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 Phytoremediation Technology Water soluble/ volatile $10-100 per m 3 soil $ 0.02-1.00 per m 3 compounds soil ($200-10,000 per hectare) of cropping $ 60-300 per m 3 Compounds requiring land- filling or low temp. thermal soil treatments $ 200-700 per m 3 Materials requiring special land-filling or high temp. soil thermal treatment $ 100 per m 3 soil Incineration Radionucleides $ 1000-3000 per m 3 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) Zn, Cd >2% Zn, >0.1% Cd, Thlaspi caerulescens Thlaspi spp. Zn >2% Zn >1% Cardaminopsis hallerii Se Brassica spp. Astragalus spp. Se 0.1-1% Se Atriplex spp. Pb <1% (~0.8%) Thlaspi rotundifolium Cu 1.3% Aelloanthus subacaulis Co Up to 1 % Haemaniastrum spp. 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 mg/kgdw Pb 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

Sesba bani nia in soil supplemented with Pb Root Pb 17500 2 weeks 15000 4 weeks 12500 [Pb] (mg/k 10000 7500 5000 2500 0 D 1.25 H 1.25 N 1.25 C 1.25 E 1.25 Pb 2.5 10 2.5 10 2.5 10 2.5 10 2.5 10 5 5 5 5 5 Chelator (mmol/kg soil)

Sesba bani nia in soil supplemented with Pb Shoot Pb 6000 2 weeks 5000 4 weeks 4000 [Pb] (mg/k 3000 2000 1000 0 5 5 5 5 5 5 5 5 5 5 b 0 0 0 0 0 2 5 2 5 2 5 2 5 2 5 . . . . . 1 1 1 1 1 P . 2 . 2 . 2 . 2 . 2 1 1 1 1 1 E D H N C Chelator (mmol/kg soil)

Estimated total Pb removed from soil by several plants (Ruley 2004) Species Chelators Soil Pb Shoot Pb Biomass Est. total Source (mg/kg) (%) (t/ha/yr) Pb extr. (kg/ha/yr) Zea mays 5.8 mmol/kg Huang et al. 2500 1.06 5-6 53-64 HEDTA 1997 1.34 g/kg Huang et al. Pisum 2450 0.897 3-4 27-36 EDTA 1997 sativum Sesbania 10 mmol/kg 7500 0.42 10-15 43-63 drummondii EDTA Brassica 10 mmol/kg Blaylock et 600 1.6 1-1.5 16-24 juncea EDTA al. 1997 Triticum 5 mmol/kg Begonia et 2000 0.92 2.5 23 aestivum EDTA+5 al. 2002 mmol/kg acetic acid

1400 Mercury concentrations in s 1200 Shoot 1000 -1 dw) 800 (mg kg 600 400 Mercury uptake 200 by 0 Sesbania 50000 45000 Mercury concentrations in 40000 Root 35000 -1 dw) 30000 (mg kg 25000 20000 15000 10000 5000 0 0 10 20 30 40 50 100 Hg concentrations (mg/l)

1800 1600 shoot Cu concentration in sh 1400 1200 -1 dw) 1000 (mg kg 800 600 400 200 Copper uptake 0 By Sesbania 35000 30000 root Cu concentration in r 25000 -1 dw) 20000 (mg kg 15000 10000 5000 0 0 25 50 100 150 300 Cu concentration (mg/l)

3500 Cr concentration in the s 3000 Shoot 2500 dw) 2000 -1 (mg kg Chromium 1500 uptake 1000 500 By Sesbania 0 6000 Root Cr accumulation in 5000 dw) 4000 roots -1 (mg kg 3000 2000 1000 0 0 25 50 100 200 Cr concentration (mg/l)

Gold uptake by Sesba bani nia 100 140 Shoot Au (mg/kg DW) Shoot Au (mg/kg DW) 120 80 100 60 80 60 40 40 20 20 0 0 0 25 50 100 200 0 d 1/2 d 1d 6d KAuCl 4 in solution (mg/L) Days

Scanning Electron Microscopy of Plant shoot grown in metals Pb root Cu shoot Cr shoot 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 180 160 140 120 Frequency 100 80 60 40 20 0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 Diameter (nm) 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.

XANES Spectra of Sesba bani nia (ET & C 23, 2068, 2004) 2.5 2.0 A B 2.0 Normalized Absorption 1.5 Normalized Absorption Lead Leaves Treatment 1.5 Lead(II) Sulfate 1.0 Lead Roots Treatment 1.0 Lead(IV) Oxide Lead(II) Nitrate 0.5 0.5 Lead(II) Sulfide Lead(II) Acetate 0.0 0.0 0.0 0.0 12.95 13 13.05 13.1 12.95 13 13.05 13.1 Energy [keV] Energy [keV] A) L III 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)

EXAFS Spectra of Sesba bani nia 5.0 5.0 Fourier Transform Magnitude Fourier Transform Magnitude Lead(II) Sulfide Root Sample 4.0 4.0 Lead(IV) Oxide 3.0 3.0 Leaves Sample Lead 2.0 2.0 Lead(II) Sulfate 1.0 Lead(II) Acetate 1.0 Lead(II) Nitrate 2 4 6 0 2 4 6 R [Å] R [Å]

XANES and EXAFS data of Pb-treated Sesba bani nia (Environ. Toxicol. Chem. 23, 2068-2073, 2004) Samples Pb(NO 3 ) 2 PbSO 4 Pb metal PbS Pb acetate % % % % % Leaves 7.6 25.8 0 14.2 52.4 Roots 10.1 0 8.8 20.2 60.9

Au(CH 3 COO) 3 A 2.0 AuOH KAuCl 4 A. XANES of gold model 1.5 Au 2 S Normalized Absorption compounds: gold acetate, Au(0) 1.0 gold hydroxide, potassium tetrachloroaurate, gold sulfide, and gold metal. 0.5 B. XANES of gold-laden 11.9 11.95 12 B plant samples. Energy [keV] 2.0 Roots 50 ppm Roots 100 ppm 1.5 Shoots 100 ppm Normalized Absorption 1.0 0.5 11.9 11.95 12 12.05 Energy [keV]

XANES of gold accumulated in Sesba bani nia Samples % % % % % Au(0) AuOH KAuCl 4 Au(CH 3 COO) 3 Au 2 S Au 0 0 18.4 81.6 0 50 ppm roots Au 0 0 16.4 83.6 0 100 ppm roots Au 0 0 14.2 84.4 1.4 100 ppm shoots