1-acre, lined, land treatment units and subsequently

expanded to 12 acres of unlined treatment area (10).

Laboratory treatability studies indicated that prepared bed land treatment at the

Libby site was an effective method for reducing contaminant levels and residual toxicity

(6, 10). However, the treatment process affected by the environment was slow. For

pyrene, a four-ring PAH, the half life was beyond one year. In order to increase the

degradation rate, accelerated bioremediation, a bioremediation process accelerated

beyond the normal actions of the naturally occurring microbial community and chemical

and geological conditions, usually by the addition of amendments or specialized microbes,

has been proposed.

ACCELERATED BIOREMEDIAITON

Accelerated biodegradation is achieved by adding either amendments or specialized

microbes. In the case of adding supplemental carbon sources or other nutrients to

stimulate the activity of indigenous or inoculated PAH degrading microbial strains, the

process is categorized as biostimulation (5). In the case of adding microbial strains with

desired degradative capacities, the process is defined as bioaugmentation (5).

5 Mycobacterium sp. KMS was isolated from the vadose zone soil of the Libby

prepared bed land treatment system. It was found to be a Mycobacterium species based

on Gram staining, electron microscopy, and 16S rDNA-sequencing. It has the potential to

degrade pyrene, benzo[a]pyrene, and other PAHs (7). Humic acid (HA), which is

polyfunctional, can act as a binding agent and detoxicant, sobent, and flushing agent,

redox mediator of abiotic and biotic reactions, nutrient carrier, and growth-stimulator (9).

The possibilities of applying Mycobacterium sp. KMS for bioaugmentation and HA for

biostimulation will be discussed in this dissertation. This work adds to the current body

of knowledge involving Mycobacterium species for PAH biodegradation and HA

addition for faster and more complete bioremediation.

HYPOTHESES

The following hypotheses were tested in this research:

Hypothesis 1: The pyrene degradation pathway by Mycobacterium sp. KMS is

different to the pathway used by other bacteria.

Hypothesis 2. Mycobacterium sp. KMS degradation of pyrene and pyrene-4,5-dione

are initiated by certain enzymes induced by the two chemicals.

Hypothesis 3: Standard Elliot soil humic acid (ESHA) increases pyrene’s solubility

and mineralization rate.

Hypothesis 4: Addition of standard ESHA to the Libby soil increases the formation

of bound residues.

Hypotheses 1, 2, 3, and 4 are the subjects of Chapter 2, 3, 4, and 5, respectively.

Specific objectives, methods, and results of each study are presented in the individual

6chapters. These studies provide a better understanding of the chemical and biological

aspects of PAH-degrading Mycobacterium sp. KMS.

REFERENCES

1. Ahn, Y., J. Sanseverino, and G. Sayler. 1999. Analyses of polycyclic aromatic

hydrocarbon-degrading bacteria isolated from contaminated soils. Biodegradation 10:149-157.

2. Cerniglia, C. E. 1984. Microbial metabolism of poycyclic aromatic hydrocarbons.

Adv. Appl. Microbiol. 30:31-71. 3. Cerniglia, C. E. 2003. Recent advances in the biodegradation of polycyclic

aromatic hydrocarbons by Mycobacterium specie, p. 51-73. In V. Sasek (ed.), The utilization of bioremediation to reduce soil contamination: problems and solutions. Kluwer Academic Publishers, Netherlands.

4. Harvey, R. G. 1991. Polycyclic aromatic hydrocarbons: chemistry and

carcinogenicity. Cambridge University Press, Cambridge, MA. 5. Herwijnen, R. V., B. Joffe, A. Ryngaert, M. Hausner, D. Springael, H. A.

Govers, S. Wuertz, and J. R. Parsons. 2006. Effect of bioaugmentation and supplementary carbon sources on degradation of polycyclic aromatic hydrocarbons by a soil-derived culture. FEMS Microbiol. Ecol. 55:122-135.

6. Huling, S. G., D. F. Pope, J. E. Matthews, J. L. Sims, R. C. Sims, and D. L.

Sorensen. 1995. Land treatment and the toxicity response of soil contaminated with wood preserving waste. Remediation J. 5(2):41-56.

