Investigation of mature biofilm populations in the distribution of drinking water with attention to bacteria of hygienic relevance [Elektronische Ressource] / Bianca Conradi. Betreuer: Ulrich Szewzyk

Kallemann

Sujets

• Biologie

Informations

• Publié par: technische_universitat_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Technische Universität Berlin
Investigation of mature biofilm populations in the
distribution of drinking water with attention to bacteria of
hygienic relevance
vorgelegt von
Diplom-Biologin Bianca Conradi
aus Greetsiel
von der Fakultät III-Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktorin der Naturwissenschaften
-Dr. rer. nat.-
genehmigte Dissertation
Promotionsausschuss
Vorsitzender: Prof. Dr. rer. nat. Wolfgang Rotard, TU Berlin
Berichter: 1. Prof. Dr. rer. nat. Ulrich Szewzyk, TU Berlin
Berichterin: 2. Prof. Dr. rer. nat. Isolde Röske, TU Dresden
Tag der wissenschaftlichen Aussprache: 31.03.2011
Berlin 2011
D83meiner Großmutter E. Hübel1 INTRODUCTION ....................................................................................................................................... 1
2 MATERIAL AND METHODS................................................................................................................... 7
2.1 REACTOR SYSTEMS AT DIFFERENT LOCATIONS ...................................................................................... 7
2.1.1 Reactor system Berlin (Germany) ................................................................................................... 7
2.1.1.1 Setup and function................................................................................................................................. 7
2.1.1.2 Sampling of material coupons ............................................................................................................... 9
2.1.2 Reactor systems Duisburg (Germany)............................................................................................. 9
2.1.3 Reactor system Lundtofte (Denmark)............................................................................................ 10
2.2 PIPE SECTIONS FROM THE DRINKING WATER DISTRIBUTION SYSTEM.....................................................11
2.2.1 Free water samples of the distribution system .............................................................................. 12
2.2.2 Treatment of pipe deposits on the inner surface............................................................................ 12
2.2.2.1 Treatment of PVC pipes ...................................................................................................................... 12
2.2.2.2 Treatment of metallic pipes ................................................................................................................. 13
2.3 ANALYSIS OF BACTERIAL POPULATIONS BY CULTIVATION TECHNIQUES ............................................... 13
2.3.1 Aerobic cultivation on modified R2A medium............................................................................... 13
2.3.2 Heterotrophic plate counts according to DIN EN ISO 6222 ......................................................... 13
2.3.3 E. coli and coliform bacteria according to DIN 38 411 K 6.......................................................... 14
2.3.4 Aerobic cultivation of P. aeruginosa according to DIN EN 12780................................................ 14
2.4 INVESTIGATION OF THE BACTERIAL POPULATION BY CULTURE INDEPENDENT METHODS ..................... 15
2.4.1 Total cell counts (TCC) determined by DAPI staining.................................................................. 15
2.4.1.1 Staining of biofilm suspensions........................................................................................................... 15
2.4.1.2 Staining on filter membrane ................................................................................................................ 15
2.4.1.3 Staining on coupons ............................................................................................................................ 16
2.4.1.4 Microscopic examination .................................................................................................................... 16
2.4.2 Fluorescence in situ hybridization (FISH) .................................................................................... 16
2.4.2.1 Fixation of biofilm coupons, suspensions, and pure cultures .............................................................. 16
2.4.2.2 Hybridization procedure...................................................................................................................... 17
2.4.2.3 Development of a new oligonucleotide probe ..................................................................................... 19
2.4.2.4 Oligonucleotide probes used in this study ........................................................................................... 19
2.4.3 Extraction of total DNA from biofilm suspensions ........................................................................ 20
2.4.3.1 Simple preparations of DNA from formaldehyde fixed and non-fixed biofilm suspensions ............... 20
2.4.3.1.1 M IV (formaldehyde fixed) and BWB III (non-fixed) serial diluted .............................................. 20
2.4.3.1.2 Alkaline lysis of formaldehyde fixed M IV.................................................................................... 20
2.4.3.1.3 Alkaline lysis, enhanced sample volume, and ethanol precipitation .............................................. 21
2.4.3.1.4 Alkaline lysis, enhanced sample volume, and isopropanol precipitation ....................................... 21
2.4.3.1.5 Addition of bovine serum albumin (BSA) ..................................................................................... 22
2.4.3.2 FastDNA Spin Sample Kit for soil ...................................................................................................... 23
2.4.3.2.1 Extraction of DNA from biofilm suspensions ................................................................................ 23
2.4.3.2.2 Evaluation of extraction efficiency ................................................................................................ 24
2.4.3.3 CTAB (hexadecyltrimethylammonium bromide) extraction ............................................................... 252.4.3.3.1 General extraction procedure ......................................................................................................... 25
2.4.3.3.2 Variations of the protocol ............................................................................................................... 27
2.4.3.4 DNA extraction with QIAamp DNA Mini Kit..................................................................................... 27
2.4.3.5 Extraction and purification by Qiagen Genomic-tips 20 ..................................................................... 28
2.4.3.6 Verification of the extraction success .................................................................................................. 30
2.4.3.6.1 Control PCR................................................................................................................................... 30
2.4.3.6.2 Measurement of DNA.................................................................................................................... 32
2.4.4 Sequencing of bacterial 16S rDNA ............................................................................................... 33
2.4.4.1 Alkaline lysis....................................................................................................................................... 33
2.4.4.2 Sequencing reaction ............................................................................................................................ 33
2.4.5 RFLP analysis of the 16S rRNA gene of isolates........................................................................... 34
2.4.6 Phylogenetic analysis.................................................................................................................... 35
2.4.6.1 Pipe sample isolates............................................................................................................................. 35
2.4.6.2 Representative reactor sample isolates obtained from RFLP analysis................................................. 35
2.5 STATISTICAL ANALYSIS........................................................................................................................ 36
3 RESULTS ................................................................................................................................................... 37
3.1 PIPE SAMPLES TAKEN FROM THE DISTRIBUTION SYSTEMS IN BERLIN AND THE RUHRGEBIET .............. 37
3.1.1 Macroscopic description of deposits on the inner pipe surface .................................................... 39
3.1.2 Total cell counts and culturable bacteria on the different pipe materials ..................................... 39
3.1.3 Phylogenetic bacterial groups detected in the pipe samples......................................................... 45
3.2 RESULTS REACTOR SYSTEM BERLIN.................................................................................................... 56
3.2.1 Operation of the Berlin reactor system ......................................................................................... 56
3.2.2 P. aeruginosa in the bulk water phase of the reactor system in Berlin.......................................... 57
3.2.2.1 Specificity of the DIN EN 12780 ........................................................................................................ 58
3.2.2.2 Growth of isolated P. aeruginosa on