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Online monitoring of biofilm in microchannels with thermal lens microscopy [Elektronische Ressource] / Tobias Roßteuscher

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Published 01 January 2009
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TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Analytische Chemie
Institut für Wasserchemie und Chemische Balneologie


Online monitoring of biofilm in microchannels with
thermal lens microscopy

Tobias Roßteuscher


Vollständiger Abdruck der von der Fakultät für Chemie der Technischen
Universität München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.




Vorsitzender: Univ.-Prof. Dr. M. Schuster
Prüfer der Dissertation: 1. Univ.-Prof. Dr. R. Nießner
2. Univ.-Prof. Dr. H. Horn




Die Dissertation wurde am 16.04.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 28.05.2009 angenommen.






Parts of this thesis have been published in journals or are in preparation for publication:

T. T. J. Rossteuscher, A. Hibara, K. Mawatari, T. Kitamori, Lateral spatial resolution of
thermal lens microscopy during continuous scanning for non-staining biofilm imaging,
Journal of Applied Physics, in press (DOI 10.1063/1.3116137)












































Acknowledgements

This thesis was financially supported by the Japanese Ministry of Education, Culture,
Sports, Science and Technology (MEXT) during the first two years of experimental
research and by the Japanese Society for the Promotion of Science (JSPS) from April
2008 until February 2009.
Activated sludge biofilm as well as Sphingomonas paucimobilis biofilm were gratefully
supported by Ohgaki-Katayama Laboratory, Department of Urban Engineering, The
University of Tokyo. MPC copolymer was provided by Ishihara-lab, Department of
Material Engineering, The University of Tokyo. Access to the 3D-reflection microscope
used in this thesis was provided by the Center of NanoBio Integration (CNBI), The
University of Tokyo.
First of all I would like to thank Professor Dr. Reinhard Niessner for giving me the
possibility to conduct the research on this very interesting topic abroad at The University
of Tokyo, Japan, and supporting me during my research track through discussions and
with helpful information. Also I want to thank him for the fast and helpful action during
my application for Monbukagakusho as well as JSPS scholarship.
I also would like to thank my supervising professor, Professor Dr. Takehiko Kitamori,
who supported me by providing equipment and access to the instruments in his laboratory,
as well as with useful help during discussions about experimental procedure and the
direction of laboratory research. Also he helped me with contacting the right people at the
right time to get my research going and on the right way.
Professor Dr. Akihide Hibara and Dr. Kazuma Mawatari were the main supervisors
regarding the experimental procedure of research and provided numerous informations
and help on experimental setups as well as technical problems. Also, proofreading of
numerous reports and papers including this thesis is gratefully acknowledged.
Professor Dr. Shinichiro Ohgaki provided useful help during the determination of the
direction for biofilm experiments inside the microchannel. He also provided me with
access to biofilm for my research.
Professor Dr. Hiroyuki Katayama was a great supporter during the whole research period,
either by providing biofilm samples whenever needed on short notice as well as by

manifold useful discussions regarding the next steps in the ongoing biofilm research
inside the microchannel.
Dr. Dai Simazaki from the Japanese Institute of Public Health was very helpful in
providing information during discussions regarding the setup of the biofilm pumping
system and the biofilm experiments in microchannels. He also provided the Japanese pipe
materials used as references for the channel modification experiments.
Dr. Christoph Haisch was a helpful contact regarding research and procedures back in
Germany apart from Professor Niessner and provided information and useful discussions
about experimental progress and the conductance of the thesis.
Dr. Takehiko Tsukahara introduced me to the field of microfluidics and explained to me
the problems and ideas for chip connections.
Dr. Kae Sato helped me with a lot of Japanese documents and application forms,
especially during my last year as a JSPS scholar.
Dr. Kiyohito Shimura provided a lot of very helpful discussions and suggestions
regarding the pressure-driven setup for biofilm experiments in a microchannel.
Dr. Arata Aota helped me during my first days in Tokyo to get used to the new
environment and during the application procedures which would have been not at all
understandable to me as most documents are still provided in Japanese with a lot of
characters I cannot understand even up to today. Also, he provided useful discussions
regarding flow in microchannels and pressure drop.
Dr. Yo Tanaka supported me throughout my stay with assistance in ‘difficult Japanese’,
especially regarding ordering of new material
Dr. Ki-Hoon Jang introduced me to surface modification with MPC copolymer, helped
me a lot with day-to-day problems in a world where Japanese is the predominant
language (it is interesting to re-learn how to use an ATM) and provided me with a good
time in Tokyo. Also he was the one who kept me doing some sports. I wonder how much
weight I would have gained without continued attempts to do sports.
Dr. Adelina Smirnova was also of great help during my first year of continued
experiences in a new environment where I was happy to have some European researcher
who could relate to my problems.