7. Miller, C. D., K. Hall, Y. N. Liang, K. Nieman, D. L. Sorensen, B. Issa, A. J.

Anderson, and R. C. Sims. 2004. Isolation and characterization of polycyclic aromatic hydrocarbon-degrading Mycobacterium isolates from soil. Microb. Ecol. 48:230-8.

8. Miller, E., and J. Miller. 1985. Some historical perspectives on the metabolism

of xenobiotic chemicals to reactive electorphiles, p. 3-28. In M. Anders (ed.), Bioactivation of foreign compounds. Academic Press, Orlando, FL.

9. Perminova, I. V., N. Y. Grechishcheva, and V. S. Petrosyan. 1999.

Relationships between structure and binding affinity of humic substances for polycyclic aromatic hydrocarbons: relevance of molecular descriptors. Environ. Sci. Technol. 33:3781-3787.

7 10. USEPA. 1996. Champion International Superfund Site, Libby, Montana:

bioremediation field performance evaluation of the prepared bed land treatment system. EPA-600/R-95/156. United States Environmental Protection Agency.

11. White, J. C., M. Alexander, and J. J. Pignatello. 1999. Enhancing the

bioavailability of organic compounds sequestered in soil and aquifer solids. Environ. Toxicol. Chem. 18:182-187.

8

CHAPTER 2

IDENTIFICATION OF PYRENE-4,5-DIONE AS A NOVEL PYRENE

DEGRADATION METABOLITE BY MYCOBACTERIUM SP. KMS1

ABSTRACT

Mycobacterium sp. KMS, isolated from vadose zone soil at the Champion

International Superfund Site in Libby, Montana, has demonstrated the ability to degrade

pyrene and other PAHs including benzo[a]pyrene. Pyrene degradation pathway

experiments revealed intermediates including: pyrene-4,5-dione, cis-4,5-pyrene-

dihydrodiol, phenanthrene-4,5-dicarboxylic acid, and 4-phenanthoic acid. This is the first

study to report pyrene-4,5-dione as a pyrene degradation intermediate in a Gram-positive

bacterium. Pyrene-4,5-dione, which accumulates as an end product in some Gram-

negative bacterial cultures, can be further utilized and degraded by Mycobacterium sp.

KMS. This study provides new information on pyrene-4,5-dione formation and

degradation by Mycobacterium KMS, which occurs at PAH-contaminated sites and is

involved in soil bioremediation processes.

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) have been detected in air, water, soil,

and food and are ubiquitous contaminants in nature (24). High-molecular-weight (HMW)

PAHs are hydrophobic, very stable, and relatively inert molecules in the environment

(34). However, their transformation products are bioactive and have been shown to be

both acutely and chronically toxic (1, 5, 7, 10, 11, 17, 31, 39). The mechanisms of PAH

1Coauthored by Yanna Liang, Dale R.Gardner, Charles D. Miller, Anne J. Anderson, and Ronald C. Sims.

9

carcinogenesis have recently been reviewed and include, 1) the dihydrodiol epoxide

mechanism, 2) the radical-cation mechanism, 3) the quinone mechanism, and 4) the

benzylic oxidation mechanism (15, 16). Therefore, it is important to understand how

PAHs are degraded, what kinds of products are produced, and whether the metabolites

are toxic.

Pyrene degradation pathways of Mycobacterium species including vanbaalenii

PYR-1, flavescens, RJGII-135, KR2, and AP1 have been studied and are proposed to be

similar (4, 6, 23, 32, 35, 38). Pyrene is first oxidized in the K-region by a dioxygenase to

form cis-4,5-pyrene-dihydrodiol, which is rearomatized to form 4,5-dihydroxy-pyrene by

dihydrodiol dehydrogenase. 4,5-Dihydroxy-pyrene is subsequently cleaved to yield

phenanthrene-4,5-dicarboxylic acid. Following loss of a carboxyl group, 4-phenanthroic

acid is formed and enters into the phenanthrene degradation pathway. Two additional

pathways have been proposed. One proposes that pyrene hydroxylation takes place at the

1, 2 positions, leading to the formation of 4-hydroxy-perinaphthenone, which is a dead-

end product and so far has been only found in Mycobacterium vanbaalenii PYR-1

cultures (4). Another pathway involves the accumulation of 6,6’-dihydroxy-2,2’-

biphenyl-dicarboxylic acid in Mycobacterium sp. AP1 (38).

