Plant-associated bacteria:

Plant-associated bacteria:
An important key to a successful application of TCE phytoremediation
Nele Weyens and Jaco Vangronsveld
Phytoremediation of organic contaminants
Plantgeassocieer
de
bacteriën
in
bodemsane
ring, van
labo tot
Plantgeassocieer
de
bacteriën
in
bodemsane
ring, van
labo tot
veldschaal
veldschaal
Evapotranspiration?
Degradation
Plantgeassocieer
de
bacteriën
in
bodemsane
ring, van
labo tot
Xylem vessels
Rhizosphere
veldschaal
Can the tree reach the contaminants?
Can the contaminants reach the tree?
Phytoremediation of organic contaminants
Plantgeassocieer
de
bacteriën
in
bodemsane
ring, van
labo tot
Plantgeassocieer
de
bacteriën
in
bodemsane
ring, van
labo tot
Evapotranspiration?
Phytotoxicity?
veldschaal
Degradation
Plantgeassocieer
de
bacteriën
in
bodemsane
ring, van
labo tot
veldschaal
Xylem vessels
Rhizosphere
veldschaal
Can the tree reach the contaminants?
Can the contaminants reach the tree?
A tree is much more than you can see!
Plantgeassocie
erde
bacteriën
in
bodemsa
nering,
van labo
tot
Plantgeassocie
erde
bacteriën
in
bodemsa
nering,
van labo
tot
veldschaal
veldschaal
Marlene Cameron and Sheng-Yang He
Michigan State University
Plantgeassocie
erde
bacteriën
in
bodemsa
nering,
van labo
tot
veldschaal
Hardoim et al., 2008
Bacteria: you can find them everywhere!
First impression…
?
Bacteria: you can find them everywhere!
Bacterial cells are much smaller than human cells.
There are at least 10 times as many bacteria as
human cells in our body!!
In average, half a kilo
of our body weight are
bacteria!!!
Bacteria:
the fingerprint of the future?
Bacteria: humans probiotics!
Bacteria: plants probiotics!
Root mass (g)
Leaf mass (g)
Bacteria can promote plant growth
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
5.00
Controle
W619
W619 + gfp
Controle
W619
W619 + gfp
4.00
3.00
2.00
1.00
0.00
Bacteria: plants probiotics!
Direct growth promotion:
- Increase the availability of:
N (e.g. Rhizobium)
P
Fe
 Biofertilizers
N2
- Production of plant growth hormones
Auxins, gibberilins, cytokinins
- Production of stress reducing enzyms
e.g. ACC-deaminase
Indirect growth promotion:
compitition between
plant growth promoting bacteria
and pathogens
NH4+
Bacteria: important players during phytoremediation
of organic contaminants
How can bacteria assist their hostplant?
Evapotranspiration?
• Growth promotion
• Production of surfactants,
organic acids, siderophores
Phytotoxicity?
Xylem vessels
• Degradation of the contaminant
Degradation
Rhizosphere
Can the tree reach the contaminants?
Can the contaminants reach the tree?
- Rhizosphere:
high diversity vs short contact time
-Xylem vessels:
lower diversity vs long contact time
Rhizosphere
Rhizosphere + Endosphere
CO2 2
CO
CO
CO
CO
CO
2222
TCE
TCE
TCE
TCE
TCE TCE
TCE
TCE
CO2
CO2
TCE
TCE
TCE
TCE
TCE TCE
TCE
TCE
TCE
TCE
TCE
TCE
TCE
TCE
TCE TCE
TCE
TCE
CO2
CO2
TCE
TCE
TCE
TCE
TCE TCE
TCE
TCE
TCE
TCE
TCE
What is the ideal bacterial strain?

Degradation genes on
mobile DNA: plasmids
Endophyte
Degrader
 Long contact time between
contaminant and degrader
 Easier to introduce
PGPB
How to create and introduce the ideal bacterial strain?
What do we have?
Soil bacteria with
degradation plasmid
Introduction by means of inoculation
PGP endophytes
 The ideal strain can be created by natural conjugation
What is the ideal bacterial strain?
 For phytoremediation of TCE using poplar
TCE degrading soil bacterial strain:
Burkholderia cepacia BU61
Plant growth promoting poplar endophyte:
Pseudomonas putida W619
Natural gene transfer
The TCE degrading,
plant growth promoting
poplar endophyte
Pseudomonas putida W619-TCE
TCE genes
Endophyte-enhanced TCE phytoremediation:
From the lab to the field
Endophyte-enhanced TCE phytoremediation:
From the lab to the field
How to measure TCE evapotranspiration?
