Characterization of methanogenic Archaea communities in biogas reactors by quantitative PCR

187 Pages·2012·2.29 MB·English

Characterization of methanogenic Archaea communities in
biogas reactors by quantitative PCR

vorgelegt von
Ingo Bergmann
aus Parchtitz auf Rügen

Von der Fakultät III - Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
Dr. rer. nat.

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr.-Ing. Sven-Uwe Geißen
Berichter: Prof. Dr. rer. nat. Ulrich Szewzyk
Berichter: PD Dr. tech. Elisabeth Grohmann
Berichter: Dr. rer. nat. Michael Klocke

Tag der wissenschaftlichen Aussprache: 19.09.2011

Berlin 2012
D-83
Zwei Dinge sind zu unserer Arbeit nötig:
Unermüdliche Ausdauer und die Bereitschaft,
etwas, in das man viel Zeit und Arbeit gesteckt hat,
wieder wegzuwerfen…

Albert Einstein
TABLE OF CONTENTS

Table of contents

1. Abbreviations .................................................................................... 6
2. Abstract ........................................................................................... 10

3. Zusammenfassung ......................................................................... 12
4. Introduction ..................................................................................... 14
4.1 The politics .................................................................................................. 14
4.2 The practice ................................................................................................. 16
4.3 The process ................................................................................................. 17
4.3.1 The four-stage pathway of anaerobic degradation of organic material to
biogas ........................................................................................................... 17
4.3.2 Methanogenesis .................................................................................. 19
4.4 The producers ............................................................................................. 20
4.5 The technique ............................................................................................. 23
4.5.1 The basics of Q-PCR applications ....................................................... 24
4.5.2 Fluorescent probes and dyes ............................................................... 27
4.5.3 Application of Q-PCR in environmental microbiology .......................... 30
4.5.4 Factors for a successful Q-PCR run .................................................... 31
4.5.5 The target genes .................................................................................. 33
4.6 The aims ...................................................................................................... 35
5. Materials and Methods .................................................................... 37
5.1 Model biogas reactors ................................................................................ 37
5.1.1 System 1 .............................................................................................. 37
5.1.2 System 2 .............................................................................................. 37
5.1.3 System 3 .............................................................................................. 38
5.1.4 System 4 .............................................................................................. 38
5.2 Agricultural biogas plants .......................................................................... 42
5.3 Physical and chemical analyses of the biogas and the reactor content 44
5.3.1 Determination of the pH value ............................................................. 44
5.3.2 Calculation of the gas composition ...................................................... 44
5.3.3 Determination of the acid composition ................................................. 44

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TABLE OF CONTENTS

5.4 DNA-based analysis of the archaeal community structure ..................... 45
5.4.1 Used strains ......................................................................................... 45
5.4.2 DNA extraction and purification ........................................................... 45
5.4.3 DNA quantification ............................................................................... 48
5.4.4 Analysis of the DNA purity ................................................................... 49
5.4.5 Quantitative real-time PCR (Q-PCR) ................................................... 49
6. Results ............................................................................................. 65
6.1 Establishment and application of a Q-PCR assay for the detection of
methanogenic Archaea in biogas plants by the use of the 16S rRNA gene 65
6.1.1 Optimization of the PCR conditions of the group-specific 16S rRNA
gene assays for quantitative real-time PCR .................................................. 65
6.1.2 Influence of DNA isolation on Q-PCR-based quantification of
methanogenic Archaea in biogas fermenters ................................................ 67
6.1.3 Accuracy of the real-time PCR assays and influence of PCR interfering
substances on Q-PCR-based quantification of methanogenic Archaea in
biogas fermenters ......................................................................................... 75
6.1.4 Application of the 16S rRNA gene real-time PCR assays for analyzing
the composition and development of the methanogenic Archaea in meso- and
thermophilic biogas reactors ......................................................................... 84
6.2 Development of group-specific primer sets for the detection of
methanogenic Archaea in biogas plants by the use of the metabolic
mcrA gene ....................................................................................................... 107
7. Discussion ..................................................................................... 123
7.1 Evaluation and optimization of the PCR conditions for amplifying the
16S rRNA gene by using the real-time PCR assay of Yu et al. (2005a) ...... 123
7.2 Design and testing of group-specific Q-PCR primers based on the
mcrA gene for the quantification of methanogenic communities .............. 125
7.3 The influence of different DNA isolation methods on the quantification
of methanogenic Archaea in biogas reactors by real-time PCR................. 127
7.4 Influences of PCR interfering substances on Q-PCR-based
quantification of methanogens in biogas reactors ...................................... 131

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TABLE OF CONTENTS

7.5 Determination of methanogenic Archaea abundances in
semi-continuous fermentation and acidification by overloading in a
short-run experiment ...................................................................................... 136
7.6 Methanogenic population dynamics in semi-continuous fermentation
and acidification by overloading under mesophilic and thermophilic
conditions in a long-run experiment ............................................................. 139
7.7 Determination of the methanogenic community in biogas reactors with
different substrates for anaerobic digestion under mesophilic and
thermophilic conditions ................................................................................. 143
7.8 Determination of the methanogenic Archaea in agricultural biogas
plants ............................................................................................................... 145
8. Outlook .......................................................................................... 148
References ......................................................................................... 149
List of figures .................................................................................... 168
List of tables ...................................................................................... 171
Publication list .................................................................................. 174
Funding .............................................................................................. 178
Acknowledgments ............................................................................ 179
Appendix............................................................................................ 182

