Microbial community metabolic concurrence involved in toluene degradation [Elektronische Ressource] : effect of oxygen availability on catabolic gene expression of aerobic and anaerobic toluene degrading bacteria / by Paula Martínez Lavanchy

Mpaula

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2009
• Nombre de lectures: 18
• Poids de l'ouvrage: 1 Mo

Extrait

Microbial community metabolic concurrence
involved in toluene degradation

Effect of oxygen availability on catabolic gene expression
of aerobic and anaerobic toluene degrading bacteria





Dissertation
to obtain the academic grade
doctor rerum naturalium
(Dr. rer. nat.)



Presented to the Faculty of Biology-Pharmacy
Friedrich-Schiller University Jena



by
Paula Martínez Lavanchy

born on 21.03.1978 in Stockholm





Microbial community metabolic concurrence
involved in toluene degradation

Effect of oxygen availability on catabolic gene expression
of aerobic and anaerobic toluene degrading bacteria






Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)



vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena



von
Paula Martínez Lavanchy

geboren am 21.03.1978 in Stockholm


Table of contents

Table of contents
ABBREVIATIONS III
SUMMARY V
ZUSAMMENFASSUNG VII
1. INTRODUCTION 1
1.1. ENVIRONMENTAL POLLUTANTS: BTEX COMPOUNDS 1
1.2. REMEDIATION OF CONTAMINANTS: MICROBIAL DEGRADATION 3
1.3. DETECTION OF MICROBIAL ACTIVITY IN THE ENVIRONMENT 4
1.4. OXIC-ANOXIC GRADIENTS IN THE ENVIRONMENT 6
1.5. MICROBIAL TOLUENE DEGRADATION 9
1.5.1. Toluene degradation by Pseudomonas putida mt-2 9
1.5.2. Toluene degradation by Thauera aromatica K172 13
2. GOAL OF THE THESIS 18
3. MATERIALS AND METHODS 19
3.1. SOLUTIONS AND MEDIA 19
3.1.1. P. putida agar plates media 19
3.1.2. Thauera aromatica K172-Medium (TAM media) 20
3.2 BACTERIAL STRAINS AND GROWTH CONDITIONS 21
3.2.1. Pseudomonas putida mt-2 21
3.2.2. Growth of P. putida mt-2 in Fermentor 21
3.2.3. Thauera aromatica K172 23
3.3. ANALYTICAL METHODS 23
3.3.1. Toluene 23
3.3.2. Dissolved Oxygen measurement 24
3.3.3. ATP 25
3.3.4. Nitrate and Nitrite concentrations 27
3.4. MOLECULAR BIOLOGY METHODS 27
3.4.1. Real Time PCR 27
3.4.2. RNA extraction 29
3.4.3. RNA quantification 30
3.4.4. cDNA synthesis 31
3.4.5. Primer optimisation 31
3.4.6. Real Time PCR reaction 33
4. RESULTS 34
4.1. OPTIMISATION OF GROWTH CONDITIONS AND REAL TIME PCR 34
4.1.1. Bacterial growth conditions 34
4.1.2. Primer selection and calibration curves 36
4.2. CATABOLIC GENE EXPRESSION OF P. PUTIDA MT-2 AND T. AROMATICA K172 DURING TOLUENE
DEGRADATION 38
4.2.1. Gene expression of xylM and xylE during toluene degradation in aerobic conditions in batch
cultures 38
4.2.2. Gene expression of xylM and xylE during a batch fermentation using toluene as carbon source
42
4.2.3. Gene expression of bssA and bcrA during toluene degradation in anaerobic conditions 45
- I -
Table of contents
4.3. EFFECT OF OXYGEN AVAILABILITY ON CATABOLIC GENE EXPRESSION OF TOLUENE DEGRADING
BACTERIA 50
4.3.1. Pseudomonas putida mt-2 50
4.3.2. Thauera aromatica K172 56
4.4. EFFECT ON CATABOLIC GENE EXPRESSION IN P. PUTIDA MT-2 DURING OXYGEN OSCILLATIONS 59
4.4.1. Batch shaking cultures 60
4.4.2. Continuous Fermentation 64
4.5. EFFECT OF OXYGEN FLUCTUATION ON CATABOLIC GENE EXPRESSION IN A MIXED CULTURE FORMED BY
P. PUTIDA MT-2 AND T. AROMATICA K172 68
5. DISCUSSION 75
6. REFERENCES 86
ACKNOWLEDGEMENTS 96
DECLARATION OF INDEPENDENT WORK 98
CURRICULUM VITAE 99



