Isolation and Characterization of New Triethylamine and Ethyl Acrylate Degrading Bacteria

136 Pages·2013·2.61 MB·English

Isolation and Characterization of New
Triethylamine and Ethyl Acrylate
Degrading Bacteria

VOM PROMOTIONSAUSSCHUSS DER
TECHNISCHEN UNIVERSITÄT HAMBURG-HARBURG
ZUR ERLANGUNG DES AKADEMISCHEN GRADES
DOKTOR(IN) DER NATURWISSENSCHAFTEN (DR. RER. NAT.)
GENEHMIGTE DISSERTATION

VON

Rita Issa Numeir Alayoub
aus Ramallah

2012
Vorsitzender des Prüfungsausschusses: Prof. Dr.-Ing. Wolfgang Calmano
1. Gutachter: Prof. Dr. rer. nat. Rudolf Müller
2. Gutachter: Prof. Dr. Dr. h.c. Garabed Antranikian

Tag der mündlichen Prüfung: 27.01.2012
In Loving Memory of My Father
Acknowledgements
There are many persons and institutions that I would like to thank for their support during my
Ph.D. studies. I would like to begin with the KAAD (Katholischer Akademischer Ausländer
Dienst) for awarding me the scholarship to carry out my doctoral studies at the Hamburg
University of Technology (TUHH) and for giving me the chance to come to Germany and
experience a new life.
I would like to express my deepest gratitude to Professor Rudolf Müller for his patient
guidance, thorough advice, wholehearted support and for giving me the opportunity to carry
out this research work at his institute at the Hamburg University of Technology. I would also
like to acknowledge the support of Professor Volker Kasche, Dr. Boris Galunsky, Professor
Andreas Liese as well as my colleagues at the Department of Technical Biocatalysis. Special
thanks are also due to Dr. Sugima Rappert for her continuous help and advice in the
laboratorial work. I also wish to thank Maren Breuer, Uta Naefken and Dip.-Ing. Gerd
Schietke for providing expert technical assistance, especially with the chronically problematic
equipment. I am also grateful to Gisela Görk for assisting me with the various bureaucratic
formalities at the University and for supporting me in the process of learning the German
language.
I deeply thank Professor Wolfgang Calmano for accepting to chair the doctoral examination
committee and Professor Garabed Antranikian for reviewing the manuscript and for being
part of the examination committee.
My sincere appreciation goes to the Department of Biomechanics at the Hamburg University
of Technology under the chairmanship of Professor Michael Morlock for the exceptionally
friendly working environment as well as for the additional support throughout the duration of
this work. I especially thank Dr. Anne Gebert de Uhlenbrock and Dr. Nick Bishop for the
many helpful tips and fruitful discussions. I also thank Dip.-Ing. Matthias Vollmer for the
indispensable technical support.
I could have not finished this work without the support of my friends in Hamburg, and those
living somewhere else but keeping in touch with me during the last years. Special thanks go
to Professor Hans Bienlein and his wife Melanie, Dr. George Zimmerer, his wife Hannelore,
their daughter Bettina, her husband Marcus and their daughters Johanna and Marlene.
Warm thanks go to my closest friends Cesar Lazo, Athina and Maria Pantoula, Corinna
Wellige, Georgios Papantoniou, Davide Moncalvo, Urs Lübbert, Manfred Glaser and
Christine Völkel.
For the spiritual support and guidance, I would like to express my deep appreciation to
Professor Said Zeedani from Palestine.
I am highly indebted to my beloved father, Issa Numeir Ayoub, who passed away on 25
September 2009, to my Mom, Farha Abbasi Ayoub, to my brothers, Marwan, Munther and
Mazen, to my sister Rima, to my nieces Rawan and Batoul and to my nephew Issa for their
affection, understanding and support throughout the years of my Ph.D. studies in Germany.
Table of Contents
CHAPTER 1 INTRODUCTION ..................................................................... 1
1.1 The revolution of biotechnology ................................................................................ 1
1.2 Odour and odorous compounds ............................................................................... 2
1.2.1 Definitions ........................................................................................................... 2
1.2.2 Sources .............................................................................................................. 3
1.2.3 Food processing ................................................................................................. 3
1.2.4 Volatile organic compounds ................................................................................ 4
1.2.5 Exposure to VOCs .............................................................................................. 4
1.2.6 Odour treatment ................................................................................................. 5
1.2.7 Analysis technique .............................................................................................. 6
1.3 Ethyl acrylate ........................................................................................................... 6
1.3.1 Properties ........................................................................................................... 6
1.3.2 Occurrence ......................................................................................................... 7
1.3.3 Applications ........................................................................................................ 7
1.3.4 Preparation ......................................................................................................... 7
1.3.5 Exposure ............................................................................................................ 8
1.3.6 Toxicity ............................................................................................................. 10
