phytoremediation: from the molecular to the field scale
TRANSCRIPT
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Phytoremediation: From the Molecular to the Field Scale
B. Van Aken, J. M. Yoon, C. L. Just, S. Tanake, L. Brentner, B. Flokstra & J.L. Schnoor*
Department of Civil and Environmental Engineering &W. M. Keck PhytoTechnologies Laboratory
The University of Iowa, Iowa City, IA 52242 USA
Presented to the International Phytotechnologies ConferenceApril 20, 2005
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Tree Hugger…
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Phytoremediation
Uptake
Photodegradation
hν
Translocation
Phytovolatilization
N
N
NO2NNO2
NO2NO2
O2N
O2N
CH3
N N
NNNO2
NO2O2N
O2N
Cl Cl
Cl
Cl
Cl
O
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl ClCl
ClCl
Cl
Cl
Cl
Phytotransformation
Introduction
Rhizodegradation
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Powers of Ten: from the molecular nanoscale(<100 nm) to the field (1000 m), more than 10
orders of magnitude!
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Plant materials for different purposes:
Tissue CulturesWhole intact plants Cell cultures
“Nodules”
Research into Contaminant Transformations
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From the Laboratory to the Greenhouse to the Field and back
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Where is Phytoremediation going? What is it?
Phyto
Molecular Biological Research
Conventional treatment technologies
Ecological
Restoration
Inno-vativeTech-nology
Nowhere?
A mature technology?
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Phytoremediation—the use of plants to help clean the environment…
• Advantages– Natural– Green, growing– Aesthetically pleasing– Cost-effective for large
land areas where other technologies are not feasible
– Sensible, appropriate, sustainable technology
• Disadvantages– Long clean-up times– Uncertain performance– Not for every site
(deep wastes, anaerobic soils, etc)
– Regulatory hurdles
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Recent Advances in PhytoTechnology
• New evidence of phytoremediation effectiveness (for PAHs, RDX, ClO4
-, other)• Progress with transgenic plants
– First use in the field for selenium removal in CA (Norman Terry, Gary Banuelos et al., ES&T)
– As and Hg progress also (Meagher, Rugh, others)
• Plume delineation by tree corings (Burken, Compton, and others)
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Recent Advances in PhytoTechnology
• Emergence of Populusgenomic database for plant functional genomics– Populus EST resource
published (Sterky et al., PNAS 101:38, 13951-13956
– Genome shotgun sequences by DOE (www.jgi.doe.gov
– International Genome Consortium (www.ornl.gov/ipgc)
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Use Arabadopsis to learn about poplar
The high similarity between Populus and Arabidopsis will allow Populus to directly benefit from Arabadopsis detailed functional genomic information. Enzymes can be identified by molecular techniques…A. thaliana (At1g17170) GST: 27.5 x increase after TNT exposure (Ekman et al., 2003, Plant Physio 133:1397-1406)
Blast Corresponding Protein (aa) Sequence in Poplar Genome Project
Find DNA poplar sequence corresponding to the Arabidopsis protein
Score = 179 bits (383), Expect(3) = 6e-79Identities = 68/109 (62%), Positives = 88/109 (80%)Frame = -1
Query: 1 MADEVILLDFWASMFGMRTRIALAEKRVKYDHREEDLWNKSSLLLEMNPVHKKIPVLIHN 60M D V LL FW S + MR ++ALAEK ++Y+ RE++L +KS LLLEMNPVHK IPVLIHN
Sbjct: 926 MEDRVTLLIFWPSPWAMRVKVALAEKGIEYESREQNLIDKSPLLLEMNPVHKTIPVLIHN 747Query: 61 GKPVCESLIQIEYIDETWPDNNPLLPSDPYKRAHAKFWADFIDKKVNVT 109
GKP+CES ++YIDE W D +PLLPSDPY+R+ A+FWAD+IDKK +++Sbjct: 746 GKPICESHNIVQYIDEVWKDKSPLLPSDPYQRSQARFWADYIDKKASIS 600
Arabidopsis
Populus
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Past Applications have shown the promise of phytoremediation for soils and sediments
Amana, Iowa, 1992: Riparian zone buffer strip to control pesticides, nutrients and soil runoff
• Groundwater and soil concs. improved for nitrate and pesticides after only 4 years
• This could still be a growth area!
