Transcript PHYTOREMEDIATION: Principles & Applications, Merits & Limitations Pedro J. Alvarez, Ph.D., P.E., DEE.

PHYTOREMEDIATION:
Principles & Applications,
Merits & Limitations
Pedro J. Alvarez, Ph.D., P.E., DEE. F.ASCE
Acknowledgements
“Water, water everywhere, nor any drop to drink”
The Rime of the Ancient Mariner, Samuel Taylor Coleridge
OBJECTIVE
To understand and utilize vegetation in the
remediation of hazardous waste sites (metals
and organics contamination of surface soils)
volatilization
Phytoremediation
Mechanisms
Plant
metabolism
•Phytoextraction
Plant uptake
•Phytostabilization
(xylem)
•Phytotransformation
•Phytovolatilization
Root
•Phytophotolysis
absorption
•Rhizofiltration
•Bioremediation in the rhizosphere
O2, Substrates
(phloem)
Contaminants
Remember the Alamo
• Perennial and hardy (tolerant to flooding, salt, and high
concentrations of toxic substances)
• Extremely fast growing and adaptable (Canada to
Mexico)
• Phreatophytes, roots penetrate to water table for
extraction of contaminated groundwater (1,000 gal/year)
• Easy planting from cuttings (male clones) and coppicing
trait (regrowth from cut stump)
Nitrate removal from groundwater flowing
Through the root zone of a poplar tree buffer
ADVANTAGES
• Relatively low cleanup cost
• Easily implemented and maintained
• Several removal mechanisms for different compounds
• Environmentally friendly and aesthetically pleasing
• Reduces landfilled wastes
• Harvestable plant material
Five-Year Cost Comparison: Phytoremediation with Poplars vs. Pump & Treat
1.
Phytoremediation
Design and Implementation$ 50,000
Monitoring Equipment
Capital
10,000
Installation
10,000
Replacement
5,000
5-Year Monitoring
Travel and administration 50,000
Data collection
50,000
Reports (annual)
25,000
Sample analysis
50,000
TOTAL $ 250,000
2.
Pump and Treat (3 wells & Reverse Osmosis)
Equipment
$ 100,000
Consulting
25,000
Installation/Construction
100,000
5-Year Costs
Maintenance
105,000
Operation (electricity)
50,000
Waste disposal & liability 280,000
TOTAL $ 660,000
DISADVANTAGES
• Longer remediation times than more aggre$$ive
alternatives such as excavation and incineration.
• Climate dependent (poor performance after leaves fall)
• Effects on food web might be unknown
• Ultimate contaminant fates might be unknown
• Results are variable (difficult to establish plants in highly
contaminated sites)
CONTAMINANTS FOR
PHYTOREMEDIATION
• Organics (BTEX, PAHs, PCBs, TNT, RDX,
chlorinated aliphatics, dioxane, pesticides)
• Metals (Pb, Cd, Zn, Ni, Cu, As, Cr, Se,
radionuclides)
• Nutrients (nitrate, ammonium, phosphate)
APPLICATIONS
Phytotransformation
Rhizosphere bioremediation
Phytostabilization
Phytoextraction
Rhizofiltration
Application
Phytotransformation
Rhizosphere bioremediation
Phytostabilization
Phytoextraction
Rhizofiltration
Media
Contaminant(s)
S, GW, WW
S, WW
S
S, WW
WW, GW
ORGS
ORGS
Metals, ORGS
Metals
Metals, ORGS
S = soil; GW = groundwater; WW = wastewater
Plants Used in Phytoremediation
• Salix family (poplars, cottonwood, willow)
• Phenolic releasers (mulberry, apple, Osage orange)
• Grasses (fescue, rye, sorghum, reed canary grass)
• Legumes (alfalfa, alsike clover, cowpeas)
Metals Accumulating Plants*
Sunflowers
Helianthus annus
Indian mustard
Brassica juncea
Crucifers
Thlaspi caerulescens
Thlaspi elegans
Violet
Viola calaminaria
Serpentine plants
Alyssum bertolonii
Other: corn, nettles, dandelions
*Reeves, Baker, Brooks (1995) Mining Environ. Mngmnt.
Metals Hyperaccumulators
Defn: Conc in plant 20-200 times higher than
other plants growing in the same soil
(Reeves, Baker, Brooks; 1995)
Zn
>
10,000 mg/kg in plant tissue
Cd
>
100
Pb
>
1,000
Se
>
100
Wetland Plants
Emergents - Bullrush, cattail, coontail,
arrowroot, pondweed, duckweed
Submerged - algae, stonewort,
Potamogeton spp., parrot feather,
eurasion water milfoil, Hydrilla spp.