1.3.7 Metabolism ....................................................................................................... 11
1.3.8 Sampling and analysis ...................................................................................... 11
1.4 Amines ................................................................................................................... 11
1.4.1 Definition .......................................................................................................... 11
1.4.2 Preparation ....................................................................................................... 12
1.4.3 Applications ...................................................................................................... 12
1.5 Triethylamine ......................................................................................................... 13
1.5.1 Properties ......................................................................................................... 13
1.5.2 Applications ...................................................................................................... 14
1.5.3 Exposure .......................................................................................................... 15
1.5.4 Metabolism ....................................................................................................... 18
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1.6 Degradation of environmental pollutants ................................................................ 18
1.6.1 Biodegradation of amines ................................................................................. 20
1.6.2 Dimethylamine .................................................................................................. 22
1.6.3 Trimethylamine ................................................................................................. 23
1.6.4 Diethylamine ..................................................................................................... 24
1.6.5 Triethylamine .................................................................................................... 25
1.7 Aim of the study ..................................................................................................... 26
CHAPTER 2 MATERIALS AND METHODS .................................................. 27
2.1 Overview ................................................................................................................ 27
2.2 Equipment, chemicals and media ........................................................................... 27
2.2.1 Growth media and solutions ............................................................................. 29
2.2.2 Buffers .............................................................................................................. 32
2.3 Bacteria that degrade triethylamine ........................................................................ 34
2.3.1 Isolation procedure ........................................................................................... 34
2.3.2 Anaerobic growth .............................................................................................. 34
2.4 Bacteria that degrade ethyl acrylate ....................................................................... 35
2.4.1 Isolation procedure ........................................................................................... 35
2.4.2 Anaerobic growth .............................................................................................. 35
2.5 Analytical procedures ............................................................................................. 35
2.6 Enzyme assay ........................................................................................................ 37
2.6.1 Spectrophotometric assay with p-nitrophenyl acetate (-NPA) as substrate ..... 37
2.7 Purification and characterization of esterase from 2Cbei cells ................................ 37
2.7.1 Bacterial strain cultivation and native crude enzyme extraction ......................... 37
2.7.2 Purification of esterase from 2Cbei cells ........................................................... 38
2.7.3 Influence of pH and temperature on esterase activity and thermostability ......... 38
2.7.4 Effect of various compounds on esterase activity ............................................. 38
2.7.5 Substrate specificity .......................................................................................... 41
2.8 Biochemical methods ............................................................................................. 41
2.8.1 Determination of protein concentration ............................................................. 41
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2.9 Disposal of waste materials .................................................................................... 42
CHAPTER 3 RESULTS ........................................................................... 43
3.1 Bacteria that degrade triethylamine ........................................................................ 43
3.1.1 Isolation procedure ........................................................................................... 43
3.1.2 Identification of the triethylamine degrading bacteria RA1 and RA2 .................. 43
3.1.3 Determination of growth characteristics of Pseudomonas citronellolis RA1 and
Mycobacterium diernhoferi RA2 ........................................................................ 47
3.1.4 Substrate spectra of Pseudomonas citronellolis RA1 and Mycobacterium
diernhoferi RA2 ................................................................................................. 51