Endophyte-enhanced TCE phytoremediation:
From the lab to the field  LAB experiments
TCE phytotoxicity
Control
25
**
**
20
*
Shoot mass (g)
Shoot mass (g)
W619
15
10
5
0
200
400
30
***
**
0
200
400
0
200
400
25
***
Root mass (g)
Root mass (g)
0
9
8
7
6
5
4
3
2
1
0
90
80
70
60
50
40
30
20
10
0
20
15
10
5
0
0
200
TCE exposure (mg l-1)
400
TCE exposure (mg l-1)
Endophyte-enhanced TCE phytoremediation:
From the lab to the field  LAB experiments
TCE evapotranspiration
control
ng TCE cm-2 h-1
4
W619
3
3
2
***
2
1
1
0
200
400
TCE exposure (mg l-1)
Endophyte-enhanced TCE phytoremediation:
From the lab to the field  application in the field
Site background
Planted poplar trees
Mixed wood of english oak and common ash
All cultivable oak- and ash-associated bacteria were isolated,
identified and tested for TCE tolerance and degradation
TCE μg/l
TCE-contamination
Endophyte-enhanced TCE phytoremediation:
From the lab to the field  application in the field
TCE tolerance and degradation
3%
82%
Is this TCE degradation enough to prevent TCE evapotranspiration?
Ash: 10.84*10-3 ± 1.17*10-3 ngTCE/cm²h
Oak: 6.35*10-3 ± 0.18*10-3 ngTCE/cm²h
Endophyte-enhanced TCE phytoremediation:
From the lab to the field  application in the field
Poplar trees were planted on
the TCE contamination plume
Planted poplar trees
Mixed wood of english oak and common ash
TCE μg/l
TCE-contamination
The trees were provided with a drainage tube for inoculation
Which bacterial strain was inoculated?
The TCE degrading,
plant growth promoting
poplar endophyte
Pseudomonas putida W619
TCE genes
Endophyte-enhanced TCE phytoremediation:
From the lab to the field  application in the field
The in situ TCE evapotranspiration was measured
10
10
Before inoculation
9
9
8
10-2 ng cm-2 h-1
8
10-2 ng cm-2 h-1
3 months after inoculation
7
6
5
4
3
2
7
6
5
4
3
2
1
1
0
0
Control
W619
Control
W619
Endophyte-enhanced TCE phytoremediation:
From the lab to the field  application in the field
Were the inoculated strain and its degradation genes introduced successfully?
3 months after inoculation
Stem:
The inoculated P. putida W619
with the TCE genes could not
be re-isolated
In situ inoculation
But
The inoculated TCE genes
Could be found in other, natural
abundant stem endophytes
P. putida W619
Horizontal gene transfer
TCE genes
Roots:
The inoculated P. putida W619 with the TCE
genes could be re-isolated in very high numbers
Conclusions
Evapotranspiration?
1) Degradation capacity is present
in the natural abundant population
Phytotoxicity?
Degradation
Degradation genes
Can the tree reach the contaminants?
Can the contaminants reach the tree?
Conclusions
Evapotranspiration?
2) Degradation capacity is NOT (or not
enough) present in the natural
abundant population
 INOCULATION
Phytotoxicity?
Degradation
Degradation genes
Can the tree reach the contaminants?
Can the contaminants reach the tree?
Conclusions
Evapotranspiration?
2) Degradation capacity is NOT (or not
enough) present in the natural
abundant population
 INOCULATION
Phytotoxicity?
Degradation
Degradation genes
Can the tree reach the contaminants?
Can the contaminants reach the tree?
Enrichment of the inoculated strain
Conclusions
Evapotranspiration?
2) Degradation capacity is NOT (or not
enough) present in the natural
abundant population
 INOCULATION
Phytotoxicity?
Degradation
Degradation genes
Can the tree reach the contaminants?
Can the contaminants reach the tree?
Transfer of the degradation genes
Thank you for your attention!
Contact
Plantgeassocieer
de
bacteriën
in
information:
bodemsane
ring, van
labo tot
Dr. Ir. Nele Weyens
veldschaal
Universiteit Hasselt
Centrum voor Milieukunde
[email protected]
+32 11 26 83 16
Prof. Dr. Jaco Vangronsveld
Plantgeassocieer
Universiteit Hasselt
de
Centrum voor Milieukunde
bacteriën
in
[email protected]
bodemsane
+32 11 26 83 31 ring, van
labo tot
veldschaal
Plantgeassocieer
de
bacteriën
in
bodemsane
ring, van
labo tot
veldschaal