Radiolabel uptake by plant
Uptake Model
U = (TSCF) (T) (C)
U=
Contaminant uptake rate, mg/day
TSCF = Transpiration Stream Concentration Factor,
a measure of uptake efficiency.
T=
Transpiration rate, L/day
C=
Contaminant concentration in pore water, mg/L
Uptake of 14C- atrazine in sandy soil
Uptake of 14C- atrazine in clay-rich soil
14C-
metabolites in poplar tissue, after 80 days
The Green Liver Concept
•
Metabolism of xenobiotics by plants
generally proceeds in three phases:
1. Transformation
2. Conjugation
3. Compartmentation.
•
The participating enzymes on these
phases have numerous similarities to
those used in mammalian livers.
•
Plants may be considered as a 'green
liver', acting as a global sink for many
environmental pollutants.
Detoxification Pathways in Plants
Pha se III
Compartmentation
Binding to
cell wall
components
Vacuole
Xenobiotic
Endoplasmic
Reticulum
Pha se III
Transformation
by P450
Pha se I
Cytosol
Pha se II
Phase I: Transformation by Cytochrome P450
• 51 Plant P450 Families
• Monooxygenation reaction
• Atypical activities:
– Dealkylations
– Dimerizations
– Isomerizations
– Dehydrations
– Reductions
Tertiary structure of P450 protein
http://genomebiology.com/2000/1/6/reviews/3003/figure/F2
Example of Xenobiotic Oxidative Metabolism
Hydroxylation of the pesticide methoxychlor by
cytochrome P450 isoforms (CYP).
Phase II: Conjugation by Various Transferases
Binding of Phase-1 metabolite by
• Glycosyltransferases
• Acyltransferases
• Glutathione S-transferases
Cut away view of GST’s active site,
highlighting amino acids important for
herbicide specificity (Thom et al., 2002).
Glutathione S-transferases (GSH + R-X  GSR + HX)
GST detoxifies endobiotic and xenobiotic compounds by covalently linking glutathione (GSH) to a
hydrophobic substrate (R-X), forming less reactive and more polar glutathione S-conjugate (GSR).
Phase III: Vacuole and Apoplast Storage
(visualized with immunochemical techniques)
Let’s take a break
Mineralization of 14C- Atrazine by Soil Bacteria
Occurs Faster and to a Greater Extent in Planted Soil
Rhizoplane bacteria on a root hair
1 µm
Effect of (7-Year Old) Poplar Rhizosphere on
Microbial Populations Important for Pollution Control
a) TOTAL HETEROTROPHS
b) DENITRIFIERS
2.0E+10
MPN per gram soil
MPN per gram soil
8.0E+10
6.0E+10
4.0E+10
2.0E+10
0.0E+00
1.5E+10
1.0E+10
5.0E+09
0.0E+00
Total Heterotrophs
Rhizosphere
Bulk soil
DenitrifiersBulk soil
Rhizosphere
c) PSEUDOMONADS
d) BTX DEGRADERS
2.0E+03
MPN per gram soil
MPN per gram soil
1.0E+09
7.5E+08
5.0E+08
2.5E+08
0.0E+00
1.5E+03
1.0E+03
5.0E+02
0.0E+00
Pseudomonas
Rhizosphere
Bulk soil
Jordahl, Foster, Schnoor, and Alvarez (1997). Environ. Tox. and Chem. 16(6):1318-1321
BTX Degraders
Rhizosphere
Bulk soil
Close-up of a root
Root Hair
100 µm
Bioreporter Strain: P. fluorescens HK44
Gary Sayler (UTK) fused a reporter gene (lux) with nahG gene
Salicylate
(inducer)
reporter gene (lux)
catabolic enzymes (NDO)
catabolic genes (nah)
promoter/operator
reporter signal (e.g., light)
reporter protein (e.g., luciferase)
When nah is induced, the reporter gene is also expressed producing
luciferase, which emits light (luminometer)
Effect of naphthalene on HK44 specific bioluminescence
Uninduced
Induced
120
100
(RLU· OD600-1)
Specific Bioluminescence
Effect of naphthalene on HK44 specific bioluminescence
80
60
40
20
0
0
2
4
6
Naphthalene (mg·L-1)
Light response is proportional to inducer concentration
?
Screening for Potential Inducers
Which root-derived substrates/extracts induce nahG?