3.1.5 Determination of the degradation pathway for triethylamine .............................. 55
3.1.5.1 Growth of RA1 on the different substrates in a nitrogen-free mineral
medium 1.10 M (YE-N). ............................................................................ 55
3.1.5.2 Growth of RA2 on the different substrates in a nitrogen-free mineral
medium 1.10 M (YE-N). ............................................................................ 57
3.1.5.3 Growth of RA1 on the different substrates in minimal mineral medium
with an ammonium source (1.10 M (-YE+N)). ........................................... 59
3.1.5.4 Growth of RA2 on the different substrates in minimal mineral medium
with an ammonium source (1.10 M (-YE+N)). ........................................... 61
3.1.5.5 Growth of RA1 on the different substrates in minimal mineral medium
without an ammonium source (1.10 M (-YE-N)). ....................................... 63
3.1.5.6 Growth of RA2 on the different substrates in minimal mineral medium
without an ammonium source (1.10 M (-YE-N)). ....................................... 65
3.2 Bacteria that degrade ethyl acrylate ....................................................................... 67
3.2.1 Isolation procedure ........................................................................................... 67
3.2.2 Identification of the ethyl acrylate degrading bacteria 2C and 2Cbei ................. 67
3.2.3 Determination of growth characteristics of Defluvibacter lusatiensis 2C and
2Cbei ................................................................................................................ 70
3.2.4 Determination of the degradation pathway for ethyl acrylate ............................. 76
3.2.4.1 Growth of 2C on the different substrates in a mineral medium containing
0.1% yeast extract (1.10 M (+YE)). ........................................................... 76
3.2.4.2 Growth of 2Cbei on the different substrates in a mineral medium
containing 0.1% yeast extract (1.10 M (+YE)). .......................................... 77
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3.2.5 Detection and characterization of an esterase from Defluvibacter lusatiensis
2Cbei ................................................................................................................ 80
3.2.5.1 Substrate spectrum .................................................................................. 80
3.2.5.2 Inhibitors, detergents, solvents and metal ions ......................................... 80
3.2.5.3 pH (optimum) ............................................................................................ 83
3.2.5.4 Temperature (optimum) ............................................................................ 84
3.2.5.5 Purification ................................................................................................ 85
CHAPTER 4 DISCUSSION ....................................................................... 87
4.1 Bacteria that degrade triethylamine ........................................................................ 87
4.1.1 Isolation, characterization and identification of new triethylamine degrading
bacteria, RA1 and RA2 ..................................................................................... 87
4.1.1.1 The species, Pseudomonas citronellolis, desription: ................................. 88
4.1.1.2 The species, Mycobacterium diernhoferi, description: .............................. 89
4.1.2 Determination of growth characteristics of Pseudomonas citronellolis RA1 and
Mycobacterium diernhoferi RA2 ........................................................................ 90
4.1.3 Substrate spectra of Pseudomonas citronellolis RA1 and Mycobacterium
diernhoferi RA2 ................................................................................................. 91
4.1.4 Determination of the degradation pathway for triethylamine .............................. 92
4.2 Bacteria that degrade ethyl acrylate ....................................................................... 95
4.2.1 Isolation, characterization and identification of new ethyl acrylate degrading
bacteria, 2C and 2Cbei ..................................................................................... 95
4.2.1.1 Description of the genus Defluvibacter gen. nov. : .................................... 95
4.2.1.2 Description of the type species Defluvibacter lusatiae sp.nov. .................. 96
4.2.2 Determination of growth characteristics of Defluvibacter lusatiensis, 2C and
2Cbei ................................................................................................................ 96
4.2.3 Determination of the degradation pathway for ethyl acrylate ............................. 97
4.2.4 Detection and characterization of an esterase from Defluvibacter lusatiensis
2Cbei ................................................................................................................ 98
4.3 Concluding remarks and further prospects ........................................................... 100
CHAPTER 5 SUMMARY ........................................................................ 101
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CHAPTER 6 ZUSAMMENFASSUNG ......................................................... 104
CHAPTER 7 REFERENCES ................................................................... 107
7.1 General References ............................................................................................. 107
7.2 Online References ............................................................................................... 119

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