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Phyto Applications that really work!
• MNA + something more!• Petrochemical wastes and
rhizodegradation• Plume control and
degradation (TCE, MTBE, RDX, ClO4
-)• Landfill cap and closures• Dewatering contaminated
sludges/sediments cdfs• Created wetlands
(rhizofiltration) • Phytostabilization
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Former Refinery and Tank Farm, Cabin Creek WV
• Highly contaminated soil >5,000 mg/kg TPH significantly cleaned in 4 years
• Planted with DN-34 hybrid poplar and grasses in 1999
• Growth is shown after 3 seasons; soil concs. improving, g.w. slow
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Iowa Army Ammunition Plant
• RDX & TNT concs. in soil/groundwater up to 1 mg/L; excavation followed by phyto…
• Constructed wetlands phytoremediation full scale
• Meets RDX discharge permit of 2 ppb partly due to photolysis
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TCE in surficial groundwater plume, at major chip manufacturer, Myrtle Beach SC
• Groundwater plume of TCE was migrating off the plant property
• Two acres of hybrid poplar were planted w/ roots into gw table
• Phytoremediation has decreased the TCE in gw and soils
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Hydraulic capture of MTBE by hybrid poplars after (a) 1 month and (b) 8 months of growth at Houston, TX, 1999.
(a) (b)
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Elevation, cm
0 5000 10000 15000 20000Distance, cm, along gw flow gradient
-200-100
0100200
Constant Surface Boundary and PET ConditionsDeep Infiltration by Precipitation: ~ 6 cm/year (5% of precip.)Average (Yearly Basis) PET: ~ 3 mm/dayAverage (Yearly Basis) AET: ~ 2.2 mm/day (constant LAI, = 2)Left-Hand Side Boundary Condition No FlowRight-Hand Side Boundary Condition Constant Head
Trees TreesElevation, cm
0 5000 10000 15000 20000Distance, cm, along gw flow gradient
-200-100
0100200
(b)
(a)
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NO2O2N
NO2
CH3 ?
Inside the plant...
Introduction
NH2O2N
NO2
CH3Plant enzyme
Phase IActivation
H
OH
H
OH
HH
O
OH
CH2OH
OH
H
H
OH
HH
O
OH
CH2OH
OH
HCH3
NH NO2
NO2
Phase IIConjugation
Phase IIISequestration
“Green liver” modelColeman et al. 1997 Trend Plant Sci 2:144-151
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Analysis
UptakeInitial pollutant Translocation
Storage
PhytotransformationMetabolites Conjugation
Enzymes involved
HPLC14C,rad15N,13C, GC-MSLC-MSLC-MS-MS
TNT Metabolite Analysis
NO2O2N
NO2
CH3
NO2O2N
NH2
CH3NH2O2N
NH2
CH3
Non-extractable
Nitroreductase
Glutathione-S-transferase
The case of TNT
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N N
N
O2N
NO2
NO2
H
N N
H
NO2
O
H
N N
N
O2N NO2
H N N N
O
H H
NO2
NO2
H2O
N2OCH2O
4-nitro-2,4-diazabutanal
Volatilizationof
nitrous oxide
Suspected formaldehydeconjugation by
S-formylglutathione
RDX
NO2-
other intermediates
Phytophotolysisof RDX by plants (Just and Schnoor, ES&T, 2004)
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Mineralization of 14C-RDX and HMX by DN-34 Populus deltoides x nigra Nodules
• Nodules grown in Murashigeand Skoog liquid culture medium are capable of mineralizing RDX and HMX to 14CO2 with high yields
(Van Aken & Schnoor, ES&T, 2004) T im e ( d )
0 2 0 4 0 6 0 8 0
Rad
io-A
ctiv
ity (%
)
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
Min
eral
izat
ion
(%)
0
4
8
1 2
1 6
2 0
R D X ( 2 0 m g / l)R D X ( 2 0 m g / l) C o n t r o l 1R D X ( 2 0 m g / l) C o n t r o l 2
T i m e ( d )0 2 0 4 0 6 0 8 0
Rad
io-A
ctiv
ity (%
)
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
Min
eral
izat
ion
(%)
0
5
1 0
1 5
2 0
2 5
H M X ( 2 . 5 m g / l )H M X ( 2 . 5 m g / l ) C o n t r o l 1H M X ( 2 . 5 m g / l ) C o n t r o l 2
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Ecotoxicology Phyto Research
Ecotoxicology of plant materials after phytoremediation using leaching tests, Microtox, earthworm, and C. elegans microarray
Fate of (a) TNT; (b) RDX; (c) HMX in plants following uptake…
TNT RDX
TNT & Transformed
productsBound Residue
RDX
HMX
HMX
Transformed products
Litter Litter Litter
Leachate(Transformed product)
Leachate(parent HMX)
(a) (b) (c)
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Plants and plant tissues ‘infected’ by Methylobacterium populi sp. BJ001
Developments – Ecology of Endophytes
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What do the plants do for the bugs and vice versa? And how does this affect remediation?
