Hydrogen Metabolizers: Drivers of Anaerobic Degradation Processes in Peatlands and Earthworm ...

248 Pages·2016·8.09 MB·English

Hydrogen Metabolizers: Drivers of Anaerobic Degradation
Processes in Peatlands and Earthworm Guts

Dissertation

To obtain the Academic Degree
Doctor rerum naturalium
(Dr. rer. nat.)
Submitted to the Faculty of Biology, Chemistry, and Geosciences
of the University of Bayreuth

by
Oliver Schmidt

Bayreuth, April 2016

This doctoral thesis was supervised by Prof. Harold L. Drake and prepared at the Department
of Ecological Microbiology, University of Bayreuth, from Januar 2010 until April 2016.

This is a full reprint of the dissertation submitted to obtain the academic degree of Doctor of
Natural Sciences (Dr. rer. nat.) and approved by the Faculty of Biology, Chemistery and
Geosciences of the University of Bayreuth.

Date of submission: 07.04.2016

Date of defence: 14.06.2016

Acting dean: Prof. Dr. Stefan Schuster

Doctoral committee:

Prof. Harold Drake, PHD (1st reviewer)
Prof. Dr. Ortwin Rabenbauer (2nd reviewer)
Prof. Dr. Dirk Schüler (chairman)
Prof. Dr. Egbert Matzner

“Thermodynamics sets the frame, evolution draws the picture.”
Barbara Schoepp-Cothenet (2013)

CONTENTS I

CONTENTS
CONTENTS ................................................................................................................. I
FIGURES ................................................................................................................... VI
TABLES .................................................................................................................. VIII
APPENDIX TABLES ................................................................................................. IX
EQUATIONS .............................................................................................................. X
ABBREVIATIONS ..................................................................................................... XI
1. INTRODUCTION .................................................................................................. 1
1.1. Peatlands: sources and sinks for greenhouse gases .......................................................... 1
1.1.1. Formation and classification of peatlands ............................................................................2
1.1.2. Vegetation and its effect on the microbial community in peatlands .......................................3
1.1.3. Flow of carbon and reductant in peatlands...........................................................................4
1.2. Earthworms: engineers that promote soil fertility ............................................................... 6
1.2.1. Earthworm ecotypes............................................................................................................6
1.2.2. Digestive system of Lumbricus terrestris ..............................................................................7
1.2.3. Activation of anaerobic soil microbes during gut passage ....................................................8
1.3. Microbes involved in the anaerobic degradation of organic matter in peat and the
gut of earthworms ................................................................................................................. 9
1.3.1. Primary fermenters ..............................................................................................................9
1.3.2. Secondary and syntrophic fermenters................................................................................ 10
1.3.3. Methanogens .................................................................................................................... 11
1.3.4. Acetogens ......................................................................................................................... 14
1.3.5. Alternative anaerobic respiratory processes ...................................................................... 15
1.3.6. Aerobic processes ............................................................................................................. 16
1.4. Hydrogenases and their contribution to the energy metabolism of H producers
2
and H consumers ............................................................................................................... 17
2
1.5. Hypotheses and objectives ................................................................................................. 20
2. MATERIALS AND METHODS ........................................................................... 22
2.1. Sampling site Fen Schlöppnerbrunnen .............................................................................. 22
2.1.1. Location and Sampling ...................................................................................................... 22
2.1.2. Microcosms experiments with peat soil and roots of Carex rostrata derived from the
Fen Schlöppnerbrunnen .................................................................................................... 23
2.1.2.1. Preparation and incubation conditions of anoxic microcosms in cellulose
degradation experiments (3.2.1)................................................................................. 23
II CONTENTS