Pyrene-4,5-dione was identified to be a pyrene degradation intermediate of several

bacteria. First, it was observed as a pyrene metabolite by Sphingomonas yanoikuyae

strain R1 by Kazunga and Aitken (20). Formation of pyrene-4,5-dione probably proceeds

by metabolism of cis-4,5-pyrenedihydrodiol, as strains R1 converted cis-4,5-pyrene-

dihydrodiol to pyrene-4,5-dione essentially stoichiometrically and resulted in pyrene-4,5-

dione accumulation in the culture. Second, Mycobacterium vanbaalenii strain PYR-1

10

formed significant amounts of pyrene-4,5-dione when it was incubated with a high

concentration of cis-4,5-pyrene-dihydrodiol, although it was not reported as an

intermediate when Mycobacterium vanbaalenii strain PYR-1 grew on pyrene (20). Third,

pyrene-4,5-dione was identified to be a pyrene metabolite in the phagemid clone My6-

pBK-CMV containing a dioxygenase gene when it was incubated with pyrene (22).

Mycobacterium sp. KMS was isolated from vadose zone soil of the Champion

International Superfund Site in Libby, Montana and has the ability to degrade pyrene and

other PAHs (29). It was found to have the dioxygenase gene for PAH degradation (13,

29). Due to the toxicity of pyrene-4,5-dione (30) and its possible presence during pyene

degradation in Mycobacterium cultures, it is important to identify this metabolite and

determine its fate during pyrene degradation, as it was observed as an end product in

some Gram-negative cultures and may result in toxicity increase during in-situ soil

bioremediation (20). The objectives of this work reported here were to: 1) determine the

pyrene degradation pathway used by Mycobacterium sp. KMS by isolating and

identifying the metabolites, and 2) determine the capability of Mycobacterium sp. KMS

to degrade pyrene-4,5-dione.

MATERIALS AND METHODS

Chemicals Pyrene (99%) was purchased from Fluka, Switzerland. Cis-4,5-pyrenedihydrodiol

was kindly provided from Dr. Michael Aitken at the University of North Carolina at

Chapel Hill. Pyrenol (1-hydroxypyrene, 99%), phthalic acid (99%) were purchased from

Aldrich Chemical Company, Inc. Radio-labeled [4,5,9,10-14C] pyrene (95% purity,

11

specific activity = 56 mCi/mmol) was purchased from Amersham International

(Burkinghamshire, England). All solvents (methanol, acetonitrile, ethyl acetate) used

were HPLC grade or the equivalent and were purchased from Sigma-Aldrich, St. Louis,

MO. Basal Salt Medium (BSM) and Luria Broth (LB) are the same as described by

Miller (29). Deuterated solvents, methanol-D4 (99.8%) was purchased from Sigma-

Aldrich, St. Louis, MO and dimethyl sulfoxide–D6 (DMSO) was purchased from Acros

Organics, Morris Plains, NJ.

Pyrene-4,5-dione and phenanthrene-4,5-dicarboxylic acid were synthesized based on

Yong and Funk’s procedure (40). Briefly, pyrene was regioselectively oxidized to 4,5-

phenanthrene-dicarboxylic acid by using hydrogen peroxide and tungstic acid. The

carboxylated ion generated with sodium bicarbonate is alkylated with iodomethane in

dimethyl formamide to produce dimethyl 4,5-phenanthrenedicarboxylate. Treatment of

the diester with excess sodium in refluxing tetrahydrofuran resulted in a good yield of

pyrene-4,5-dione. The purity and authenticity of synthesized pyrene-4,5-dione and

phenanthrene-4,5-dicarboxylic acid were determined after analysis by high pressure

liquid chromatography (HPLC) and 1H nuclear magnetic resonance (NMR) spectroscopy.

Bacteria and growth condition Mycobacterium sp. KMS cells were grown in BSM+ (a 9:1 mixture of BSM and LB)

for five days to stationary phase, pelleted and washed twice with sterile distilled water.

The suspension was used as an inoculum for the following experiments.

Pyrene mineralization in liquid culture