DEGRADATION OF BRANCHED CHAIN ALIPHATIC AND AROMATIC PETROLEUM HYDROCARBONS BY MICROORGANISMS I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Le Thi Nhi Cong geboren am 18/02/1980 in Thanhhoa, Vietnam Greifswald, September 2008 Dekan: Prof. Dr. Klaus Fesser Mathematisch-Naturwissenschaftlichen Fakultät Ernst-Moritz-Arndt-Universität Greifswald 1. Gutachter: Prof. Dr. Frieder Schauer Institut für Mikrobiologie Ernst-Moritz-Arndt-Universität Greifswald 2. Gutachter: Prof. Dr. Ulrich Klinner Biologie Fakultät IV (Mikrobiologie und Genetiks) Universität Aachen Tag der Promotion: 14 November 2008 Acknowledgements The work described here was carried out between July 2005 and September 2008, in the Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald (EMAU) - Germany in partial fulfilment of the requirements for the Degree of Ph. D. I wish to express my deepest gratitude to my supervisor Prof. Dr. Frieder Schauer for suggesting the topic of this thesis as well as for his professional guidance and constructive criticism. I am also greatly indebted to Dr. Annett Mikolasch, whose worthful advice, excellent suggestions and permanent motivating support were in valuable. Sincere thanks are also due her for her knowledgeable correction of this work and for her encouragement to me in hard times. My sincerest thanks go to Prof. Dr. Robert Jack, Institute for Immunology, for his careful correction in English writing. My thanks also go to Dr. Peter-Hans Klenk, DSMZ - German Collection of Microorganisms and Cell Cultures, for his help in bacterial identification. I wish to express my honest gratitude to Ms. Anne Rheinhard, Ms. Brigite Fricke for providing microorganisms and medium throughout this work and for their kindness. My thanks are also due to Susanne Awe for her help with protocols and her guidance with all experimental techniques as well as her experience in HPLC and GC/MS analyses. For the friendship, helpful comments, professional advice and pleasant working environment, I wish to thank Dr. Rabea Sietmann, Doreen Waldau, Veronica Hahn, Jahn Kabisch and other members of Prof. Schauer´s group. I would like to thank all students working in this group for sharing with me late evening and weekend work. My deepest thanks are also extended to Assoc. Prof. Dr. Lai Thuy Hien, head of Petroleum Microbiology laboratory, Institute of Biotechnology, Vietnam for advice and encouragement to me during the preparation of this work. I gratefully thank Vietnamese Ministry of Education and Training (MOET) and German Academic Exchange Service (DAAD) as well as Overseas Organization Office, Greifswald for financial support. I would like to thank the organizers of the Joint Graduate Education Program (JGEP) for giving me the opportunity to be a Ph. D. student in EMAU Greifswald, Germany. I am grateful to my friends in Germany as well as in Vietnam and other lands for being near me and for encouraging me. I wish to extend my sincere thanks to everybody who helped me and promoted me during this study. Last but not least, I would like to take this opportunity to express my deeply gratitude to my mother and my family for loving me and supporting me endlessly. CONTENTS Contents 1 INTRODUCTION ....................................................................................... 1 1.1 Biodegradation and bio-remediation......................................................1 1.2 Degradation of branched chain alkanes.................................................2 1.3 Degradation of pristane ...........................................................................3 1.4 Degradation of alkylbenzene ...................................................................5 2 MATERIAL AND METHODS .................................................................... 8 2.1 Microorganisms ........................................................................................8 2.2 Culture media ...........................................................................................8 2.3 Culture conditions ..................................................................................11 2.3.1 Strain storage ...................................................................................................11 2.3.2 Determination of biomass................................................................................11 2.3.3 Culture condition of pristane-degrading bacteria ........................................12 2.3.3.1 Selection and isolation of pristane-degrading bacteria ......................................12 2.3.3.2 Toxicity tests......................................................................................................12 2.3.3.3 Growth kinetics and degradation of pristane by bacteria...................................13 2.3.4 Culture conditions of aromatic-degrading microorganisms........................13 2.3.4.1 Preculture ...........................................................................................................13 2.3.4.2 Toxicity tests......................................................................................................14 2.3.4.3 Biotransformation experiments for aromatic compounds..................................14 2.4 Extraction and derivatization methods ................................................15 2.5 Chemical analyze and measurement procedure..................................16 2.6 Chemicals ................................................................................................18 3 RESULTS................................................................................................ 20 3.1 Pristane-degrading microorganisms.....................................................20 3.1.1 Selection of pristane-degrading microorganisms..........................................20 3.1.2 Identification ....................................................................................................22 3.1.3 Pristane degradation........................................................................................25 3.1.3.1 Kinetics of pristane degradation ........................................................................25 3.1.3.2 Detection of possible degradation products.......................................................27 3.2 Transformation of alkyl-substituted aromatics...................................32 3.2.1 Toxicity of aromatic compounds on microorganisms ..................................32 CONTENTS 3.2.2 Screening microorganisms transforming aromatics.....................................35 3.2.3 Biotransformation of iso -pentylbenzene........................................................37 3.2.3.1 Identification of biotransformation metabolites of iso -pentylbenzene by HPLC and GC/MS ...............................................................................................................37 3.2.3.1.1 iso -Pentylbenzene biotransformation metabolites by M. neoaurum 109 ...............37 3.2.3.1.2 iso -Pentylbenzene biotransformation metabolites by N. cyriacigeorgica 1472.....42 3.2.3.1.3 iso -Pentylbenzene biotransformation metabolites by R. ruber SBUG 82..............44 3.2.3.1.4 iso -Pentylbenzene biotransformation metabolites by T. mucoides SBUG-Y 801..46 3.2.3.2 Kinetics of the iso -pentylbenzene transformation metabolites..........................48 3.2.3.3 Transformation of transformation metabolites ..................................................50 3.2.4 Biotransformation of sec -octylbenzene..........................................................52 3.2.4.1 Biotransformation of sec-octylbenzene by N. cyriacigeorgica SBUG 1472.....52 3.2.4.2 Kinetics of the sec-octylbenzene transformation by N.cyriacigeorgica 1472...57 3.2.4.3 Transformation of N. cyriacigeorgica 1472 with intermediate products ..........58 3.2.4.3.1 Biotransformation of 3-phenylbutyric acid ............................................................58 3.2.4.3.2 Biotransformation of ß methylcinnamic acid.........................................................60 3.2.4.3.3 Biotransformation of acetophenone .......................................................................61 3.2.4.4 Biotransformation of sec-octylbenzene by M. neoaurum 109...........................62 3.2.4.5 Kinetics of the sec-octylbenzene transformation by M. neoaurum 109 ............64 3.2.4.6 Transformation of M. neoaurum 109 with intermediate products.....................65 3.2.4.6.1 Biotransformation of 3-phenylbutyric acid ............................................................65 3.2.4.6.2 Biotransformation of ß-methylcinnamic acid.........................................................67 3.2.4.6.3 Biotransformation of 2-phenylpropionic acid ........................................................67 3.2.4.6.4 Biotransformation of acetophenone .......................................................................68 3.2.4.6.5 Biotransformation of benzoic acid .........................................................................68 3.2.4.7 Biotransformation of sec-octylbenzene by R. ruber 82.....................................69 3.2.4.8 Kinetics of the sec-octylbenzene transformation by R. ruber 82.......................72 3.2.4.9 Transformation of R. ruber 82 with intermediate products ...............................73 3.2.4.9.1 Biotransformation of 3–phenylbutyric acid............................................................73