Poplar callus
Symbioticred bacterium
Some remaining research questions…
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Plant cell ‘infected’ by Methylobacteriumpopuli sp. BJ001
Optical microscope (1,000x), safranine
Healthyplant cell
‘Infected’plant cell
Developments
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Gene Expression Research
NO2O2N
NO2
CH3 Extraction
DNA
mRNA cDNAGene expression(Active gene)
RT
Realtime-PCR
Gene identification(Blueprint)
(Identification,Quantification)
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Exposed plants show induced genes…
Populus trichocarpa genes induced by TNT and identified
Glutathione S-Transferase (2 isozymes)Nitrilase 2Monodehydroascorbate ReductaseIsocitrate LyaseIndole-3-Acetate B-Glucosyl TransferaseRAP2-like Transcription Factor12-Oxophytodienoate Reductase (OPR1)Cyto P450 - CYP71A12Cyto P450 - CYP706A2Cyto P450 - C4H - CYP73A5Cyto P450 - CYP89A6Cyto P450 - CYP81D11
Calibrators
ActinCyclophilinGlyceraldehyde 3-Phosphate Dehydrogenase (GAPDH)18S rDNA
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Glutathione S
-Transfe
rase (
GST10)
Glutathione S
-Transfe
rase (
GST13)
Cytoch
rome P
450 (
CYP89A6)
Monodehyd
roas
corb
ate R
educta
se
RAP2-Like
Transc
ritpion Fac
tor
Indole-3-A
cetat
e b-G
lucosy
l Tran
sferas
e
Isocit
rate L
yase
Nitrila
se 2
Cytoch
rome P
450 (
CYP81D11
)
12-O
xophyto
dienoate
Red
uctase
Cytoch
rome P
450 (
CYP706A
2)
Cytoch
rome P
450 (
C4H - C
YP73A5)
Nor
mili
zed
Expr
essi
on0
10
20
30
40
50
60
70
80
160
200
240
Normilized using 18S rDNA
Enzymes Induced by TNT exposure
Gene Expression – Results
P. trichocarpa tissue culturesexposed to TNT 20.0 mg L-1 for 24 h
Normalized using “housekeeping” gene 18S rDNA
Expressed by reference to non-exposed tissues
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Gene Expression – Results
P. trichocarpa tissue cultures exposed to TNT 20.0 mg L-1 show a higher expression (induction) of potential “detoxification” genes
-Glutathione S-transferases (conjugation with GSH)-Cytochromes P-450 (oxidative transformation, e.g. hydroxylation)-Reductases, nitrilase (nitro group reduction)-Glucosyl transferase (conjugation with sugar)
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Phyto has “morphed” into many innovative technologies including green roofs and low impact development..
James Patchett, PresidentConservation Design Forum, Inc.Elmhurst, IllinoisFebruary 4, 2004
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Before
After
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Chicago City Hall 1999
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Conclusions• PhytoTechnologies are
evolving…– It’s innovative but also
becoming part of mainstream treatment technologies (constructed wetlands, landfill caps, and ecological restoration)
– Research is becoming more molecular to understand which plants might be used to degrade which chemicals opening new sustainable development technologies
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Sachiyo Tanaka
Brittany Folkstra Dr. Jong Moon Yoon
Laura Brentner
Acknowledgements
The Great Students:
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-SERDP (Strategic Environmental Research and Development Program)
-W. M. Keck Foundation
-NSF (National Science Foundation)
Acknowledgements