Biological and Chemical Degradation of Azo Dyes under Aerobic Conditions , Jack T. Spadaro B.S., Worcester Polytechnic Institute, 1987 A dissertation submitted to the faculty of the Oregon Graduate Institute of Science & Technology in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biochemistry July 1994 The dissertation "Biological and Chemical Degradation of Azo Dyes under Aerobic Conditions" by Jack T. Spadaro has been examined and approved by the following Examination Committee: Dr. V. Renganathan Advisor and Associate Professor Dr. Michael H. Gold Institute Professor Dr. David R. Boone Professor Dr. Paul G. Tratnyek Assistant Professor Dedicated to my parents, Richard and Vera,for their depotion to my upbringing and education, and also to all those in the world who strive to make a positive difuence ACKNOWLEDGMENTS Foremost, I wish to thank my scientific mentors, Dr. V. Renganathan, at the Oregon Graduate Institute, and Dr. William D. Hobey, at Worcester Polytechnic Institute, for their fine and extensive efforts in my training. From OGI, I must also thank: Dr. Michael Gold, for his great interest in and guidance of my work; Dr. James Pankow, for the use of his cryofocusing GC-MS system; Drs. David Boone and Paul Tratnyek for their advice as members of my thesis committee; Lorne Isabelle and Gerry Boehme, for their good natures and their abilities to keep vital instrumentation functioning; Nancy Christie, for her excellent secretarial and management skills, and for her friendship; Drs. Muralikrishna Chivukula, Hiro Wariishi, and Khadar Valli, for sharing so many laboratory techniques and tricks, as well as for their interest in my work. Also of note are the many fine laboratory colleagues and support personnel that I have spent long hours with at OGI, especially Dr. Wenjun Bao, with whom I had the pleasure of sharing a laboratory for more than 5 years, Ciro DiMeglio, a friend through my days at WPI and OGI, and Bruce Godfrey. There have been many teachers throughout my life, especially in school and Scouting, whose understanding, support, and wisdom have meant a great deal to me. They include Mr. Reynard Bums, Mr. Theodore Scalzo, and Mr. David Kling. On the personal front, my sister Nancy, my entire extended family, and many close friends have all contributed greatly to my situation. I also wish to thank Linda Steinle, my companion, for her wonderful support these past four years. TABLE OF CONTENTS List of Tables viii List of Figures ix Abstract xi Chapter One - Introduction I Azo Dyes - General Aspects A. Historical development B. Azo dye synthesis C. Azo dye classes, structures, applications II. Azo Dyes - Environmental and Health Aspects A. Introduction of dyes to the environment 1. Dye manufacture and application 2. Waste dye treatment 3. Azo dye pollution B. Azo dye metabolism 1. Bacterial metabolism a. Metabolism under anaerobic conditions b. Metabolism under aerobic conditions 2. Mammalian azo dye metabolism III. Fungal Degradation of Wood and Pollutants A. The fungus Phanerochaete chrysosporium B. Wood C. Physiology of lignin degradation D. Fungal degradation of pollutants TV. Peroxidases A. Peroxidase history and the catalytic cycle B. Peroxidases and lignin degradation C. Peroxidases and pollutant degradation V. Chemical Oxidation A. Advanced oxidation processes B. Fenton chemistry C. Mechanisms of chemical oxidation D. Pollutant degradations VI. Thesis Outline VII. References Chapter Two - Degradation of azo Dyes by the Lignin-degrading Fungus Phanerochaete chrysosporium I. Introduction II. Materials and Methods III. Results ZV. Discussion V. References Chapter Three - Peroxidase-catalyzed Oxidation of Azo Dyes: Mechanism of Disperse Yellow 3 Degradation I. Introduction 11. Materials and Methods III. Results ZV. Discussion V. References Chapter Four - Peroxidase-catalyzed Oxidation of Azo Dyes with Phenylazo Substitutions Generates Benzene I. Introduction II. Materials and Methods DI. Results IV. Discussion V. References Chapter Five - Degradation of Azo Dyes by Hydroxyl Radicals: Evidence for Benzene Generation I. Introduction 11. Materials and Methods m. Results IV. Discussion V. References Chapter Six - Conclusion I. Introduction TI. Fungal Degradation of Azo Dyes III. Azo Dye Degradation by Peroxidases A. Decolorization studies B. Proposed mechanisms IV. Azo Dye Degradation by Chemical Oxidation V. Future Work VI. References Biographical Sketch vii LIST OF TABLES ms Chapter Two Table I. Mineralization of 14C-labeled azo dyes 51 Table II. Recovery of 14C label 53 Chapter Three Table I. Quantification of NDY3 degradation products 65 Chapter Four Table I. Benzene generated during dye degradation 78 Table II. Benzene and 12NQS produced during degradation of dye I1 under varying