1 hour
15 minutes
Light
Light
HK44 washed
and rested in
Phosphate Buffer
1 mL substrate
(50 mg/L)
+
2 mL HK44 in PB
Induction Assay
Inducer if bioluminescence is significantly higher than control
Tested 22 Substrates, 7 Root Extracts
Sugars
glucose, lactose
Organic Acids
acetate, adipate, citrate, lactate, malate, oxaloacetate,
pyruvate, succinate
Amino Acids
glutamate, phenylalanine, tryptophan
Aromatics
acetyl salicylate, l-carvone, p-cymene, indole-acetic acid,
methyl salicylate, o-coumaric, p-aminobenzoate, salicylic acid
Root Extracts
Kou, Milo, Mulberry, Osage orange, Poplar, Switch grass, Willow
Of these, only 3 induced nahG:
O
O
C – OCH3
C – OH
OH
OH
Methyl Salicylate
O
Salicylate
C - OH
Acetyl Salicylate
O
O
CH3
Most substrates and all root extracts repressed nahG
Control (no substrate)
Naphthalene
glucose + nap
Sugars
lactose + nap
acetate + nap
succinate + nap
Organic Acids
lactate + nap
glutamate + nap
phenylalanine + nap
Amino Acids
tryptophan + nap
salicylate + nap
acetyl salicylate + nap
Aromatic Substrates
o-coumaric + nap
methylsalicylate + nap
0
50
100
Relative Specific Bioluminescence
150
Relative Specific Bioluminescence (%)
Repression Assays:
Mulberry Root Extracts + Naphthalene (3 mg/L)
120
100
80
60
40
20
0
0
50
100
150
200
250
Concentration of Mulberry Extract (mg/L)
300
Naphthalene Degradation Assays (pure culture)
Naphthalene concentration
2 hours
Growth Media
+
HK44
+
2.7 mg-naphthalene L-1
+
30 mg-C root extract L-1
OD600
Light
Light
Naphthalene Degradation
Naphthalene (mg•L-1)
2.5
Sterile Control
2.0
Naphthalene only
1.5
1.0
Mulberry Root Extract
+
Naphthalene
0.5
0.0
6
8
Time (hr)
10
12
Naphthalene degradation rate normalized to control
Co-substrate
rd
None
1 ± 0.08
Kou (30 mg-C·L-1)
1.12 ± 0.14
Milo (20 mg-C·L-1)
1.43 ± 0.41
Mulberry (30 mg-C·L-1)
1.77 ± 0.28*
Osage Orange (30 mg-C·L-1)
1.73 ± 0.10*
Poplar (30 mg-C·L-1)
1.36 ± 0.29
Switch grass (20 mg-C·L-1)
1.97 ± 0.37*
Willow (20 mg-C·L-1)
1.41 ± 0.10*
Initial OD600 = 0.001, Naphthalene = 3.56 ± 0.15 mg L-1
Biomass growth during naphthalene degradation
30
Biomass (mg·L-1)
Mulberry Root Extract
25
+ Naphthalene
20
15
10
Naphthalene only
5
0
6
8
Time (hr)
10
Specific Bioluminescence during degradation
350
(RLU mg·L-1)
Specific Bioluminescence
400
300
250
Naphthalene only
200
150
100
Mulberry Root Extract + Naphthalene
50
0
6
8
Time (hr)
10
Total Bioluminescence (RLU)
Total Bioluminescence during degradation
600
500
400
Mulberry Root Extract
300
+ Naphthalene
200
Naphthalene only
100
0
6
8
Time (hr)
10
Effect of Rhizodeposition on Naphthalene Degradation
Naphthalene only
Naphthalene (mg-L-1)
2.5
A
2.0
1.5
1.0
0.5
25
20
15
10
5
600
B
500
400
300
200
100
400
C
Specific Bioluminescence
((RLU mg- biomass 1 L)
L-
-1
Biomass (mg - L )
30
Naphthalene and Mulberry Root Extract
Total Bioluminescence (RLU)
Sterile control
D
350
300
250
200
150
100
50
0
0
6
8
Time (hr)
10
6
8
Time (hr)
10
Kamath R, JL Schnoor, PJJ Alvarez (2004). Effect of root-derived substrates on the expression of nah-lux genes in Pseudomonas fluorescens HK44
implications for PAH biodegradation in the rhizosphere. Environ. Sci. Technol. 38:1740-1745
Soil Microcosm Experiments
Contaminated
Rhizosphere
Reactors
Contaminated
Bulk Soil
Reactors
Contaminated soil
Contaminated soil
20 mg-C·L-1 RE
MSB
MSB
Fate of Phenanthrene in Soil Matrix
Rhizosphere
Sorbed/
Bound
