Investigation of microbial groups involved in the uptake of atmospheric trace gases in upland soils [Elektronische Ressource] / Jennifer Pratscher. Betreuer: Ralf Conrad

Pratscher

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2011
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

Investigation of microbial groups involved in the uptake of
atmospheric trace gases in upland soils






Dissertation


zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.)

dem Fachbereich Biologie der Philipps-Universität Marburg

vorgelegt von








Jennifer Pratscher
aus Jülich




Marburg/Lahn 2010 Die Untersuchungen zur folgenden Arbeit wurden von Januar 2008 bis Oktober 2010 am
Max-Planck-Institut für terrestrische Mikrobiologie in Marburg unter Anleitung von Prof. Dr.
Ralf Conrad durchgeführt.






























Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation angenommen
am: 28.10.2010

Erstgutachter: Prof. Dr. Ralf Conrad
Zweitgutachter: Prof. Dr. Erhard Bremer

Tag der Disputation: 13.01.2011 Publication list

Die in dieser Dissertation beschriebenen Ergebnisse sind in folgenden Publikationen
veröffentlicht bzw. zur Veröffentlichung vorgesehen:

1. Pratscher, J., Dumont, M.G., Conrad, R. (2010) Assimilation of acetate by the
putative atmospheric methane oxidizing clade USC α. (submitted to Nature
Geoscience in October 2010)

2. Pratscher, J., Dumont, M.G., Conrad, R. (2010) Ammonia oxidation coupled to
CO fixation by Archaea and Bacteria in an agricultural soil. (submitted to 2
Proceedings of the National Academy of Sciences of the United States of
thAmerica on 30 July 2010)

3. Constant, P., Chowdhury, S.P., Pratscher, J., Conrad, R. (2010)
Streptomycetes contributing to atmospheric molecular hydrogen soil uptake are
widespread and encode a putative high-affinity [NiFe]-hydrogenase. Environ
Microbiol 12: 821-829.









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Contents


Contents 


Abbreviations .................................................................................................. 4
Zusammenfassung ......................................................................................... 6
Summary.......................................................................................................... 8

I. Introduction ................................................................................................ 10
I.1 General introduction......................................................................................................10
I.2 Methane in the atmosphere and its uptake into upland soils.........................................13
I.3 CO fixation by ammonia oxidizing prokaryotes in terrestrial environments ..................15 2
I.4 Biological uptake of atmospheric H ..............................................................................18 2
I.6 Objectives of this study.................................................................................................20
I.6 References ...................................................................................................................21

II. Materials and methods............................................................................. 26
II.1 Chemicals and gases...................................................................................................26
II.2 Cultures and media......................................................................................................26
II.3 Environmental samples and sampling procedures.......................................................28
II.3.1 Marburg forest soil (MF) .......................................................................................28
II.3.2 Rauischholzhausen agricultural soil (RH) .............................................................28
II.4 Chemical analyses.......................................................................................................28
II.4.1 Determination of pH in soil samples .....................................................................28
II.4.2 Gravimetric determination of soil moisture content ...............................................28
II.4.3 Determination of ammonium.................................................................................29
II.4.4 Analysis of nitrate and nitrite.................................................................................29
II.4.5 Quantification of acetate .......................................................................................30
II.4.7 Calculation of the atmospheric methane oxidation rate (V )...............................31 atm
13II.4.8 Determination of CO .........................................................................................32 2
II.5 Nucleic acid extraction.................................................................................................32
II.5.1 Marburg forest soil................................................................................................32
II.5.2 Rauischholzhausen agricultural soil (SDS) ...........................................................34
II.5.3 Pure cultures ........................................................................................................35
II.5.4 Purification of RNA ...............................................................................................35
II.6 Amplification of DNA and cDNA by PCR......................................................................36
II.6.1 Amplification of pmoA...........................................................................................37
II.6.2 Amplification of amoA...........................................................................................38
II.6.3 Amplification of accA ............................................................................................38
II.6.4 Amplification of hydB ............................................................................................39
II.6.5 Amplification of archaeal and bacterial 16S rRNA genes and transcripts .............39
II.6.6 Gelelectrophoresis and purification.......................................................................39
II.6.7 Quantification........................................................................................................40
II.6.8 T-RFLP analysis ...................................................................................................40
II.7 Stable isotope probing (SIP) of nucleic acids...............................................................40
II.7.1 Incubation for SIP .................................................................................................41
II.7.2 RNA stable isotope probing ..................................................................................42
II.7.3 DNA stablerobing ..................................................................................45
II.8 Quantitative PCR (qPCR) ............................................................................................46
II.8.1 Solutions for qPCR ...............................................................................................46
II.8.2 qPCR assays........................................................................................................47
II.9 Phylogenetic analyses .................................................................................................50
II.9.1 Cloning .................................................................................................................50
II.9.2 Sequencing...........................................................................................................52
1Contents


II.9.3 Phylogenetic analyses..........................................................................................53
II.10 Fluorescence in situ hybridization (FISH)...................................................................53
II.10.1 Generation of RNA polynucleotide probes – in vitro transcription .......................53
II.10.2 Generation of expression clones as controls for FISH ........................................54
II.10.3 Separation of microbial cells from soil.................................................................55
II.10.4 Fixation of cells and environmental samples.......................................................56
II.10.5 RING-FISH targeting pmoA of USC α..................................................................57
II.10.6 CARD-FISH........................................................................................................64
II.10.7 Fluorescence microscopy ...................................................................................71
II.11 Enrichment of Upland Soil Cluster α ..........................................................................72
II.11.1 Soil substrate mem