2.1.2.2. Preparation and incubation conditions of anoxic root-free peat soil microcosms
and soil-free root microcosms (3.2.2) ......................................................................... 23
2.1.2.3. Preparation and incubation conditions of anoxic microcosms in syntrophic
oxidation experiments (3.2.2) ..................................................................................... 24
2.2. Experiments with gut contents of the earthworm Lumbricus terrestris ........................... 24
2.2.1. Source of samples used for the amplification of hydrogenase transcripts and genes
(3.3.1) ............................................................................................................................... 24
2.2.2. Preparation and incubation conditions of anoxic Lumbricus terrestris gut content
microcosms ....................................................................................................................... 25
2.2.2.1. Earthworms................................................................................................................ 25
2.2.2.2. Preparation of Saccharomyces cerevisiae cell lysates ................................................ 25
2.2.2.3. L. terrestris gut content and control microcosms ......................................................... 25
2.3. Solutions and growth media ............................................................................................... 26
2.3.1. Anoxic water ..................................................................................................................... 26
2.3.2. Anoxic mineral medium ..................................................................................................... 26
2.3.2.1. Mineral salt solution ................................................................................................... 27
2.3.2.2. Trace element solution ............................................................................................... 27
2.3.2.3. Vitamin solution.......................................................................................................... 27
2.3.3. Phosphate buffer ............................................................................................................... 28
2.3.4. SOC medium .................................................................................................................... 28
2.3.5. LB (lysogeny broth) agar plates ......................................................................................... 28
2.3.5.1. LB agar plates with ampicillin ..................................................................................... 29
2.3.5.2. LB agar plates with ampicillin/IPTG/X-Gal .................................................................. 29
2.3.6. Oxic S. cerevisiae growth medium ..................................................................................... 29
2.4. Analytical methods .............................................................................................................. 29
2.4.1. pH measurements ............................................................................................................. 29
2.4.2. Dry weight and moisture content of soils ............................................................................ 29
2.4.3. Gases ............................................................................................................................... 30
2.4.4. Soluble organic compounds .............................................................................................. 31
2.5. Molecular methods .............................................................................................................. 32
2.5.1. Extraction of nucleic acids ................................................................................................. 32
2.5.2. Purification and precipitation of nucleic acids ..................................................................... 33
2.5.2.1. Isopropanol precipitation ............................................................................................ 33
2.5.2.2. Gel extraction ............................................................................................................. 33
2.5.3. Quantification of nucleic acids ........................................................................................... 33
2.5.3.1. NanoDrop-based quantification .................................................................................. 33
2.5.3.2. Pico- and Ribogreen-based quantification .................................................................. 33
2.5.4. Agarose gel electrophoresis .............................................................................................. 34
2.5.5. 16S rRNA-based stable isotope probing (SIP) ................................................................... 34
2.5.5.1. Density gradient centrifugation ................................................................................... 34
2.5.5.2. Fractionation .............................................................................................................. 35
2.5.5.3. Determination of the CsTFA buoyant density of fractions ............................................ 35
2.5.5.4. RNA precipitation ....................................................................................................... 36
2.5.6. Reverse transcription of RNA into cDNA ............................................................................ 37
2.5.7. Polymerase chain reaction (PCR) ...................................................................................... 38
2.5.7.1. Amplification of inserts in vector plasmids of (M13-PCR) ............................................ 38
2.5.7.2. Bacterial and archaeal 16S rRNA PCR for cloning ...................................................... 38
2.5.7.3. Bacterial 16S rRNA PCR for Illumina sequencing ....................................................... 39
2.5.7.4. Hydrogenase specific PCR......................................................................................... 40
2.5.8. Construction of clone libraries ........................................................................................... 41
CONTENTS III

2.5.8.1. Ligation ...................................................................................................................... 42
2.5.8.2. Transformation ........................................................................................................... 42
2.5.8.3. Blue/white screening .................................................................................................. 42
2.5.9. Sequencing by chain-termination ....................................................................................... 43
2.5.10. Sequencing by synthesis ................................................................................................... 43
2.6. Sequence analyses and phylogenetic calculations ........................................................... 43
2.6.1. Analyses of sequences derived from clone libraries ........................................................... 43
2.6.1.1. Alignment of 16S rRNA sequences and check for chimeric sequences ....................... 44
2.6.1.2. Clustering of 16S rRNA sequences into OTUs ............................................................ 44
2.6.1.3. Alignment of hydrogenase gene and transcript sequences ......................................... 44
2.6.1.4. Calculation of similarity correlation plots ..................................................................... 45
2.6.1.5. Clustering of hydrogenase gene and transcript sequences into OTUs ........................ 45
2.6.2. Analyses of Illumina sequencing-derived data ................................................................... 45
2.6.3. Coverage and rarefaction analyses ................................................................................... 46
2.6.4. Calculation of phylogenetic trees ....................................................................................... 46
2.6.5. Nucleotide sequence accession numbers .......................................................................... 47
2.7. Calculations and statistical analyses ................................................................................. 47
2.7.1. Carbon and electron balances ........................................................................................... 47
2.7.1.1. Recoveries of carbon and reductant in cellulose-supplemented peat soil
microcosms (3.2.1) .................................................................................................... 47
2.7.1.2. Recoveries of carbon and reductant in soil-free root and root-free soil
microcosms (3.2.2) .................................................................................................... 47
2.7.1.3. Recoveries of carbon and reductant in peat soil microcosms supplemented with
ethanol, butyrate, or propionate (3.2.3)....................................................................... 47
2.7.1.4. Recoveries of carbon and reductant in earthworm gut content microcosms
supplemented with S. cerevisiae cell lysate ................................................................ 48
2.7.2. Thermodynamic calculations ............................................................................................. 49
2.7.3. Average and standard deviation ........................................................................................ 50
2.8. Chemicals, gases, and labware .......................................................................................... 51
2.9. Contribution of other workers to this dissertation............................................................. 51
2.9.1. Hydrogenase primer design and hydrogenase gene and transcript sequence analyses ..... 51
2.9.2. Experiments with peat soil microcosms ............................................................................. 51
2.9.2.1. Cellulose degradation experiments ............................................................................. 51
2.9.2.2. Syntrophic oxidation experiments ............................................................................... 52
2.9.2.3. Experiments with root-free soil and Carex roots .......................................................... 52
2.9.3. Experiments with earthworm gut contents.......................................................................... 52
2.9.3.1. Glucose supplemented gut content microcosms ......................................................... 52
2.9.3.2. Gut content microcosms supplemented with S. cerevisiae cell lysate .......................... 52
3. RESULTS ........................................................................................................... 53
3.1. Hydrogenases as molecular markers for H -producing and H -consuming
2 2
prokaryotes .......................................................................................................................... 53
3.1.1. Design of PCR primers specific for hydrogenase genes ..................................................... 53
3.1.2. Criteria for establishing hydrogenase OTUs ....................................................................... 54
3.2. Anaerobic mineralization of plant-derived organic carbon and associated
prokaryotic taxa in peatlands.............................................................................................. 57
3.2.1. Degradation of cellulose by peat soil anaerobes ................................................................ 57
IV CONTENTS

