Phytoremediation, a novel strategy for the removal of toxic metals - - PowerPoint PPT Presentation

Phytoremediation, a novel strategy for the removal of toxic metals from the environment: biochemical and molecular mechanisms

Shivendra V. Sahi, Ph.D. Department of Biology Western Kentucky University

Outline

 Introduction of phytoremediation  Sesbania drummondii

• Metal uptake
• Microscopic evidence of metal transport
• Biotransformation of metal compounds
• Stress enzymes
• Gene identification/expression
• Long term goal
• Conclusion

Phytoremediation

Use of vegetation for the in situ treatment of contaminated sites

 A fast emerging environmental clean up strategy  Immense promise for remediation of contaminated

sites (soil, ground water, waste water)

 Effective against

• inorganic (toxic metals and nutrients)
• organic pollutants (BTEX)
• chlorinated solvents, ammunition wastes

Phytoextraction Process

Background

 Sources of pollution:

 Mining and smelting, municipal wastes, sewage

sludge, landfill leachates, fertilizers, pesticides, nuclear accidents

 Dimension of the problem:

 1980 Statute recognized over 40,000 Superfund sites

endangering human health

 >10,000 sites remain active today (Superfund

Accomplishment Figures-FY 2003)

 40% of these sites have problems of heavy metal (Pb,

Cd, Cr, As, Zn etc.) contamination

Conventional remediation strategies against metal contaminations

 Excavation and reburial of contaminated soils

to another site

 Soil flushing/washing  Solidification/stabilization  Vitrification  Electro-kinetics

Cost Analysis

• Conventional engineering technology v/s Phytoremediation

(TIBTECH, 13, 1995)

Contaminants Conventional Technology Phytoremediation Water soluble/ volatile compounds $10-100 per m3 soil $ 0.02-1.00 per m3 soil ($200-10,000 per hectare) of cropping Compounds requiring land- filling or low temp. thermal treatments $ 60-300 per m3 soil Materials requiring special land-filling or high temp. thermal treatment $ 200-700 per m3 soil Incineration $ 100 per m3 soil Radionucleides $ 1000-3000 per m3 soil

Benefits

 Economically feasible  Socially desirable  Environment friendly  Improves soil health  Effective

Phytoremediation approaches

• 1. Phytoextraction: to remove contaminants directly from

soil/water

• 2. Phytostabilization: use of vegetation and soil

amendments to reduce contaminant availability and movement.

• 3. Rhizofiltration: plant root system is directed to extract

pollutants from water bodies

• 4. Phytomining: for extraction and concentration of valuable

metals

Prerequisites for Phytoremediation

Hyperaccumulators

 Accumulate 100 times more metals than the non-

accumulators

• Conc. Criterion (% Shoot DW)

Cd (>0.01), Co, Cu, Cr and Pb (>0.1), Ni and Zn (>1), Hg (0.001)

 Should have good biomass

Terrestrial Hyperaccumulators (Brooks, 1998)

Plant Metal % metal in shoot (DW) Thlaspi caerulescens Zn, Cd >2% Zn, >0.1% Cd, Thlaspi spp. Zn >2% Cardaminopsis hallerii Zn >1% Brassica spp. Se Astragalus spp. Se 0.1-1% Atriplex spp. Se Thlaspi rotundifolium Pb <1% (~0.8%) Aelloanthus subacaulis Cu 1.3% Haemaniastrum spp. Co Up to 1 % Brake fern As >1.5% (Nature,409,2001)

Aquatic Hyperaccumulators

The water hyacinth (Eichhornia crassipes)

Rate of removal of heavy metals from aqueous phase Element mg/g DW biomass/day g/ha/day Cd 0.67 400 Co 0.57 340 Pb 0.18 90 Hg 0.15 110 Ni 0.50 300 Ag 0.44 260

Gold Hill Mesa Corp. (CO Springs) - water hyacinth for removal of Au from Au tailings.

Sesba bani nia d drummond ndii

• A high biomass plant
• Common name: Rattlebox
• Native to Southeastern U.S.

