bioremediation technology for heavy metal pollution · 3 environmental remediation challenges in...
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Bioremediation Technology for Heavy Metal Pollution
Dr. Surasak SiripornadulsilDepartment of Microbiology,
Faculty of Science, Khon Kaen University
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2Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
Environmental PollutantsType of Pollutant ExamplesInorganic
Metals Cd, Hg, Ag, Co, Pb, Cu, Cr, FeRadionuclideNitrates, nitrites, phosphatesCyanidesAsbestos
OrganicBiodegradable Sewage, domestic agricultural,
and process wastePetrochemical Oil, diesel, BTEXSynthetic Pesticides, organohalogens, PAHs
BiologicalPathogens Bacteria, viruses, molds
GaseousGases SO2, CO2, N2O, CH4Volatiles CFCs, VOCs
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Environmental Remediation ChallengesIn USA
About 5,700 individual contaminant sitesAt Nevada Test site 1.5 million m3
At Fernald site 0.71 Km3
Savannah River site 7.8 Km3
Hanford site 18 Km3
Unknown quantities of buried waste at numerous sites.Costs $142 billion to locate and character contamination, restore, and dispose of wastes over the next 35 years.
In UKAbout 100,000 individual contaminant sitesCosts between £10,000 and £20,000 million to clean up.
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Arsenic in Ground-Water Resources of the United States
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http://www.epa.gov/
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Superfund sites in the United States
http://www.pollutionissues.com/
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Heavy Metalsa group of metals with density higher than 5.0 g/cm3
B C N O F Ne
He
Al Si P S Cl Ar
Se Br Kr
I Xe
At Rn
HLi Be
VNa Mg
K Ca Sc TiRb Sr YCs Ba La*
Ac**
Lanthanides*
Actinide**
Fr Ra Rf Db Sg Bh Hs Mt
Cr
Zr Nb Mo
Mn
Tc
Fe
Hf Ta W Re OsRu Rh
Co Ni Cu Zn Ga
PtIr
Pd Ag Cd InAu
Sb
As
SnGe
Hg Tl
Te
Lr
Lu
Bi
Gd Dy Er
PoPb
Tm YbTb Ho
MdFm NoEsCfBkCm
Ce Pr Nd PmPa Np Pu Am
Sm EuTh U
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The global distribution of cadmium output in 2005 as a percentage of the top producer (China - 3,000 tonnes)
Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
Cadmium output in 2005 shown as a percentage of the top producer (China)
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http://www.csr-asia.com/
Gold Peak, Cadmium and “Grey Babies”
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The relative importance of various cadmium sources to human exposure (Van Assche, 1998)
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www.jamesmbrown.co.uk/cd_pigments/green.htm
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EPA Maximum Contaminant Levels for Drinking Water
Metals PPB μMolarBarium 2,000.0 15.00Copper 1,300.0 21.00 Chromium 100.0 2.00Arsenic 50.0 0.60Selenium 50.0 0.60Lead 15.0 0.07 Cadmium 5.0 0.04Mercury 2.0 0.01Thallium 2.0 0.01Organics PPBBenzene 5.0Atrazine 3.0PCBs 0.5Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
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Toxic substances in the food chain(Bioaccumulation)
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Molecular Mechanisms of Heavy Metal Toxicity
• Replace essential metals in metalloenzymes
• Bind to sulfhydryl groups
• Facilitate formation of free radicals and reactive oxygen species
• Alter redox status of cells
• Interfere with essential metal uptake and transport
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Cadmium ToxicityCarcinogenDevelopmental ToxicantReproductive ToxicantCardiovascular or Blood ToxicantEndocrine ToxicantImmuno ToxicantKidney ToxicantNeuro ToxicantRespiratory Toxicant
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15Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
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Heavy Metal Pollution in Thailand
11: Nakhon Si Thammarat (1986) Arsenic
2
2: Kanchanaburi (1998) Lead
3
3: Tak (2002) Cadmium
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Cd Contamination in Baan Pha Te, Mae Sot, Tak Province
Simmons, RW, et al. (2005) Environ. Geochem. & Health, 27: 501-511.
