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Degradation of branched chain aliphatic and aromatic petroleum hydrocarbons by microorganisms [Elektronische Ressource] / vorgelegt von Le Thi Nhi Cong

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149 pages
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.
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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
3.2.4.9.2 Biotransformation of ß–methylcinnamic acid........................75
3.2.4.9.3 Biotransformation of 2-phenylpropionic acid76
3.2.4.9.4 Biotransformation of acetophenone .......................................................................77
3.2.4.9.5 Biotransformation of benzoic acid.........79
3.2.4.10 Biotransformation of sec-octylbenzene by T. mucoides SBUG-Y 801 .........80
4 DISCUSSION .......................................................................................... 81
CONTENTS
4.1 Pristane degradation ..............................................................................81
4.2 Degradation of aromatic compounds....................86
4.2.1. Biotransformation of iso-pentylbenzene........................................................87
4.2.2. Biotransformation of sec-octylbenzene..........................................................92
5 REFERENCES ...................................................................................... 101
6 APPENDIX ............................................................................................ 109
SUMMARY
Summary
The overall aim of the work was to investigate the ability of several Gram-positive
bacteria including Mycocbacterium neoaurum SBUG 109, Nocardia cyriacigeorgica
SBUG 1472 and Rhodococcus ruber SBUG 82 and the yeast Trichosporon mucoides
SBUG-Y 801 to degrade and transform branched chain hydrocarbons which occur in
petroleum and its fraction products such as gasoline or gas oil and which are known as
important and recalcitrant environmental pollutants. Pristane, iso-pentylbenzene and sec-
octylbenzene were used in this work as model compounds. These compounds represent
significant groups of petroleum constituents (branched chain alkanes and aromatic
hydrocarbons).
Three bacteria and the yeast T. mucoides SBUG-Y 801 were selected in a screen of
16 hydrocarbon-utilizing strains in the SBUG collection and from 21 isolated hydrocarbon-
utilizing strains from oil-contaminated habitats of Saudi Arabian Desert and of Vietnam.
The bacteria were identified in cooperation with DSZM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen) as M. neoaurum SBUG 109, N. cyriacigeorgica
SBUG 1472, R. ruber SBUG 82. These bacterial and yeast strains were shown to possess
high potential for degrading and transforming pristane, iso-pentylbenzene and sec-
octylbenzene.
The intermediates produced by these bacteria during incubation with pristane were
analyzed by GC and GC/MS. The products 4-methyl pentanoic acid; methyl butanedioic
acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid and
2,6,10,14–tetramethyl-pentadecan–3–one were detected in M. neoaurum cultures. In R.
ruber, methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4-
methyl heptanedioic acid; 2,6,10–trimethylundecanoic acid; 3,7-dimethyl decanedioic acid
and 2,6,10,14–tetramethyl–pentadecan–3-one were identified. In N. cyriacigeorgica, 2-
methylpentanedioic acid; 4,8-dimethylnonanedioic acid; 2,6-dimethylheptanedioic acid
and pristanic acid were found.
The detection of 11 intermediates during pristane degradation by the three Gram-
positive bacteria provided sufficient information to elucidate in detail three degradative
pathways of pristane involving mono-, di- and sub-terminal oxidations. The sub-terminal
oxidation by M. neoaurum and R. ruber was demonstrated for the first time. This

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