Sesba bani nia drummondii dii

& Lead

Demonstrated as lead hyperaccumulator

• Tolerates up to 1,000 ppm in

hydroponic solution

• Accumulated >4% (DW) Pb

in shoots in hydroponic conditions

• Roots showed 6% (DW)

accumulation

• EDTA and low pH increased

accumulation further

(EST 36, 4676-4680, 2002).

mg/L Pb mg/kgdw Pb

Sesba bani nia in soil supplemented with Pb

Root Pb

2500 5000 7500 10000 12500 15000 17500 Pb E 1.25 2.5 5 10 D 1.25 2.5 5 10 H 1.25 2.5 5 10 N 1.25 2.5 5 10 C 1.25 2.5 5 10

Chelator (mmol/kg soil) [Pb] (mg/k 2 weeks 4 weeks

Sesba bani nia in soil supplemented with Pb

Shoot Pb

1000 2000 3000 4000 5000 6000 P b E 1 . 2 5 2 . 5 5 1 D 1 . 2 5 2 . 5 5 1 H 1 . 2 5 2 . 5 5 1 N 1 . 2 5 2 . 5 5 1 C 1 . 2 5 2 . 5 5 1

Chelator (mmol/kg soil) [Pb] (mg/k 2 weeks 4 weeks

Species Chelators Soil Pb (mg/kg) Shoot Pb (%) Biomass (t/ha/yr)

• Est. total

Pb extr. (kg/ha/yr) Source

Zea mays 5.8 mmol/kg HEDTA

2500 1.06 5-6 53-64

Huang et al. 1997 Pisum sativum 1.34 g/kg EDTA

2450 0.897 3-4 27-36

Huang et al. 1997 Sesbania drummondii 10 mmol/kg EDTA

7500 0.42 10-15 43-63

Brassica juncea 10 mmol/kg EDTA

600 1.6 1-1.5 16-24

Blaylock et

• al. 1997

Triticum aestivum 5 mmol/kg EDTA+5 mmol/kg acetic acid

2000 0.92 2.5 23

Begonia et

• al. 2002

Estimated total Pb removed from soil by several plants

(Ruley 2004)

200 400 600 800 1000 1200 1400 Mercury concentrations in s (mg kg

• 1dw)

5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 10 20 30 40 50 100

Hg concentrations (mg/l)

Mercury concentrations in (mg kg

• 1 dw)

Shoot Root

Mercury uptake by Sesbania

200 400 600 800 1000 1200 1400 1600 1800 Cu concentration in sh (mg kg

• 1 dw)

5000 10000 15000 20000 25000 30000 35000 25 50 100 150 300

Cu concentration (mg/l)

Cu concentration in r (mg kg

• 1 dw)

shoot root

Copper uptake By Sesbania

500 1000 1500 2000 2500 3000 3500 Cr concentration in the s (mg kg

• 1

dw) 1000 2000 3000 4000 5000 6000 25 50 100 200

Cr concentration (mg/l)

Cr accumulation in roots (mg kg

• 1

dw)

Chromium uptake By Sesbania

Shoot Root

Gold uptake by Sesba bani nia

20 40 60 80 100 120 140 25 50 100 200 KAuCl4 in solution (mg/L) Shoot Au (mg/kg DW)

20 40 60 80 100 0 d 1/2 d 1d 6d Days Shoot Au (mg/kg DW)

Scanning Electron Microscopy of Plant shoot grown in metals

Cu shoot Cr shoot Pb root Control

X-ray microanalysis (EDS) of Sesba bani nia tissue

Cu Cr Pb

Control

Transmission Electron Microscopy of Sesba bani nia tissues with metals

Pb Au

Gold nanoparticles in Sesba bani nia

20 40 60 80 100 120 140 160 180 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43

Diameter (nm) Frequency

ES & T (accepted)

Transport of Pb in Sesba bani nia

 Scanning Electron Microscopy

• Transport of metals via different cell types

 Transmission Electron Microscopy

• Pb particles in intercellular spaces, cell

membranes and cell walls.

• Au particles (nanoparticles) are inside the

cell.

• Some deposits were also located within

the tonoplast.

Biotransformation of Metals

(Using XAS Technology)

Types of XAS

 XANES (X-ray absorption near edge structure)

– determines the oxidation state and atomic geometry of a bound metal.

 EXAFS (Extended X-ray absorption fine

structure) – traces the ligand involved in the metal binding by measuring the distance from the X-ray-absorbing atom to the next nearest atom.

0.0 0.5 1.0 1.5 2.0 0.0 12.95 13 13.05 13.1 Energy [keV] Lead(II) Sulfide Lead(IV) Oxide Lead(II) Sulfate Normalized Absorption

0.0 0.5 1.0 1.5 2.0 2.5 0.0 12.95 13 13.05 13.1 Normalized Absorption Energy [keV] Lead(II) Nitrate Lead(II) Acetate Lead Roots Treatment Lead Leaves Treatment

XANES Spectra of Sesba bani nia

(ET & C 23, 2068, 2004)

A) LIII XANES of lead laden plant samples, lead(II) nitrate, and lead(II)