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Crop tissue Zn, Cd and Fe concentrations (mg/kg) and tissue Cd:Zn and Cd:Fe ratios
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• Some trace metals are essential nutrients (e.g., copper, cobalt, etc.) and others are toxic (e.g., cadmium, lead, mercury).
• Trace metals are active in their elemental form or as salts.
• Trace metal pollutants can not be decomposed, unlike organic pollutants, but must be sequestered from the environment.
• Ideally, trace metal recovery should be selective without interference from other metals.
Impediments to Remediation of Trace Metal Contaminants
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A Problem of the PastA Problem of the Past,,A Solution for the FutureA Solution for the Future
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21Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
http://biogeochemistry.plantbiology.msu.edu
Biogeochemical Cycle
Microbes
Microbes
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Bioremediation is the process of using naturally occurring plants and microbes to digest and convert
waste material into harmless substances.
Select a strategy of Bioremediation
Identify the problem
Develop a solution
Microbes and plants detoxify waste
Problem solved
Life Cycle of a pollution problem
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Bioremediation Strategies1. Use the indigenous microbial population.2. Stimulation of indigenous microbial growth.3. Bioaugmentation: the addition of adapted
or designed inoculants.4. Addition of genetically modified
microorganisms. 5. Phytoremediation.
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ESSENTIAL FACTORS FOR MICROBIAL BIOREMEDIATION
Factor Desired Conditions• Microbial Suitable kinds of organisms that can biodegrade
population all of the contaminants• Oxygen Enough to support aerobic biodegradation
(about 2% oxygen in the gas phase or 0.4 mg/liter in the soil water)
• Water Soil moisture should be from 50–70% of the water holding capacity of the soil
• Nutrients Nitrogen, phosphorus, sulfur, and other nutrients to support good microbial growth
• Temperature Appropriate temperatures for microbial growth (0–40˚C)
• pH Best range is from 6.5 to 7.5
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LIMITATIONS OF BIOREMEDIATION• Contaminant type and concentration• Environment• Soil type• Condition and proximity of groundwater• Nature of organism• Cost/benefit ratios: cost versus overall
environmental impact• Does not apply to all surface• Length of bioremediation process• Capabilities of bioremediation
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Microbes effectively sequester heavy metals:• Microbes are the primary means of heavy metal
deposition in terrestrial/aquatic environments.• Tolerant of high heavy metal concentrations. • High heavy metal binding capacity. • Synthesize high-affinity, heavy metal binding proteins in
response to toxic heavy metal concentrations.• Bioimmobilization
Applications in terrestrial/aquatic environments:• Easily grown and harvested. • Unicellular, high surface to volume ratio.• Can be genetically engineered to potentially improve
metal recovery.
Microbes as Heavy Metal Remediation
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1. Heavy Metal-Induced Gene Expression in Chlamydomonas
2. Expression of Plant “Stress Genes” in Transgenic Chlamydomonas: Their Effects on Heavy Metal Tolerance and Binding
3. Expression of Surface-Displayed Heavy Metal Binding Proteins: Their Effects on Heavy Metal Tolerance and Binding
4. Cadmium-Resistant Bacteria/ Cadmium Contaminated Soil
Research Programs
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Analysis of Early Cadmium-Induced Transcripts by Differential Display
• Cells exposed to 25 μM cadmium for two hours
• mRNA isolated from WT and cadmium exposed cells and reverse transcribed
• cDNA amplified using GC-biased sets of primers and products displayed on sequencing gel
• Cadmium induced transcripts confirmed by reverse northern blot analysis (3X)
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Representative Early, Cadmium-Induced Differential Display Result
No Cd 2-hr Exposureto 25 μM CdCl2
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1. Thirteen non-redundant cadmium-induced cDNAsidentified by differential display.
