Characterization of the microbial community in a biogas reactor supplied with organic residues [Elektronische Ressource] / Frank Bengelsdorf

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2012
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 11 Mo

Extrait

Characterization of the microbial
community in a biogas reactor
supplied with organic residues






Dissertation



Submitted for the fulfillment of the requirements for the doctoral degree Dr. rer. nat
at the Faculty of Natural Sciences, University of Ulm







By Frank Robert Bengelsdorf from Wismar
2011


































The current study was prepared at the Institute of Microbiology and Biotechnology,
University of Ulm.


Dekan: Prof. Dr. Axel Groß

1. Reviewer: Prof. Dr. Peter Dürre
2. Reviewer: Prof. Dr. Marian Kazda

Graduation date: 15-Dec.-2011
Contents I
Contents
Abbreviations ........................................................................................................................... IV
1 Introduction ............................. 1
2 Materials and Methods ........................................................................................................... 5
2.1 Biogas plant .......................................................................................................................... 5
2.2 Sampling ............................... 6
2.2.1 Standard sampling for genomic DNA extraction ............................................................ 6
2.2.2 Sampling for 454 Pyrosequencing .................................................. 7
2.2.3 Sampling for microscopy ................................ 8
2.3 DNA isolation and purification ........................................................................................... 9
2.3.1 Phenol chloroform extraction and ethanol precipitation ............... 10
2.3.2 Preparation of plasmid DNA from E. coli by alkaline lysis ......................................... 10
2.3.4 Nucleic acid enrichment and purification ..................................... 11
2.4 Amplification of genes ....................................................................... 12
2.4.1 Genes for construction of clone libraries ...................................... 13
2.4.2 Colony PCR .................................................................................. 15
2.4.3 Denaturing gradient gel electrophoresis (DGGE) ......................... 15
2.4.4 454 Pyrosequencing ...................................................................................................... 16
2.5.1 Direct cloning of amplified genes ................. 18
2.5.2 Competent E. coli DH5α cells ...................................................................................... 19
2.5.3 Heat shock transformation ............................ 20
2.5.4 Blue white screening ..................................................................................................... 20
2.5.5 Restriction fragment length polymorphism (RFLP) ..................................................... 20
2.6 DNA sequencing ................................................................................. 21
2.7 Phylogenetic analysis ......................................... 21
2.7.1 Clone library data .......................................................................... 21
2.7.2 Nucleotide sequences accession numbers ..... 22
2.7.3 Pyrosequencing data ..................................... 22II Contents


2.8 DGGE .................................................................................................................................. 23
2.8.1 Internal classification standard (ICS) ........... 25
2.8.2 Gel image analysis ........ 25
2.9 Microscopy .......................................................................................................................... 25
2.9.1 Fluorescence microscopy .............................. 25
2.9.2 Fluorescence in situ hybridization (FISH) .................................................................... 26
2.9.2.1 Standard slide preparation and probe hybridization protocol 28
2.9.2.2 Enhancing hybridization signals ............ 30
3 Results .................................................................................................................................... 32
3.1 Analysis of clone libraries .. 32
3.1.1 Molecular analysis ........................................................................................................ 32
3.1.2 Phylogenetic analysis of sequences .............. 33
3.2 DGGE .................................................................................................................................. 39
3.3 454 Pyrosequencing ............ 44
3.3.1 Species richness and diversity indices .......................................................................... 44
3.3.2 Phylogenetic distribution of dominating OTUs ............................ 47
3.4 Microscopic analyses .......................................................................................................... 53
3.4.1 Number of prokaryotic and eukaryotic organisms ....................... 53
3.4.2 Fluorescence in situ hybridization (FISH) .... 56
4 Discussion ............................................................................................................................... 57
4.1 Enumeration of prokaryotic and eukaryotic organisms in biogas reactor content ..... 57
4.2 Actual state and long-term stability of the microbial community in an anaerobic .........
biogas reactor utilizing food residues .............................................................................. 59
4.3 The biofilm-forming microbial community attached to straw improves the ...................
utilization of food residues in an anaerobic biogas reactor ............................................ 71 Contents I
5 Summary ................................................................................................................................ 85
6 Zusammenfassung ................................................................................................................. 87
7 References .............................................................................................................................. 89
8 Curriculum vitae ................................................................................................................. 102
9 Publications .......................... 103
10 Danksagung (Acknowledgments) .................................................................................... 104
11 Statement ........................................................... 106

IV Abbreviations

Abbreviations ddH O double distilled water 2
Dec. December
A
DGGE denaturing gradient gel
A adenine
electrophoresis
α alpha
DMSO dimethyl sulfoxide
AO acridine orange
DNA desoxyribonucleic acid
Ap ampicillin
dNTP deoxyribonucleotide
APS ammonium persulfate
triphosphate
ATP adenosine triphosphate
dsrA gen coding for
Aug. August
dissimilatory sulfite
Arc archaea
reductase


B
E
Bac bacteria
E. coli Escherichia coli
bap biofilm attached to particles
EDTA ethylenediaminetetraacetic
bas biofilm attached to straw
acid
β beta
et al. and others (et alii)
BLAST Basic Local Alignment

Search Tool
F
bp base pairs
Feb. February

Fig. Figure
C
FISH fluorescence in situ
C carbon
hybridization
°C degree Celcius
FITC fluoresceinisothiocyanat
CoA coenzyme A
frc fluid reactor content
Co. Company

conc. concentration
G

γ gamma
D
g gram
δ delta
G guanine
DAPI 4',6'-Diamidino-2-

phenylindole
H
DF DNA free
h hour Abbreviations V
H' Shannon index
no. number
I Nl norm litre
ICS internal classification Nov. November
standard
Inc. Incorporation O
IPTG isopropyl β-D Oct. October
thiogalactopyranoside OD optical density
OTU operational taxonomic
K units
k kilo
kWh kilowatt per hour P
PCR polymerase chain reaction
L PFA paraformaldehyde
l liter pH negative decimal
LB Luria Broth logarithm of the hydrogen
Ltd. Limited company ion activity
PIPES 1,4-Piperazinediethane-
M sulfonic acid
Mar. March pmoA gen coding for membrane-
-6µ micro, 10 bound methane
M molar monooxygenase
min minute
MSA multiple sequence R
alignment ® registered
mxaF gen coding for methanol RDP Ribosomal Database
dehydrogenase Project
rDNA ribosomal DNA
N RFLP restriction fragment length
-9n nano, 10 polymorphism
N nitrogen RNA ribonucleic acid
NCBI National Center for RNase ribonuclease
Biotechnology Information rRNA ribosomal RNA
database RT room temperature VI Abbreviations
S X
SDS sodium dodecyl sulfate X