29.6%
Unrecovered
8%
Mineralized
580.9%
Bulk soil
Sorbed/
Bound
34.9%
Mineralized
530.7%
Unrecovered
8.8%
Kamath R., J.L. Schnoor, and P.J.J. Alvarez* (2005). Environ. Sci. Technol. 39:9669-9675
Primers - Naphthalene Dioxygenase
Targets
Non-Targets
N.2.A subfamily
Pseudomonas G7 (nahAc)
Marine Isolates
Burkholderia sp. (dntAc)
Burkholderia RP007(phnAc)
Pseudomonas putida U2 (nagAc)
Rhodococcus sp.(narAa)
Primers –TodC1 and bmoA
Toluene Dioxygenase
Toluene Monooxygenase
Primers - dmpN
Phenol Monooxygenase
Targets
R.2 and R.3 subfamily
HC-attacking monooxygenases
Non-Selective Proliferation of PAH Degraders
A
8.0
2.0
6.0
1.5
4.0
1.0
2.0
0.0
0.5
Contaminated
Rhizosphere
0.0
Contaminated
bulk soil
30
Total Bacteria  106 g-1
2.5
Total Bacteria
dmpN
todC1
10.0
B
25
8.0
20
6.0
15
4.0
10
2.0
5
0
dmpN/todC1  104 g-1
3.0
Uncontaminated
rhizosphere
Uncontaminated
bulk soil
0.0
dmpN/todC1  104 g-1
Total Bacteria  106 g-1
10.0
Da Silva M.L.B., Kamath R., and P.J.J. Alvarez* (2006). Environ. Toxicol. Chem. 25(2): 386–391
Summary

Higher PAH mineralization in the simulated rhizosphere

Rhizosphere did not select for PAH-degraders

Rhizodeposition encourages the fortuitous growth of
microorganisms harboring the catabolic potential
(e.g., oxygenase enzymes) for PAH biodegradation

Plant-induced (non-selective) genotypic shifts could enhance
overall PAH’s removal in aged contaminated soil during
phytoremediation
DESIGN CONSIDERATIONS
• Plant Selection
• Treatability
• Planting Design and Pattern
• Irrigation, Inputs, Maintenance
• Groundwater Capture Zone/Transpiration Rate
• Contaminant Uptake Rate/Clean-up Time
• Analysis of Failure Modes
Phytoextraction Design Criteria - Metals
1) Yield  3 tons dry matter/acre-yr in
harvestable portion
2) Accumulation Factor 100
3) 30 year design life
mg / kg plant
Acc. Factor 
mg / kg soil
Phytoremediation Metals Demonstrations
Application
Plant
Contaminants
Results
Chernobyl Pond
Rhizofiltration
Sunflowers
Helianthus annus
137Cs, 90Sr
90% reduction
in 2 wks
(Raskin, Rutgers)
Trenton, NJ Soil
Phytoextraction
Indian mustard
Brassica juncea
Pb
SITE Program
700400 mg/kg
in 1 season
(Ensley, Phytotech)
Rocky Flats, Leachate
Rhizofiltration
Sunflowers
Indian mustard
U, NO3SITE Program
Just beginning
(Rock, EPA)
San Francisco, Soil
Phytovolatilization
Indian mustard
Brassica juncea
Dearing, KS Soil
Phytostabilization
Hybrid Poplar
Populus spp.
Se
Pb, Zn, (Cd)
8,000 mg/kg
Se is volatilized, but
soil remains
contaminated
(Banuelos, USDA)
50% survival, soil
stabilization
(Pierzynski, KSU)
Organics Treatability
log Kow
Mechanisms
< 1.0
Possible Uptake & Transformation
1.0-3.5
> 3.5
Uptake, Transformation, Volatilization
Rhizosphere bioremediation or
Phytostabilization
Briggs’ Correlation
Uptake Efficiency (TSCF)
TSCF =0.784 exp {-(log Kow – 1.78)2 / 2.44}
1
0.8
0.6
0.4
0.2
0
-1
0
1
2
Log Kow
3
4
5
Recall, Uptake Rate - Organics
U = (TSCF) (T) (C)
where U =
TSCF =
T=
C=
uptake rate of contaminant, mg/day
transpiration stream concentration factor
transpiration rate, L/day
aqueous phase conc, mg/L
Transpiration Rates
3-5 acre-ft/yr
36-60 inches/yr
600-1000 gal/tree (mature)
200 gal/tree (1st three years)
Poplars: < 5,000 trees/hectare
Trees
Plume
Regional flow
Phytotransformations Demonstrations
Application
Plant
Contaminants
Results
Ogden, UT
Soil & Petroleum
wastes
Hybrid Poplar
Populus spp.
