Molecular ecology and biogeography of methanotrophic bacteria in wetland rice fields [Elektronische Ressource] / vorgelegt von Claudia Lüke

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Molecular ecology and biogeography of methanotrophic bacteria in wetland rice fields Dissertation zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg/Lahn vorgelegt von Claudia Lüke aus Recklinghausen Marburg an der Lahn | 2009 Die Untersuchungen zur folgenden Arbeit wurden von November 2006 bis Dezember 2009 am Max-Planck-Institut für terrestrische Mikrobiologie in Marburg unter der Leitung von Prof. Dr. Peter Frenzel durchgeführt. Vom Fachberich Biologie der Philipps-Universität Marburg als Dissertation angenommen am: 27.01.2010 Erstgutachter: Prof. Dr. Peter Frenzel Zweitgutachter: Prof. Dr. Wolfgang Buckel Tag der Disputation: 23.03.2010 Die in dieser Dissertation beschriebenen Ergebnisse sind in den folgenden Publikationen veröffentlicht bzw. zur Veröffentlichung vorgesehen: Krause, S., Lüke, C., and Frenzel, P. (2009) Spatial heterogeneity of methanotrophs: a geostatistical analysis of pmoA-based T-RFLP paterns in a paddy soil. Environ Microbiol Rep 1: 393-397. Lüke, C., Krause, S., Cavigiolo, S., Greppi, D., Lupotto, E., and Frenzel, P. (2010) Biogeography of wetland rice methanotrophs. Environ Microbiol. In press. Lüke, C., Bodrossy, L., Lupotto, E., and Frenzel, P.

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Molecular ecology and biogeography of
methanotrophic bacteria in wetland
rice fields




Dissertation



zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
(Dr. rer. nat.)

dem Fachbereich Biologie
der Philipps-Universität Marburg/Lahn
vorgelegt von



Claudia Lüke
aus Recklinghausen






Marburg an der Lahn | 2009
Die Untersuchungen zur folgenden Arbeit wurden von November 2006 bis Dezember
2009 am Max-Planck-Institut für terrestrische Mikrobiologie in Marburg unter der
Leitung von Prof. Dr. Peter Frenzel durchgeführt.












Vom Fachberich Biologie der Philipps-Universität Marburg als Dissertation
angenommen am: 27.01.2010

Erstgutachter: Prof. Dr. Peter Frenzel
Zweitgutachter: Prof. Dr. Wolfgang Buckel

Tag der Disputation: 23.03.2010
Die in dieser Dissertation beschriebenen Ergebnisse sind in den folgenden
Publikationen veröffentlicht bzw. zur Veröffentlichung vorgesehen:


Krause, S., Lüke, C., and Frenzel, P. (2009) Spatial heterogeneity of methanotrophs: a
geostatistical analysis of pmoA-based T-RFLP paterns in a paddy soil. Environ
Microbiol Rep 1: 393-397.

Lüke, C., Krause, S., Cavigiolo, S., Greppi, D., Lupotto, E., and Frenzel, P. (2010)
Biogeography of wetland rice methanotrophs. Environ Microbiol. In press.

Lüke, C., Bodrossy, L., Lupotto, E., and Frenzel, P. Methanotrophic bacteria associated
to rice roots: the rice cultivar effect assessed by T-RFLP and microarray analysis. In
preparation.

Lüke, C, and Frenzel, P. The pmoA gene as functional and phylogenetic marker for
methanotrophic bacteria: a sequence database analysis. In preparation.
Table of contents
Summary III
Zusammenfassung V
Introduction 1 1
1.1 Methane in the atmosphere 1
1.2 Methanotrophic microorganisms 2
1.3 The wetland rice ecosystem 11
1.4 Marker genes for studying methanotroph diversity 13
1.5 The aims of this study 15
1.6 References 17
Spatial heterogeneity of methanotrophs:
A geostatistical analysis of pmoA-based T-RFLP patterns in a paddy soil 27 2
2.1 Abstrac 27
2.Inoduction 28
2.3 Results and discussion 30
2.4Supplementary material 36
2.5 References 39
Biogeography of wetland rice methanotrophs 43 3
3.1 Abstrac 43
3.2Inoduction 44
3. Result 47 4Dicssion 53
3.5 Experimental procedures 59
3.6Supplementarymaterial 64
3.7 References 66
Methanotrophic bacteria associated to rice roots: 4 The rice cultivar effect assessed by microarray and T-RFLP analysis 73
4.1 Abstrac 73
4.2Inoduction 74
4.3 Result 7 Dicssion 83
4.5 Experimental procedures 88
4.6Supplementarymaterial 90
4.7 References 96
I The pmoA gene as functional and phylogenetic marker for methanotrophs:
A sequence database analysis 101 5
5.1 Abstract 101
5.2 Introduction 102
5.3 Results 104
5.4 Discussion 109
5.5 Experimental procedures 114
5.6 References 116
General discussion and outlook 125 6
6.1 Diversity of methanotrophs in the wetland rice ecosystem 126
6.2 Niche differentiation of wetland rice methanotrophs 128
6.3 Factors influencing methanotrophs in wetland rice fields 130
6.4 Outlook 131
6.5 References 133
Publication list
Contribution by other people
Curiculm vitae
II
SUMMARY
Methanotrophic bacteria perform a central function in our climate system
representing the only biogenic sink for the greenhouse gas methane. In wetland rice
fields, they function as bio-filters preventing methane produced in anoxic layers
escaping into the atmosphere, attenuating the potential methane emission by up to
90%. Despite intensive studies in the past, molecular approaches have barely started to
explore the full diversity of methanotrophs. Furthermore, only little is known on
their ecological niche differentiation and the factors influencing their community
structure. This thesis focuses on the aerobic methanotrophic communities in the
wetland rice ecosystem using the pmoA gene as a functional and phylogenetic marker
to detect these bacteria in the environment. A high diversity could be recorded
Methylocystis and Methylosinus species (type II) and yet uncultivated dominated by
bacteria grouping within type Ib methanotrophs. The rice paddy cluster 1 (RPC-1)
forms the largest cluster consisting entirely of sequences obtained from paddy fields
located around the world. It is only distantly related to cultivated species and might
form a new genus of methanotrophs specifically adapted to wetland rice fields.
Methanotrophic communities showed no large scale horizontal distribution
patterns within an Italian paddy field; thus, a reduced sampling effort is sufficient to
extrapolate to the field scale. However, different methanotrophic communities were
detected on the rice roots compared to the field soil and the communities in different
fields differed significantly. The rice roots were characterized by a high abundance of
type I methanotrophs and different rice cultivars were shown to have an effect on
these communities. This effect could be correlated to the plant genotype and enables
to select specific cultivars for in-depth studies.
Re-evaluating the pmoA gene as a phylogenetic marker for methanotrophs
revealed a good correlation of the pmoA to the 16S rRNA phylogeny. Nevertheless,
some exceptions suggests that methanotrophy might be evolutionary more
complicated having been even exchanged between species. Furthermore, a meta-
analysis of pmoA sequences from various environments revealed distinct correlations
of genotypes and habitats.
III IV