2. Five cDNAs identifieda. psbA - D1 protein of PSIIb. chlL - Fe-S subunit of light-independent
protochlorophyllide reductasec. CHRSAMS - SAM synthetased. CRD1 - required for PSI assembly under copper
deficiency, Fe-protein?e. H43 – mostly highly induced by cadmium, encodes a
periplasmic protein involved in iron uptake.3. Two cDNAs present in Chlamydomonas EST library, one
is high-CO2 induced.4. Currently, six unidentified cDNAs.
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Early Cadmium-Induced Genes in Chlamydomonas
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The Chlamydomonas H43 Gene Compliments Yeast fet3fet4 (low and high affinity iron uptake) Mutants.
Solid symbols: fet3fet4complemented with H43 gene
Open symbols: fet3fet4
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Comparative Growth at Different Iron Concentrations (5-8 μM Fe+3)
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H43 Expression in WAT11 (WT S. cerevisiae) Does Not Confer Cadmium Sensitivity
WA
T11::pY
ES2 (3)
WA
T11 ::H
43 (1 ,3)
WA
T11::pY
ES2 (4)
WA
T11 :: H
4 3 (2 ,2)
WA
T11::pY
ES2 (3)
WA
T11 ::H
43 (1 ,3)
WA
T11::pY
ES2 (4)
WA
T11 ::H
43 ) ( 2,2 )
No Cd 100 μM Cd
10 –1 O.D.
10 –2 O.D.
10 –3 O.D.
Cell number
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Does H43 Transport both Iron and Cadmium or Just Iron?
Cell wall
Periplasmicspace
Plasmamembrane
Fe(III)
Fe(II)
H43
Fe(III) Fe(II)
ZIP
Cd H43 Cdferric reductase
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Conclusions• Several early cadmium-induced genes involved
in Fe metabolism.• Chlamydomonas H43 is a novel protein with a
role in iron uptake◘ H43 induced in response to relatively low cadmium
exposure and iron deficit implying competition between iron and cadmium uptake
◘ The H43 gene will be a useful tool in the study of metal homeostasis and may be utilized in the development of superior iron-acquiring crops
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1. Heavy Metal-Induced Gene Expression in Chlamydomonas
2. Expression of Plant “Stress Genes” in Transgenic Chlamydomonas: Their Effects on Heavy Metal Tolerance and Binding
3. Expression of Surface-Displayed Heavy Metal Binding Proteins: Their Effects on Heavy Metal Tolerance and Binding
4. Cadmium-Resistant Bacteria/ Cadmium Contaminated Soil
Research Programs
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Expression of Plant “Stress Genes”in Transgenic Chlamydomonas:
Their Effects on Heavy Metal Binding and Tolerance
Over-expression of Proline
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Pyrroline 5 Carboxylate Synthetase (P5CS) Catalyzes a Rate-Limiting Step in Proline Synthesis
P5CS
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Southern/Western blot analysis of wild-type (CC-425) Chlamydominas and transformed cells
expressing foreign P5CS geneKb 1 2 3 4
14.88.8
5.4
4.33.8
2.42.2
Lane 1: Wild-type (CC-425)/KpnI Lane 2: P5CS-1/KpnI Lane 3: P5CS-2/KpnI Lane 4: P5CS-3)/KpnI
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1 2 3 4 5 690
70
kD
P5CS trangenic W.T.
Lane 1: 10 mg protein of P5CS-1Lane 2: 15 mg protein of P5CS-1Lane 3: 20 mg protein of P5CS-1Lane 4: 10 mg protein of CC-425Lane 5: 15 mg protein of CC-425Lane 6: 20 mg protein of CC-425
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Growth of wild-type (CC-425) and transformed Chlamydomonas expressing the P5CS gene
0 μM Cd 100 μM CdDays of Growth
0 2 4 6 8 10
Abs
orba
nce
750
nm
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
W.T. P5CS-1 P5CS-2 P5CS-3-
Days of Growth0 2 4 6 8 10
Abs
orba
nce
750
nm0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6W.T. P5CS-1 P5CS-2 P5CS-3-
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Cadmium Binding Capacity of wild-type and transformed Chlamydomonas cells expressing
foreign P5CS gene
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1μg Cd/ Abs 750 nm
W.T.