• acetate. LIII XANES of lead model compounds lead(II) sulfide, lead(II) sulfate,

and lead(IV) oxide. B)

A B

1.0 2.0 3.0 4.0 5.0 2 4 6 R [Å] Fourier Transform Magnitude Lead(II) Nitrate Lead(II) Sulfate Lead(IV) Oxide Lead(II) Sulfide 1.0 2.0 3.0 4.0 5.0 2 4 6 R [Å] Fourier Transform Magnitude Lead(II) Acetate Leaves Sample Lead Root Sample

EXAFS Spectra of Sesba bani nia

XANES and EXAFS data of Pb-treated Sesba bani nia

(Environ. Toxicol. Chem. 23, 2068-2073, 2004)

Samples

Pb(NO3)2 % PbSO4 % Pb metal % PbS % Pb acetate %

Leaves 7.6 25.8 14.2 52.4 Roots 10.1 8.8 20.2 60.9

0.5 1.0 1.5 2.0 11.9 11.95 12 Normalized Absorption Energy [keV]

A

Au(CH3COO)3 AuOH KAuCl4 Au2S Au(0)

0.5 1.0 1.5 2.0 11.9 11.95 12 12.05 Normalized Absorption Energy [keV]

Roots 100 ppm Shoots 100 ppm Roots 50 ppm

B

• A. XANES of gold model

compounds: gold acetate, gold hydroxide, potassium tetrachloroaurate, gold sulfide, and gold metal.

• B. XANES of gold-laden

plant samples.

XANES of gold accumulated in Sesba bani nia

Samples % KAuCl4 % Au(CH3COO)3 % Au2S % Au(0) % AuOH

Au 50 ppm roots 18.4 81.6 Au 100 ppm roots 16.4 83.6 Au 100 ppm shoots 14.2 84.4 1.4

XANES of Cu accumulated in Sesba bani nia

(Chemosphere 67, 2257-66, 2007)

Samples

% copper(II) acetate % copper(II) nitrate % copper(II) phthalocyanine % copper(II) gluconate % copper(II) Oxide Cu 25 mg l-1 20 2 57 21 Cu 100 mg l-1 30 14 5 18 33

Biotransform cupric sulfate to copper(II) sugar/small

• rganic acid complex and acetate in its tissue

XANES analysis of Cr in Sesba bani nia

Sesba bani nia has capability to biotransform Cr(VI) in to Cr(III) in its tissue

Antioxidant Reactions & metal Stress in Sesba bani nia

 Generally metal exposure triggers an increase in

activity of antioxidant enzymes.

 Superoxide dismutase (SOD) – catalyze dismutation of superoxide

radicals to hydrogen peroxide & oxygen

 Catalase (CAT) – catalyzes decomposition of hydrogen peroxide to

water and oxygen

 Ascorbate deroxidase (APX) – detoxifies hydrogen peroxide to

water using ascorbate as substrate

 Glutathione reductase (GR) – reduces oxidized glutathione

(GSSG) to reduced glutathione (GHS)

• maintains high GHS/GSSH to sustain role of GHS as anti-
• xidant
• also incorporating into phytochelatins
• GSH also function as free radical scavenger

Stress enzymes in Sesba bani nia in Cr

10 20 30 40 50 60 25 50 100 200

Cr concentration (mg l-1) SOD activ (Units mg

0.5 1 1.5 2 2.5 3 3.5 4 4.5 25 50 100 200

Cr concentration (mg l-1) APX activity (Units mg-1 prot

0.5 1 1.5 2 2.5 3 3.5 4 25 50 100 200

Cr concentration (mg l-1) GR activity (Units mg-1 prot

50 100 150 200 250 300 350 25 50 100 200

Cr concentration (mg l-1) GSH conten (nmol g

• 1

FW)

SOD APX GR GHS

Stress enzymes in Sesba bani nia in Hg

20 40 60 80 100 120 140 10 20 30 40 50 100

Hg concentrations (mg l-1) SOD activity (units mg

• 1 protein

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 10 20 30 40 50 100 Hg concentrations (mg l-1) APX activity (units mg

• 1 protein

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 10 20 30 40 50 100

Hg concentrations (mg l-1) GR activity (units m

• 1

SOD APX GR

Superoxide dismutase (SOD)

Pb EDTA Cont Pb+E D Pb+D

Ascorbate Peroxidase (APX)

C Pb Pb+E E

25 50 75 100 125 150 175 Control Pb EDTA DTPA HEDTA Pb+EDTA Pb+DTPA Pb+HEDTA

Glutathione (ng / mg plan

Glutathione content

Identification of lead and mercury responsive genes

Experimental Design

Growth Parameters

• Size
• Biomass

Metal accumulation RNA Isolation mRNA Suppression Subtraction Hybridization (SSH) PCR-select differential screening Sequencing analysis Sesbania Seed germination (Sahi et al. 2002)