3.2.1.1. Effect of cellulose on the flow of carbon and reductant in anoxic peat soil
microcosms at 5°C and 15°C ..................................................................................... 57
3.2.1.2. Active bacterial taxa linked to the degradation of [13C]cellulose ................................... 61
3.2.1.3. Active but not labeled bacterial taxa ........................................................................... 65
3.2.1.4. Active archaeal taxa in cellulose-supplemented anoxic peat soil microcosms at
5°C and 15°C ............................................................................................................ 66
3.2.2. Effect of roots from the peat soil covering sedge Carex rostrata on H metabolizers in
2
the rhizosphere ................................................................................................................. 69
3.2.2.1. H -metabolizing processes in formate-supplemented and unsupplemented root-
2
free soil and soil-free root microcosms ....................................................................... 69
3.2.2.2. H -metabolizing taxa associated with Carex roots ...................................................... 74
2
3.2.3. Syntrophic oxidation of ethanol, butyrate, and propionate by peat soil anaerobes .............. 82
3.2.3.1. Preincubation of anoxic peat soil microcosms............................................................. 82
3.2.3.2. Anaerobic flow of endogenous carbon and reductant in unsupplemented anoxic
peat soil microcosms ................................................................................................. 84
3.2.3.3. Effect of supplemental ethanol, butyrate, and propionate in anoxic peat soil
microcosms ............................................................................................................... 87
3.2.3.4. Thermodynamics of processes potentially linked to syntrophic methanogenesis ......... 93
3.2.3.5. Microbial community of fresh peat .............................................................................. 95
3.2.3.6. Bacteria involved in the anaerobic mineralization of endogenous carbon sources
in unsupplemented controls ....................................................................................... 96
3.2.3.7. Bacterial taxa potentially linked to syntrophic processes ............................................. 97
3.2.3.8. Archaeal taxa linked to syntrophic processes ........................................................... 100
3.3. Fermentation processes and associated taxa in the gut of the earthworm
Lumbricus terrestris .......................................................................................................... 103
3.3.1. Hydrogenase transcript analyses in glucose supplemented anoxic gut content
microcosms ..................................................................................................................... 103
3.3.1.1. [FeFe]-hydrogenase gene transcript diversity ........................................................... 103
3.3.1.2. Group 4 [NiFe]-hydrogenase gene and transcript diversity ........................................ 105
3.3.2. Bacterial taxa involved in the degradation of microbial cells in the gut of L. terrestris ........ 108
3.3.2.1. Fermentation profile of anoxic earthworm gut content microcosms supplemented
with lysed S. cerevisiae cells .................................................................................... 108
3.3.2.2. Effect of lysed S. cerevisiae cells on the composition and activity of the bacterial
community in gut content microcosms ...................................................................... 111
4. DISCUSSION ....................................................................................................115
4.1. Cooperation and competition: interactions between H -metabolizing and other
2
microbes in peatlands ....................................................................................................... 115
4.1.1. Dynamics in fermenter community compositions and product profiles in respond to
changing availabilities of plant-derived organic carbon in peatlands ................................. 115
4.1.1.1. Novel taxa replace model cellulolytic fermenters in cold, acidic, and substrate-
limited peatland soils................................................................................................ 116
4.1.1.2. Cooperation instead of competiton: a model for synergism between cellulolytic
and noncellulolytic fermenters in the fen ................................................................... 118
4.1.1.3. Growth yield vs. growth rate: how the fermentation profile in peatlands is affected
by substrate availability ............................................................................................ 120
4.1.1.4. The rhizosphere of sedges: a hotspot for H -metabolizing fermenters in
2
peatlands ................................................................................................................. 124
4.1.2. Trophic interactions between novel syntrophs and acetate- and H -scavenging
2
methanogens at 5°C and 15°C ........................................................................................ 126