Transcript No Slide Title

“ENDEGRADE”
Endophytic bacteria for improving
phytoremediation
Fiona Porteous Moore,
Colin Campbell, Edward Moore
& E.U. partners
Craigiebuckler, Aberdeen, AB15 8QH, UK
ENDEGRADE
7 project partners
SCOTLAND BELGIUM
Macaulay
Institute
LUC
VITO
IRELAND
IT Carlow
DEC-NV
(INDUSTRIAL
PARTNER)
DENMARK
DTU
NERI
PHYTOREMEDIATION
• Ideally plants must have
• Plants
• 4 methods



deep roots
perennial
large water use
 breakdown/volatilise compounds
 store them in leaves/shoots
 phytovolatilisation
 phytoextraction
 rhizofiltration
 phytostabilisation
Problems with some current remediation
strategies
• Phytoremediation
> pollutants kill the plants
> volatilisation through leaves
> partial degradation leading to toxic products
• Soil bioremediation
> indigenous population overcomes introduced
degraders
> nutrient applications often needed
> pollutants can be taken up by plants faster than
soil microbes can degrade them
Endegrade - the concept
• enhance plant survival
• enhance degradation
• reduce volatilisation
Project aims
•Isolate & identify bacteria from Willow and Poplar
•Screen for natural degradation potential for target
compounds
•Equip endophytic bacteria with degradation
plasmids
•Assess re-colonisation and phytoremediation
efficiency
•Risk assessment for field use
What is known about endophytic bacteria?
•
neutral or beneficial effects on the plant
- direct plant growth-promoting activity
- N2-fixation
- disease suppression
- enhanced pest control
•
•
•
•
exist in all plant species examined
103 - 105 cfu/gm plant tissue
highest numbers observed in the roots
bacteria remain localised in specific tissues
• We do not know…
• community dynamics & diversity
• colonisation potential
• plant specific relationships?
Pollutants problematic for
phytoremediation...
Compound
Fate in plant
(toxic, build up, or volatile)
Reference
Phenols
Toxic
Pfleeger et al., 1991
Chloro-phenols
Toxic
Pfleeger et al., 1991
TNT
Toxic, degraded to amino-dinitrotoluene
Thompson et al., 1998
Amino-dinitrotoluene
Rather persistent, toxic
Thompson et al., 1998
MTBE
Volatile
Trapp et al., 1994
BTEX
Volatile
Trapp et al., 1994
TCE
Volatile, Build-up of trichloroacetate
Trapp et al., 1994
PER
Volatile
Trapp et al., 1994
Bacterial endophyte isolates analysed to date
Code
SA
BSA
WHV
POPHV
BHV
WHZD
HZD
BHZD
VM(415-419)
VM(420-427)
VM(428-433)
Plant
Location Supplier total no
Willow
shoot
LUC
57
Willow
leaves
LUC
24
Poplar var Hoogvorst
root
LUC
14
Poplar var Hoogvorst
shoot
LUC
8
Poplar var Hoogvorst
leaves
LUC
36
Poplar var Hazendans
root
LUC
37
Poplar var Hazendans
shoot
LUC
17
Poplar var Hazendans leaves
LUC
11
Brassica napus
root
VITO
5
Brassica napus
shoot
VITO
8
Brassica napus
leaves VITO
6
Total
223
Relative frequencies of bacterial isolates, classified to the
genus level, on the basis of 16S rDNA sequence analysis
Mycetocola
Curtobacterium
Paenibacillus
Micrococcus
Bacillus (7%)
Aerococcus
Agreia
Sphingomonas (6%)
Xylophilus
Herbaspirillum
Duganella
Burk holderia (6%)
Arthrobacter (5%)
Delftia (6%)
Moraxella
Acinetobacter
Flavimonas
Klebsiella
Pantoea
Enterobacter
Xanthomonas (7%)
Pseudomonas(42%)
Relative frequencies of isolates classified as Pseudomonas
species, based upon 16S rDNA sequence analysis
P. putida
2%
P. mandelii
4%
P. fragi
2%
P. migulae
2%
P.
P. graminis
azotoformans
4%
2%
P. stutzeri
P. fulva
4%
13%
P. beteli
2% P. jessenii
7%
P. veronii
11%
P.
plecoglossida
19%
P. frederiks
13%
P. tolaasii
13%
P. rhodesiae
2%
Poplar Hazendans
Sphingomonas
isolates
Pseudomonas
leaves
Blastomonas
Xylophilus
Xanthomonas
Paenibacillus
Arthrobacter
Bacillus
shoot
Pseudomonas
Klebsiella
Herbaspirillum
Flavimonas
Enterobacter
Root
Arthrobacter
Acinetobacter
Bacillus
0
2
Pseudomonas
4
6
8
No of isolates
10
12
14
Distribution of endophytic bacterial isolates in Poplar
ROOT
SHOOT
FLAVIMONAS
BACILLUS
HERBASPIRILLUM
ACINETOBACTER
ENTEROBACTER
KLEBSIELLA
PAENIBACILLUS
ARTHROBACTER
PSEUDOMONAS
XYLOPHILUS
BLASTOMONAS
SPHINGOMONAS
XANTHOMONAS
LEAF
Effect of tree species
Multivariate analysis - presence / absence of isolates
in different species of trees.
Napthalene degradation in Pea (proof of concept)
Transpiration rate of uninoculated peas when
napthalene applied
7
Weight loss
(grams)
6
5
0 mg/l
4
5mg/l
3
10mg/l
20mg/l
2
1
0
0
10
20
30
40
50
60
70
80
Time (hrs)
Transpiration rate when inoculated with endophytic construct
7
Work by:
Germaine et al,
IT Carlow
Weight loss
(grams)
6
5
0 mg/l
4
5mg/l
10mg/l
3
20mg/l
2
1
0
0
10
20
30
40
Time (hrs)
50
60
70
80
Plant protection
• Iris & VM1450 - 2,4-D protection (Germaine et al, IT Carlow)
• Pea & PCB degraders - 4-chlorobiphenyl
protection (Germaine et al, IT Carlow)
• Lupine & VM1330- Toluene protection, reduced
volatilisation, enhanced growth (Barac et al, LUC)
• Toluene & TCE degrader constructs from Brassica
napus - to test in plantae degradation (Borremans et al,
VITO)
Re-inoculation
• K.Germain et al, FEMS Microbiol.Ecol, in press.
• 3 endophytic isolates gfp/Kan marked and
re-inoculated into Poplar (original host)
• All strains colonised roots, and 2 strains
colonised stems and leaves after 10
weeks
VM1449 (Ps. veronii) colony on
root xylem of poplar tree 10 weeks
after inoculation x1000
Poplar used in phytoremediation
field trial in Belgium
Current work
• Endophytic construct in Poplar exposed to
2,4-D - does in degrade/protect?
> 2,4-D degradation genes & colonisation
• Seasonal community dynamics of
endophytes - affects inoculation time/method
> intra/inter species variation, seasonal dynamics,
compartmentalisation
Acknowledgements
Macaulay Institute - Renate Wendler, Duncan White
NERI - Denmark - Uli Karlson
DTU - Denmark - Stefan Trapp
VITO - Belgium - Daniel van der Lelie, Brigitte Borremans
LUC- Belgium - Jaco Vangronsveld, Licy Oeyen,
Tanja Barac
IT Carlow- Ireland - David Dowling, David Ryan,
Keiran Germaine, Elaine Keogh
DEC NV- Belgium - Siegried D’Haene, Gunther de Becker