TPH, BTEX
SITE Program
Second year
(Ferro, Phytokinetics)
Carswell AFB, TX
GW Plume
Hybrid Poplar
Populus spp.
Milan, TN (TAAP)
wastewater, GW
Canary Grass
Elodeia, Bullrush
Middletown, IA (IAAP) Pondweed,
Soil, stream, GW
Coontail,
Arrowroot, Poplar
Aberdeen, MD
Soil, GW
Hybrid Poplar
Populus spp.
TCE
TNT, RDX
Eng. wetlands
TNT, RDX
Eng. wetland
and trees
TCE, PCA
Second year
(Harvey, WP AFB)
> 90% removal
anaerobic/aerobic
w/cassein addition
(Bader, Army APG)
(Schnoor et al.)
Second year
(Compton, EPA)
McMinville, OR, Holding Pond
Fuel Type
Heat Content
Units
Agricultural Byproducts
8.248
Million Btu/Short Ton
Black Liquor
11.759
Million Btu/Short Ton
Digester Gas
0.619
Million Btu/Thousand Cubic Feet
Landfill Gas
0.490
Million Btu/Thousand Cubic Feet
Methane
0.941
Million Btu/Thousand Cubic Feet
Municipal Solid Waste
9.945
Million Btu/Short Ton
Paper Pellets
13.029
Million Btu/Short Ton
Peat
8.000
Million Btu/Short Ton
Railroad Ties
12.618
Million Btu/Short Ton
Sludge Waste
7.512
Million Btu/Short Ton
Sludge Wood
10.071
Million Btu/Short Ton
Solid Byproducts
25.830
Million Btu/Short Ton
Spent Sulfite Liquor
12.720
Million Btu/Short Ton
Tires
26.865
Million Btu/Short Ton
Utility Poles
12.500
Million Btu/Short Ton
Waste Alcohol
3.800
Million Btu/Barrel
Wood/Wood Waste
9.961
Million Btu/Short Ton
Source: Energy Information Administration, Form EIA-860B (1999), "Annual Electric Generator Report - Nonutility 1999."
SUMMARY
• Metals
• Phytoextraction - uptake & harvest, goal of 1%
• Rhizofiltration - binding to roots, generally aquatic
• Physical stabilization - less transport & leaching
• Organics
• Uptake - transport to above ground, transformation?
• Rhizoremediation - increased degradation & binding
• Physical stabilization - less transport & leaching
FUTURE OF PHYTOREMEDIATION
• Hazardous wastes
– Pesticides
– Volatile organics
– Munitions wastes
• Wastewater, Drainages
– Nitrogen
– TDS/Salts
– Polishing
• Landfill
– Capping systems
– Leachate treatment
• Advantages
– Aesthetically pleasing
– Inexpensive
– Robust/Simple
CONCLUSIONS
1. Phytoremediation is an emerging technology
2. It is cost-effective and practical
3. More demonstrations are needed to prove that
it works and to understand its applications better
4. More research is needed to understand
plant/enzyme/chemical systems and the
potential impacts of GMOs
PHYTOREMEDIATION
REGULATORY ISSUES
• Can it clean-up the site to below action levels?
• Does it create any toxic products?
• Is it cost-effective?
• Does the public accept the technology?
CHALLENGES AHEAD
• Screening Methodologies
(which plant for which contaminant?)
• Ultimate Fate of Contaminants
• Does it work?
Burken & Schnoor’s Correlation for poplars
TSCF = 0.756 exp{-(log Kow – 2.50)2 / 2.58}
1.00
TCE
Toluene
Benzene
0.75
TSCF
m-Xylene
0.50
0.25
0.00
0
2
Log Kow
4
6
Results
Soil
Total Bacteria
Oxygenase
Containing Bacteria
Contaminated Soil
Rhizosphere
8.9 ± 0.64
 10
6
2.6 ± 0.32
 10
4
Bulk Soil
4.7 ± 0.45
 10
6
1.4 ± 0.22
 10
4
Rhizosphere
2.3 ± 0.15
 10
7
10.4 ± 4.1
 10
4
Bulk Soil
1.6 ± 0.31  10
7
5.9 ± 2.8
 10
4
Uncontaminated Soil