“endegrade” endophytic bacteria for improving phytoremediation
DESCRIPTION
“ENDEGRADE” Endophytic bacteria for improving phytoremediation. Fiona Porteous Moore, Colin Campbell, Edward Moore & E.U. partners. ENDEGRADE 7 project partners. SCOTLAND BELGIUM IRELAND DENMARK Macaulay IT Carlow Institute - PowerPoint PPT PresentationTRANSCRIPT
Craigiebuckler, Aberdeen, AB15 8QH, UK
“ENDEGRADE”Endophytic bacteria for improving
phytoremediation
Fiona Porteous Moore,
Colin Campbell, Edward Moore
& E.U. partners
ENDEGRADE7 project partners
SCOTLAND BELGIUM IRELAND DENMARK
Macaulay IT Carlow
Institute
LUC VITO DEC-NV DTU NERI (INDUSTRIAL
PARTNER)
PHYTOREMEDIATION
• Ideally plants must have deep roots perennial large water use
• Plants breakdown/volatilise compounds store them in leaves/shoots
• 4 methods 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
Code Plant Location Supplier total noSA Willow shoot LUC 57BSA Willow leaves LUC 24WHV Poplar var Hoogvorst root LUC 14POPHV Poplar var Hoogvorst shoot LUC 8BHV Poplar var Hoogvorst leaves LUC 36WHZD Poplar var Hazendans root LUC 37HZD Poplar var Hazendans shoot LUC 17BHZD Poplar var Hazendans leaves LUC 11VM(415-419) Brassica napus root VITO 5VM(420-427) Brassica napus shoot VITO 8VM(428-433) Brassica napus leaves VITO 6
Total 223
Bacterial endophyte isolates analysed to date
Enterobacter
Bacillus (7%)
Acinetobacter
Flavimonas
Mycetocola Sphingomonas (6%)
Xylophilus
Herbaspirillum
Duganella
Burkholderia (6%)
Delftia (6%)
Klebsiella
Pantoea
Pseudomonas(42%)
Xanthomonas (7%)
Moraxella
Arthrobacter (5%)
AgreiaAerococcus
Paenibacillus
Curtobacterium Micrococcus
Relative frequencies of bacterial isolates, classified to the genus level, on the basis of 16S rDNA sequence analysis
P. azotoformans
2%
P. fulva13%
P. beteli2% P. jessenii
7%
P. plecoglossida
19%
P. rhodesiae2%
P. tolaasii13%
P. frederiks13%
P. veronii11%
P. migulae2%
P. mandelii4%
P. fragi2%
P. putida2%
P. graminis4%
P. stutzeri4%
Relative frequencies of isolates classified as Pseudomonasspecies, based upon 16S rDNA sequence analysis
Poplar Hazendansisolates
Bacillus
Acinetobacter
ArthrobacterEnterobacter
Flavimonas
Herbaspirillum
Klebsiella
Bacillus
Arthrobacter
PaenibacillusXanthomonas
Xylophilus
Blastomonas
PseudomonasSphingomonas
Pseudomonas
Pseudomonas
0 2 4 6 8 10 12 14
Root
shoot
No of isolates
leaves
ROOT SHOOT
LEAF
HERBASPIRILLUMBACILLUS
PSEUDOMONASENTEROBACTER
SPHINGOMONAS
ACINETOBACTER
KLEBSIELLA
XANTHOMONAS
ARTHROBACTER
FLAVIMONAS
PAENIBACILLUS
XYLOPHILUS BLASTOMONAS
Distribution of endophytic bacterial isolates in Poplar
Multivariate analysis - presence / absence of isolates in different species of trees.
Effect of tree species
Napthalene degradation in Pea (proof of concept)Transpiration rate of uninoculated peas when
napthalene applied
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60 70 80
Time (hrs)
We
igh
t lo
ss
(gra
ms
) 0 mg/l
5mg/l
10mg/l
20mg/l
Transpiration rate when inoculated with endophytic construct
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60 70 80
Time (hrs)
We
igh
t lo
ss
(g
ram
s) 0 mg/l
5mg/l
10mg/l
20mg/l Work by: Germaine et al, IT Carlow
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