- II -
Abbreviations

Abbreviations
A Adenine
ATP Adenosine triphosphate
(ATP) ATP concentration of the standard St
bp basepare(s)
BTEX Benzene, Toluene, Ethylbenzene, Xylene
°C degree Celsius
C concentration
C ATP concentration of the sample ATP
cDNA complementary desoxyribonucleic acid
C threshold cycle t
D.F. dilution factor
DNA desoxyribonucleic acid
dO dissolved oxygen 2
D.W. dry weight
E efficiency
ENA enhanced natural attenuation
et al. et altera (and others)
F forward primer
Fig. figure
g gram
G Guanine
GC-FID gas chromatography with flame ionization detector
h hour
I light intensity of the sample 1
I light intensity of the sample with internal ATP standard 2
kDa kilo Dalton
L litre
M molar
mg milligram
mL millilitre
mM millimolar
MNA monitored natural attenuation
- III -
Abbreviations

mRNA messenger ribonucleic acid
mV millivolt
g microgram
l microlitre
M micromolar
NA natural attenuation
ng nanogram
nm nanometer
OD optical density at wavelength 560 nm 560
PCR polymerase chain reaction
pH potentia hydrogenii
pO partial oxygen pressure 2
R ratio relative gene expression
R reverse primer
RNA ribonucleic acid
rpm rounds per minute
rRNA ribosomal ribonucleic acid
RT reverse transcription
s second(s)
T Thymine
TAM-Medium Thauera aromatica K172 Medium
T annealing temperature ann
Taq polymerase gained from Thermus aquaticus
T melting temperature m
UV/VIS ultraviolet visible
w/v weight per volume


- IV -
Summary

Summary
Bacteria in the environment are constantly exposed to oxygen variations and
gradients as they occur, e.g., in aquifers, in microbial mats and the plant root and the
rhizosphere. Microorganisms living in polluted sites have the ability to process these
input signals in order to develop adaptive responses to survive fluctuations of external
conditions.
The relative expression of catabolic genes under oscillating oxygen conditions,
from two toluene degrader bacteria capable of aerobic toluene degradation, Pseudomonas
putida mt-2 (contains the TOL plasmid pWW0), and an anaerobic toluene degrader,
Thauera aromatica K172, was studied. The catabolic genes targeted were xylM and xylE
for P. putida and bssA and bcrA for T. aromatica.
The main results of this study can be summarised as follows:
1. Real-time PCR primer sets were developed in order to detect and quantify the
catabolic genes xylM, xylE, bssA and bcrA in pure cultures, as well as in a
binary mixed culture of Pseudomonas putida mt-2 and Thauera aromatica
K172.
2. A decrease in the expression level of xylM and xylE was observed under
oxygen limiting conditions when Pseudomonas putida mt-2 was grown on
toluene as carbon source. Thus, oxygen is needed as a kind of co-inducer for
the expression of the catabolic genes of the TOL plasmid.
3. The addition of oxygen to Thauera aromatica K172 cultures grown with
toluene as the carbon source immediately halted toluene degradation, bacterial
growth and denitrification processes. A repression of bssA and bcrA
expression was observed in the presence of oxygen.
4. Pseudomonas putida mt-2 is able to modulate the expression of xylM and xylE
according to the oxygen availability in the media. During anoxic periods these
bacteria decrease the growth rate and the expression of catabolic genes to a
level which allow them to recover the activity when oxygen is present again in
the medium.

- V -
Summary

5. In mixed binary cultures of P. putida and T. aromatica, submitted to
anoxic/oxic cycles, a regulation of catabolic genes depending on the presence
of oxygen was observed. After two oxic cycles T. aromatica showed an up-
regulation of catabolic genes once oxygen was depleted by P. putida.

This thesis showed that catabolic gene expression of T. aromatica and P. putida is
affected by the presence or lack of oxygen. Moreover, both aerobic and anaerobic
bacteria are able to modulate the expression of catabolic genes depending on the oxygen
availability. This fact allows them to react quickly with respect to catabolic gene
expression, once the favourable growth conditions are restored regarding oxygen
concentration. Additionally, bacteria showed stability and physiological fitness under
unfavourable conditions regarding oxygen availability. All these findings give the
possibility to think about new approaches for bioremediation, exploiting the idea of cyclic
aerobic-anoxic conditions to bioengineer polluted sites in order to accelerate
biodegradation processes.










- VI -
Zusammenfasung

Zusammenfassung
Bakterien sind in ihrer natürlichen Umgebung permanent mit variierenden
Sauerstoffgradienten, wie sie z.B. in Grundwasserleitern, mikrobiellen Matten und im
Wurzelraum von Pflanzen, der Rhizosphäre, existieren, konfrontiert. Schadstoff-
abbauende Mikroorganismen haben zudem die Fähigkeiten entwickelt, sich mit Hilfe
adaptiver Mechanismen und der Regulation ihrer katabolen Gene an die sich permanent
ändernden Umweltbedingungen kontaminierter Standorte anzupassen.
In dieser Arbeit wurde die relative Expression kataboler Gene unter