P5CS-1
P5CS-2
P5CS-3
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X-ray: electro-magnetic wave λ = 0.01 - 10 nm or Energy = 1100 eV - 100 KeV
Application of EXAFS :To obtain information about the arrangement of
atoms in locality of the absorbing atom.
The Useful Properties of EXAFS:1. Non-crystalline and crystalline solids can be treated
on the same basis.2. Structural information is obtained from EXASF by
a simple and direct analysis.3. The measurement is relatively easy and rapid.
EXAFS(Extended X-ray Absorption Fine Structure)
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Cd-ProlineComplex
Structure Model of Cadmium-PC Complex
S
S
SS
Cd
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43Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
Siripornadulsil, et al. 2002. The Plant Cell. 14: 2837–2847.
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Cd K-edge EXAFS curve-fitting results for Cd-Chlamydomonas
Sample atom types N R(Å) σ2 (Å2) ΔΕ0 (eV)
W.T. (CC-425) Cd-O 1.15 2.2065 0.00389 4.953
Cd-S 1.40 2.5184 0.00208 4.953
P5CS-1 Cd-S 3.24 2.5000 0.00859 -0.574
N : Coordination number, R (Å) : Interatomic distance,σ2 (Å2) : Debye-Waller factor,ΔΕ0 (eV) : The difference of the resulting absorption energy
and the constant input in the FEFF8 calculation.
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Phytochelatin (PC) Synthesis
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Summary & Conclusion1. P5CS transformants have higher cytoplasmic
proline levels (1.8X) than wild-type cells.2. Expression of the P5CS gene enhances cadmium
tolerance (1.5X) and binding capacity (4.2X).3. Cadmium is coordinated predominantly by sulfur
in P5CS transformants (reduced sulfhydryl of phytochelatin).
4. Proline enhances cadmium tolerance by directly reacting to free radical as primary antioxidant, resulting the elevated GSH level. Then high level of GSH would presumably allow for increased PC synthesis.
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Cd2+ Cd2+
Glu + Cys γGluCys GSH PCsPC
synthaseGSH
synthetaseγ -GluCys
synthetase
Cd2+
sulfide
HMW CdS-PC complex
LMW Cd-PC complexs
Cd2+
Cd2+
vacuole
GS-Cd
Chloroplast, mitochondria,
ER
1O2, •OH1O2, •OH
1O2, •OH
GSSG
Cd2+
PCsPC
synthase
Cd2+
sulfide
HMW CdS-PC complex
LMW Cd-PC complexs
Cd2+
GS-Cd
Proline
+Proline + 3O2
Proline + nitroxideProline + peroxide
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48
1. Heavy Metal-Induced Gene Expression in Chlamydomonas
2. Expression of Plant “Stress Genes” in Transgenic Chlamydomonas: Their Effects on Heavy Metal Tolerance and Binding
3. Expression of Surface-Displayed Heavy Metal Binding Proteins: Their Effects on Heavy Metal Tolerance and Binding
4. Cadmium-Resistant Bacteria/ Cadmium Contaminated Soil
Research Programs
Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
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Metallothionein-II, α− and β−Domain Polymers Fused to a Periplasmic
Membrane Protein
Expression of Surface-Displayed Heavy Metal Binding Proteins: Their Effects on Heavy Metal
Tolerance and Binding
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Rabbit Metallothionein-II
α
β
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Metallothionein-Plasma Membrane Fusion Proteins
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Cd K-edge EXAFS curve-fitting results for Cd-Chlamydomonas
Sample atom types N R(Å) σ2 (Å2) ΔΕ0 (eV)
W.T. (CC-2137) Cd-O 1.89 2.1926 0.00450 1.659Cd-S 1.29 2.5003 0.00211 1.659
W.T. (CC-425) Cd-O 1.15 2.2065 0.00389 4.953Cd-S 1.40 2.5184 0.00208 4.953
MT-9 Cd-S 3.60 2.5113 0.00778 -4.690AMT-11 Cd-S 3.41 2.5001 0.00797 -2.765BMT-5 Cd-S 3.52 2.5131 0.00799 -1.972
N : Coordination number, R (Å) : Interatomic distance,σ2 (Å2) : Debye-Waller factor,ΔΕ0 (eV) : The difference of the resulting absorption energy
and the constant input in the FEFF8 calculation.Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
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Summary & Conclusion1. Cytoplasmic expression of MT-II increases cadmium
tolerance (2X, at 40 mM CdCl2) but does not increase cadmium binding.