Asakawa-san introduced me to TLM setup and gave me instructions for aligning the laser
beams in English! Oikawa-san was helpful in continued discussions regarding TLM in
the beginning and was very helpful to set up the measuring times on the shared
instrument. Morikawa-san gave useful information regarding the use of pressure
controllers.
Many more laboratory members helped me with Japanese throughout my entire stay that
I finally got to a point where I can make myself understandable in this strange yet highly
fascinating language on a daily basis, although I have still a long way to go until I
actually understand it.
Dr. Antonin Hoel and Takahashi-san provided insight and gave continued support
regarding Labview.
Jorg Entzinger arrived at the same time as me and provided numerous support and helpful
suggestions regarding MatLab procedures and also made life in Tokyo a lot more
interesting during various activities, hikes and trips. Together with my wife he also kept
some European/Christian traditions alive during our time in Tokyo, namely a yearly
Easter picnic and a yearly interlaboratory Christmas party.
The people at the office, Ogawa-san, Kamata-san and Toyama-san helped me a lot with
all documents and all regulations regarding my research in Japan.
My FACE-volunteer Mitsuyo Hasegawa was of great help regarding procedures and
living in Japan and provided initial knowledge of biofilm specific language in Japanese.
Tokyo University International Center and Faculty of Engineering (Japanese language
education) got me to a level of Japanese where I can communicate in daily life which is
of real help and utmost importance as otherwise you would miss just too much out.
Most of the bigger problems came down to understanding Japanese in daily life as well as
in the laboratory, be it in phone calls, passing of documents, computer software or during
discussions. I would like to thank anybody I did not yet mention with providing support
in this regard. が ん ば り ま す 。 (“Ganbarimasu” means something like you keep on
trying to improve your best and do the best according to your abilities to make it work)
Last but not least I would like to thank my wife, my parents and my brother for continued
and ever fulfilling support during the long stay abroad as well as the time during the
writing of this thesis. I guess I was not always easy to handle during writing, but your

continued support helped me push through. And my wife made my stay here in Japan a
most wonderful one which I will be grateful for forever.


Table of contents

1 Introduction .....................................................................................1
1.1 Biofilm................................................................................................................ 2
1.1.1 Definition ............................................................................................................ 2
1.1.2 Biofilm development .......................................................................................... 5
1.1.3 Research fields.................................................................................................. 10
1.1.4 Experimental approaches.................................................................................. 15
1.1.5 Present problems in online biofilm monitoring ................................................ 29

1.2 Microfluidics/Microdevices............................................................................ 30
1.2.1 Production methods .......................................................................................... 31
1.2.2 Applications ...................................................................................................... 33

1.3 Thermal lens microscopy ............................................................................... 34
2 Aims and scope of the thesis .........................................................39
3 Theory.............................................................................................43
3.1 Microfluidics.................................................................................................... 43
3.1.1 Laminar flow and shear stress........................................................................... 43
3.1.2 Pressure drop..................................................................................................... 44

3.2 Thermal lens microscopy ............................................................................... 45
3.2.1 Definitions......................................................................................................... 45
3.2.2 Signal generation .............................................................................................. 46
3.2.3 Resolution ......................................................................................................... 52

4 Materials and methods..................................................................55
4.1 Biofilm.............................................................................................................. 55
4.1.1 Bacteria ............................................................................................................. 55
4.1.2 Nutrients............................................................................................................ 56
4.1.3 Staining ............................................................................................................. 57

4.2 Biofilm growth in flow cells............................................................................ 57
4.3 Biofilm growth in microchannels .................................................................. 58
4.3.1 Microchannel production.................................................................................. 59
4.3.2 Microchannel setup........................................................................................... 60
I
4.3.3 Continuous flow system.................................................................................... 64
4.3.4 Biofilm adsorption and growth studies............................................................. 70
4.3.5 Biofilm removal and microchip cleaning ......................................................... 73

4.4 Adaptation of thermal lens microscopy for biofilm observation................ 74
4.4.1 Laser spot characterization ............................................................................... 76
4.4.2 Influence of instrumental parameters................................................................ 79
4.4.3 Three-dimensional scanning ............................................................................. 82
4.4.4 Biofilm observation .......................................................................................... 84

5 Results and discussion...................................................................87
5.1 Biofilms in the microchannel ......................................................................... 87
5.1.1 Biofilm adhesion in microchannels .................................................................. 90
5.1.2 Online biofilm staining in microchannels......................................................... 91
5.1.3 Biofilm growth in microchannel....................................................................... 93
5.1.4 Biofilm removal................................................................................................ 97

5.2 Thermal lens microscopy ............................................................................. 102
5.2.1 Thermal lens scanning parameters.................................................................. 102
5.2.2 Thermal lens resolution................................................................................... 105
5.2.3 Two-dimensional scanning ............................................................................. 110

5.3 Biofilm observation with TLM .................................................................... 117
5.3.1 Biofilm grown on slides.................................................................................. 118
5.3.2 Biofilm grown in microchannel...................................................................... 121

6 Summary and outlook.................................................................129
7 Appendix.......................................................................................137
7.1 Abbreviations ................................................................................................ 137
7.2 Letters and symbols ...................................................................................... 139
7.3 Japanese pipe materials................................................................................ 141
7.4 Design drawings ............................................................................................ 145
8 References.....................................................................................149


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