Plant Growth (for 12 days)

• 500mgl-1 Pb(NO3)2 (Sahi et al. 2002)
• 50mgl-1 HgCl2 (Israr et al. 2006)
• Control (half strength Hoagland’s media)

Northern blot analysis

Suppression subtraction hybridization (SSH)

 Based on the technique called suppression

PCR

 Compare two populations of mRNA  Obtain clones of genes that are expressed in

• ne population but not in the other

Sequencing results for Pb samples

 63 clones corresponds to unigenes  49 (78 %) identified as segments of cDNAs

contained in GenBank database

 14 (22 %) were unknown (no similarity)  Clone # 7 exhibited homology to

type 2 metallothionein sequences

Northern blot analysis (Pb)

Planta 2007 (in press)

Water-stress induced gene (Clone # 29, 36, 37, 38) Cold stress-induced gene (Clone # 4, 13, 14) ACC synthase/oxidase gene (Clone # 31) Abiotic stress-induced gene (Clone # 28) Metallothionein gene (Clone # 7) EtBr stained RNA

Shoot Root Shoot Root

Control Pb treated

Sequencing results (Hg)

 87 clones corresponds to unigenes  Clone # 31 had homology to type 2

metallothionein (MT)

 Clone # 252- 70 kD Heat shock cognate

protein 3

 Clone # 4- ATFP6-metal ion binding protein

β-1,3-Glucanase gene (Clone # 110) 70 kD Heat shock cognate protein 3 Putative Rieske Fe-S protein precursor (# 47) Glutathione S-transferase (Clone # 10) WRKY82 (Clone # 135) ATFP6 Metal ion binding protein (Clone # 4) Type 2 metallothionein (Clone # 82)

Northern blot analysis (Hg)

Shoot Root Shoot Root

Control Hg treated

Bioengineering of Plants for Efficient Remediation



Arabi bidops psis transgenics constructed to express bacterial genes merB and merA. (PNAS 93, 1996) merB merA (CH3)2Hg -----------Hg2+ --------------Hg



Indian mustard transformed with ATP Sulfurylase (APS) genes demonstrate 4- fold increase in APS activity and accumulated 3X Se than the wild type.



Transgenic tomato over expressing the bacterial gene 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase demonstrated enhanced tolerance for and accumulation of Co, Cu, Ni, Pb and Zn (J. Biotech 81, 2000).



Transgenic tobacco expressing citrate synthase showed enhanced tolerance to Al toxicity (Science 276, 1997).



Transgenic Arabi bidops psis expressing phytochelatin synthase from wheat demonstrated enhanced accumulation of Cd (PNAS 100, 2003)

Sesba bani nia Transformation

 Developed in vitro regeneration system using

nodal explants

 Sesbania callus infected with Agrobacterium

containing pCambia 1305

 pCambia has GUS gene which produces beta-

glucuronidase

 GUS histochemical assay to check gene

expression.

 Expression confirmed by PCR

Sesba bani nia callus showing transformation

pCAMBIA 1301

1 2 3 4

Lane 1: S. drummondii transformed tissue with strain K289 (without plasmid), Lane 2: transformed tissue with strain K289pCAMBIA 1305.1. Lane 3: 1 kb ladder; Lane 4: GUS gene amplified from pCAMBIA 1305.1 plasmid

1 1 2 3 4

Sesba bani nia Regeneration

General Conclusion

 Phytoremediation by Sesbania is effective against a

wide variety of contaminants.

 Effective for sites with shallow contaminated soils.  A type II metallothionein gene identified - may be

involved in heavy metal detoxification

 Pb and Hg in Sesbania also induced other stress

related genes

 Slow process  Interdisciplinary approach  More research to manipulate metals accumulation

efficiency of Sesbania.

ACKNOWLEDGEMENTS

Collaborators

• Dr. J. Andersland, WKU
• Dr. K. Raghothama, Purdue U
• Dr. J. Jain, Univ. Notre Dam
• Dr. J. Gardea-Torresdey, UTEP
• Dr. D. Sarkar, UTSA
• Dr. R. Datta, UTSA

Research Associates

• Dr. N. Sharma
• Dr. M. Israr

Graduate Students

• S. Cheepala
• T. Ruley
• A. Srivastava

Undergraduate Students

• N. Bryant
• D. Starnes
• A. Small

Financial Support

NSF-EPSCoR Ogden College