conditions 80 Chapter Five Table I. Optimization of Fe(III) and H202 concentrations for dye mineralization 93 Table II. Mineralization of azo dyes by Fe(III)/H202 94 viii LIST OF FIGURES Chapter One Figure 1. Three common synthetic dye structures Figure 2. Common azo dye syntheses Figure 3. Structures for azo dye classes Figure 4a. Aerobic and anaerobic azo dye metabolism Figure 4b. Biological azo dye degradation using anaerobic and aerobic stages in succession Figure 5. Spruce lignin structure Figure 6. HRP and LiP catalytic cycle Figure 7. MnP catalyic cycle Figure 8. Lip oxidation of 1,2,4,5-tetramethoxybenzene Figure 9a. LIP, HRP, and Mn(III) oxidation of benzenesulfonic acids Figure 9b. LiP oxidation of 2,4,6-trichlorophenol Figure 10a. Hydroxylation of benzene by hydroxyl radical Figure lob. Ring-opening of benzene by Fenton reagent Chapter Two Figure 1. Mineralization profiles for Solvent Yellow 7 and Disperse Orange 3 Chapter Three Figure 1. Products identified from dye degradation Figure 2. HPK chromatograms of NDY3 degradation products Figure 3. Mass spectra of deuterated acetanilide products Figure 4. Proposed mechanism for NDY3 degradation Chapter Four Figure 1. Structures of the dyes used in HRP reactions Figure 2. Products from the degradation of dyes I and 11 Figure 3. Proposed mechanism for Solvent Yellow 14 degradation by HRP Chapter Five Figure 1. HPLC analysis of Disperse Yellow 3 and Disperse Orange 3 degradation products 95 Figure 2. HPLC analysis of acid products from 4-phenylazoaniline 97 Figure 3. Volatiles production by four dyes during the first six hours of dye degradation 98,99 Figure 4. GC-MS analysis of volatile products from N,N-dimethyl-4-phenylazoaniline 100 Figure 5. Proposed mechanism for Fe(III)/H202 degradation of azo dyes 102 Chapter Six Figure 1. Structures of several sulfonated azo dyes tested for fungal degradation Figure 2. 14C-labeled sulfonated azo dyes mineralized by P. ch ysosporiurn Figure 3. Pathway proposed for HRP-catalyzed oxidation of Sudan I Figure 4. Products observed for peroxidatic oxidation of several sulfonated azo dyes Figure 5. Two mechanisms proposed for azo cleavage Figure 6. Proposed redox and hydrolysis reactions in enzymatic azo dye oxidations Figure 7. Photocatalytic degradation of two azo dyes ABSTRACT Biological and Chemical Degradation of Azo Dyes under Aerobic Conditions By Jack T. Spadaro Oregon Graduate Institute of Science & Technology, 1994 Dr. V. Renganathan, Thesis Advisor Azo dyes represent >SO% of synthetic industrial dyes. Azo dyes are recalcitrant to aerobic bacterial degradation. Reductive cleavage of the azo linkage under anaerobic conditions yields potentially carcinogenic aromatic amines. This thesis examines aerobic azo dye degradation by the white-rot fungus Phanerochaete ch ysosporium, by peroxidases, and by hydroxyl radicals. P. ch ysosporium, a lignin-degrading basidiomycete, extensively mineralized several hydrophobic azo dyes over a 12day period. All dyes were degraded most extensively in ligninolytic cultures. Hydroxyl, acetamido, nitro, and N-alkylamino substituents enhanced dye degradation. Disperse Yellow 3 (DY3, 2-[4'-acetamidophenylazo]-4-methylphenol),a dye mineralized by P. chysosporium, yielded acetanilide as a major metabolite during fungal degradation in cultures that produce lignin and manganese peroxidases (Lip and MnP). Degradation of DY3 by Lip, Mn(III)- malonate (a MnP mimic), and horseradish peroxidase (HRP) was studied. The major products were acetanilide, 4-methyl-1,2-benzoquinone,and dimerized DY3. A mechanism for DY3 degradation is suggested. Either Mn(III) or the H202-oxidized forms of the peroxidases oxidize the phenolic ring of the dye by two electrons, producing an azo-bearing carbonium ion. Hydrolytic azo cleavage forms the quinone product and an acetamidophenyldiazene intermediate. The acetamidophenyldiazene is oxidized by metal or oxygen to produce an acetamidophenyldiazenyl radical, which cleaves homolytically to acetamidophenyl radical and molecular nitrogen. The acetamidophenyl radical abstracts a hydrogen from the surroundings, yielding acetanilide. Consistent with this mechanism, dyes containing phenylazo substitutions were degraded to quinones and benzenes by HRP. Further support for the mechanism was obtained through deuterium labeling studies. Hydroxyl radicals, produced for 24 h by reaction of ferric nitrate and hydrogen peroxide at pH 2.8, degraded large amounts of hydrophobic azo dyes to CO2 and water-soluble compounds. Products included benzene, formed during degradation of phenylazo-substituted dyes, and aliphatic acids. A mechanism resembling that for the peroxidase-catalyzed degradation of azo dyes is proposed for dye degradation by hydroxyl radical. xii CHAPTER ONE