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

Tree Hugger…

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

Powers of Ten: from the molecular nanoscale(<100 nm) to the field (1000 m), more than 10

orders of magnitude!

Plant materials for different purposes:

Tissue CulturesWhole intact plants Cell cultures

“Nodules”

Research into Contaminant Transformations

From the Laboratory to the Greenhouse to the Field and back

Where is Phytoremediation going? What is it?

Phyto

Molecular Biological Research

Conventional treatment technologies

Ecological

Restoration

Inno-vativeTech-nology

Nowhere?

A mature technology?

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

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)

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)

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

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!

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

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

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

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

Hydraulic capture of MTBE by hybrid poplars after (a) 1 month and (b) 8 months of growth at Houston, TX, 1999.

(a) (b)

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)

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

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

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)

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

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)

Plants and plant tissues ‘infected’ by Methylobacterium populi sp. BJ001

Developments – Ecology of Endophytes

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…

Plant cell ‘infected’ by Methylobacteriumpopuli sp. BJ001

Optical microscope (1,000x), safranine

Healthyplant cell

‘Infected’plant cell

Developments

Gene Expression Research

NO2O2N

NO2

CH3 Extraction

DNA

mRNA cDNAGene expression(Active gene)

RT

Realtime-PCR

Gene identification(Blueprint)

(Identification,Quantification)

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

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

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)

Phyto has “morphed” into many innovative technologies including green roofs and low impact development..

James Patchett, PresidentConservation Design Forum, Inc.Elmhurst, IllinoisFebruary 4, 2004

Before

After

Chicago City Hall 1999

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

Sachiyo Tanaka

Brittany Folkstra Dr. Jong Moon Yoon

Laura Brentner

Acknowledgements

The Great Students:

-SERDP (Strategic Environmental Research and Development Program)

-W. M. Keck Foundation

-NSF (National Science Foundation)

Acknowledgements