Biotransformation of thiomersal by naturally mercury resistant isolates and genetically engineered microorganisms [Elektronische Ressource] / von Wanda Fehr

Naturwissenschaftliche Fakultät

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2006
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Biotransformation of Thiomersal by Naturally Mercury Resistant Isolates
and Genetically Engineered Microorganisms




Von der Fakultät für Lebenswissenschaften
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig
zur Erlangung des Grades einer
Doktorin der Naturwissenschaften
(Dr.rer.nat.)
genehmigte
D i s s e r t a t i o n

















von Wanda Fehr
geboren Teheran/ Iran















































1. Referent: PD Dr. Irene Wagner-Döbler
2. Referentin oder Referent: Prof. Dr. Dieter Jahn
eingereicht am: 17.03.2006
mündliche Prüfung (Disputation) am: 24.04.2006 Vorveröffentlichungen der Dissertation
Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fachbereich für
Biowissenschaften und Psychologie, vertreten durch Mentorin Dr. habil. I. Wagner-Döbler, in
folgenden Beiträgen vorab veröffentlicht:

Publikation:

Felske AD, Fehr W, Pauling BV, von Canstein H, Wagner-Dobler I. Functional profiling of
mercuric reductase (merA) genes in biofilm communities of a technical scale
biocatalyzer. BMC Microbiol. 2003 Oct 27; 3(1):22.

Tagungsbeiträge:

Wanda Fehr and Irene Wagner-Döbler. Microbial degradation of an organic mercury
compound (Thiomersal). Roskilde, Sectoral meeting, June 14-16, 2000, Roskilde, Denmark.

Weitere Veröffentlichungen:

Brummer IH, Fehr W, Wagner-Dobler I. Biofilm community structure in polluted rivers:
abundance of dominant phylogenetic groups over a complete annual cycle. Appl
Environ Microbiol. 2000 Jul; 66(7):3078-82.

Uphoff HU, Felske A, Fehr W, Wagner-Dobler I.The microbial diversity in picoplankton
enrichment cultures: a molecular screening of marine isolates. FEMS Microbiol Ecol.
2001 May; 35(3):249-258.

Vorträge:

Irene Wagner-Döbler1, Harald von Canstein1, Andreas D.M. Felske1, Johannes Leonhäuser,
Björg V. Pauling, Wanda Fehr, Wolf-Dieter Deckwer. New tricks of old bugs. VAAM
Frühjahrstagung, March 28-31, 2001, Braunschweig, Germany.

Contents
CONTENTS
1 INTRODUCTION............................................................................................................ 1
1.1 Mercury in the Environment ...................................................................................... 1
1.2 Toxicity of Mercurial Compounds.............................................................................. 3
1.3 Thiomersal .................................................................................................................... 5
1.4 Thiomersal Utilization ................................................................................................. 7
1.5 Treatment of Mercury Contaminated Wastewater .................................................. 9
1.6 Basic Principles of Biological Mercury Decontamination...................................... 11
1.6.1 Mechanism of Microbial Resistance to Mercury ..................................................... 11
1.6.2 The Role of the merB Gene...................................................................................... 13
1.6.3 Utilization of Genetically Engineered Microorganisms........................................... 16
1.7 Aim of the Work......................................................................................................... 17
2 MATERIALS AND METHODS................................................................................... 19
2.1 Chemicals and Reagents ............................................................................................ 19
2.1.1 Preparation of Standard Solution ............................................................................. 19
2.2 Analytical Methods for Mercury Detection ............................................................. 20
2.2.1 Cold-Vapor Atomic Absorption Spectrometry ........................................................ 20
2.2.2 High Performance Liquid Chromatography (HPLC)............................................... 22
2.3 Microbiological Methods ........................................................................................... 23
2.3.1 Microorganisms........................................................................................................ 23
2.3.2 Culture Media and Buffer Solutions ........................................................................ 23
2.3.2.1 Luria Bertani Medium (Sambrook et al. 1989)................................................... 24
2.3.2.2 Inoculation Medium (von Canstein et al. 2001) ................................................. 24
2.3.2.3 M9- Pseudomonas Minimal Medium ................................................................. 24
2.3.2.4 Phosphate Buffer................................................................................................. 26
2.3.3 Substrate Utilization Profiles (BIOLOG) 26
2.3.4 Counter-Ion Selection.............................................................................................. 29
2.3.5 Biofilm Formation Assay ......................................................................................... 30
2.3.6 Carbon Source Utilization........................................................................................ 31
2.3.7 Determination of Protein Content ............................................................................ 33
2.4 Cultivation of Microorganisms ................................................................................. 33
2.4.1 Culture Conditions................................................................................................... 33
2.4.2 Growth Measurements............................................................................................. 34
I
23H23H21H21H138H138H12H12H2H2H145H145H137H137H10H10H140H140H3H3H20H20H5H5H24H24H125H125H136H136H118H118H19H19H119H119H135H135H120H120H18H18H29H29H141H141H6H6H134H134H30H30H25H25H9H9H17H17H148H148H133H133H130H130H16H16H144H144H132H132H28H28H142H142H13H13H15H15H4H4H26H26H129H129H131H131H121H121H139H139H122H122H22H22H146H146H128H128H123H123H8H8H7H7H0124H124H117H117H147H147H1H1H31H31H143H143H126H126H14H14H127H127H27H27H11H11H Contents
2.4.2.1 Determination of Cell Number in Liquid Medium ............................................. 34
2.4.2.2 Optical Density................................................................................................... 34
2.4.2.3 Determination of Growth Phase.......................................................................... 35
2.5 Deoxyribonucleic Acid (DNA) Analysis ................................................................... 38
2.5.1 DNA Extraction........................................................................................................ 38
2.5.2 DNA Gel Electrophoresis......................................................................................... 38
2.5.3 DNA Purification Techniques.................................................................................. 40
2.5.3.1 Phenol / Chloroform Extraction 40
2.5.3.2 DNA Precipitation............................................................................................... 41
2.5.3.3 Extraction of DNA from Agarose Gel ................................................................ 41
2.5.3.4 DNA Purification in Solution............................................................................. 41
2.5.4 Polymerase Chain Reaction (PCR) .......................................................................... 42
2.5.4.1 Oligonucleotide Primer....................................................................................... 42
2.5.5 Sequencing and Sequence Analysis ......................................................................... 45
2.5.6 Phylogenetic analysis............................................................................................... 46
2.6 Determination of Hg-Effect on Microorganisms..................................................... 47
2.6.1 Growth Inhibition by Thiomersal............................................................................. 47
2.6.2 Mercury Resistance Level........................................................................................ 48
2.6.3 Measurement of Thiomersal transformation rate 48
2.6.4 Calibration of Thiomersal Transformation Measurements ...................................... 49
2.6.5 Consideration of Error Propagation ......................................................................... 50
2.6.6 Lab-Scale Bioreactor................................................................................................ 51
2.6.6.1 Kinetic Model of Enzymatic Reaction................................................................ 53
3 RESULTS....................................................................