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ABBREVIATIONS

1. Abbreviations

A Adenine
ABI Applied Biosystems Instruments
AF Anaerobic filter
approx. Approximately
ARC Archaea
BA Biogas plant
BAC Bacteria
BB Brandenburg
bp Base pair
C Cytosine
Carrez I Potassium hexacyanoferrate(II)-3-hydrate
Carrez II Zinc sulphate-7-hydrate
cf. Confer
CSTR Continuously stirred tank reactor
C Threshold cycle number
T
CTAB Cetyl trimethyl ammonium bromide
DGGE Denaturing gradient gel electrophoresis
DNA Deoxyribonucleic acid
dNTP Deoxynucleoside triphosphate
DOM Dry organic matter
dsDNA Double-stranded deoxyribonucleic acid
e.g. Exempli gratia (for example)
EDTA Ethylenediaminotetraacetic acid
et al. Et alii (and others)
F primer Forward primer
FAM 6-Carboxyfluoroscein
Fig. Figure
FISH Fluorescence in-situ hybridization
FNR Fachagentur Nachwachsende Rohstoffe e.V.
FR Hydrolysis reactor
FRET Fluorescence resonance energy transfer
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ABBREVIATIONS

g Gravitational acceleration
G Guanidine
GC Gas chromatography
gDNA genomic deoxyribonucleic acid
IPTG Isopropyl β-D-1-thiogalactopyranoside
JOE 2,7-Dimethoxy-4,5-dichloro-6-carboxyfluorescein
LB Lysogeny broth
LOD Limit of detection
LOQ Limit of quantification
Mb. Methanobacterium
Mbb. Methanobrevibacter
Mbp Mega base pair
MBT Methanobacteriales
Mbt. Methanothermobacter
Mc. Methanococcus
MCC Methanococcales
Mcc. Methanococcoides
Mcd. Methanocaldococcus
MCR Methyl-coenzyme M reductase enzyme complex
Mcr. Methanocorpusculum
mcrA Methyl-coenzyme M reductase sububit α
Mcu. Methanoculleus
Mf. Methanofollis
Mg. Methanogenium
Mha. Methanohalophilus
Mm. Methanomicrobium
MMB Methanomicrobiales
Mml. Methanomethylovorans
Mpr. Methanosphaera
Mpy. Methanopyrus
Msa. Methanosaeta
Msc Methanosarcinaceae
Msp. Methanospirillum
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ABBREVIATIONS

Msr. Methanosarcina
Mss. Methanosalsum
Mst Methanosaetaceae
Mtc. Methanothermococcus
Mth. Methanothermus
Mts. Methanotorris
MV Mecklenburg-Vorpommern
NA Not analyzed
ND Not detected
OLR Organic loading rate
OTU Operational taxonomic unit
PCR Polymerase chain reaction
PET Polyethylene terephthalate
Q-PCR Quantitative real-time PCR
R primer Reverse primer
rDNA Ribosomal deoxyribonucleic acid
RFLP Restriction fragment length polymorphism
RNA Ribonucleic acid
rpm Revolutions per minute
rRNA Ribosomal ribonucleic acid
RT Retention time
S Sachsen
SA Sachsen-Anhalt
SD Standard deviation
SDS Sodium dodecyl sulphate
SSD Dilution of the standard series
ssp. Subspecies
T Thymine
TAMRA 6-Carboxytetramethylrhodamine
TET Tetrachloro-6-carboxyfluorescein
T Melting temperature
m
T-RFLP Terminal restriction fragment length polymorphism
Tris Trishydroxymethylaminomethane
8
ABBREVIATIONS

UV Ultraviolet
VFA Volatile fatty acid
X-Gal 5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside

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ABSTRACT

2. Abstract

Energy production from renewable raw material is of increasing importance. Biogas is
one of the major renewable energy sources ensuring an adequate energy supply for
the next generations. Beside technical optimization and upgrading of biogas reactors
and plants, detailed information about the diversity and composition of the
participating microbial community structure is indispensable for optimizing the
biogas-forming process. Furthermore, precise knowledge about the metabolic activity
of the microorganisms and their optimal growth conditions are of utmost importance
to ensure the maximal degradation of the substrates to biogas.

The main objective of this study was the establishment of a highly sensitive and
culture-independent approach for the detection and quantification of methanogenic
Archaea in biogas reactors and plants at the taxonomic level of orders and families.
The method of choice was the quantitative real-time PCR (Q-PCR).

Initially, DNA extraction was optimized for samples taken from biogas reactors
because Q-PCR results are strongly influenced by the DNA quality. Harsh DNA
extraction with bead-beating cell lysis was most efficient while soft DNA extraction led
to a discrimination of certain taxonomic groups like Methanosaetaceae. Hence, a
combined mechanic and chemical cell lysis was used for isolating DNA from biogas
reactor samples.

After finding the most efficient DNA extraction protocol, primer sets which were
developed for the detection of the 16S rRNA gene by Yu et al. (2005a) were
optimized. Therefore, an adaptation of the PCR protocol for the ABI system was
successfully conducted. Subsequently, this molecular genetic approach was used for
the analysis of the quantitative distribution of methanogenic Archaea in biogas
fermenters and plants.

A great variability in the composition of the methanogenes was observed at
mesophilic conditions. Depending on the chosen substrate hydrogenotrophic or
acetotrophic methanogenes were most abundant in laboratory CSTRs.
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