2. Expression of membrane anchored, periplasmic MT-II enhances cadmium tolerance (2.3X, at 100 mM CdCl2 ) and binding capacity per cell (5.1X for dimer).
3. Expression of periplasmic MT-II beta domain enhances cadmium tolerance (2.7X , at 100 mM CdCl2 ) and binding capacity per cell (4.4X for trimer).
4. Expression of periplasmic MT-II alpha domain enhances cadmium tolerance (1.6X , at 100 mM CdCl2 ) and binding capacity (4.1X for pentamer).
5. Cadmium is coordinated by sulfur in MT-II expressing cells (metallothionein cysteinyl sulfurs).
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54
1. Heavy Metal-Induced Gene Expression in Chlamydomonas
2. Expression of Plant “Stress Genes” in Transgenic Chlamydomonas: Their Effects on Heavy Metal Tolerance and Binding
3. Expression of Surface-Displayed Heavy Metal Binding Proteins: Their Effects on Heavy Metal Tolerance and Binding
4. Cadmium-Resistant Bacteria/ Cadmium Contaminated Soil
Research Programs
Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
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Cadmium-Resistant Bacteria/ Cadmium Contaminated Soil
Research in Progress
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Cd Contamination in Baan Pha Te, Mae Sot, Tak Province
Simmons, RW, et al. (2005) Environ. Geochem. & Health, 27: 501-511.
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X-ray: electro-magnetic wave λ = 0.01 - 10 nm or Energy = 1100 eV - 100 KeV
Application of EXAFS :1. To obtain information about vacant orbital, electronic
configuration and site symmetry of the absorbing atom. 2. To obtain information on the oxidation state of the
cadmium atom as well as the geometrical arrangement of the atoms surrounding the absorbing atom.
The Useful Properties of XANES:1. Non-crystalline and crystalline solids can be treated on
the same basis.2. Structural information is obtained from XANES by a
simple and direct analysis.3. The measurement is relatively easy and rapid.
XANES(X-ray Absorption Near Edeg Structure)
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
234
8834
9735
0735
1635
2635
3535
4435
5435
6335
7335
8235
9136
0136
10
Energy (eV)
Nor
mal
ized
Abs
orpt
ion
Cd(NO3)2.4H2O
CdO
CdS
S1-1
S3-2
S5-2
TS2-1
TS4-1
Thai-Chinese Joint Seminar on Phyto/ Bioremediation Technology 18 & 19 February 2008
Cadmium LIII-edge XANES3538 eV 3608 eV
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Cadmium-Resistant Bacteria
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Growth of Cadmium-Resistant Bacteria
0
0.5
1
1.5
2
2.5
0 3 6 9 12 15 18 21 24 30 36
เวลา (ชม )
Abs
orba
nce
600
nm
S2500-1
S2500-2
S2500-3
S2500-4
S2500-5
S2500-6
S2500-7
S2500-8
S2500-9
S2500-10
S2500-11
No Cd
Abs
orba
nce
600
nm
hours
0
0.5
1
1.5
2
2.5
0 3 6 9 12 15 18 21 24 30 36
(ช )
Abs
orba
nce
600
nm
S2500-1
S2500-2
S2500-3
S2500-4
S2500-5
S2500-6
S2500-7
S2500-8
S2500-9
S2500-10
S2500-11
Abs
orba
nce
600
nm
hours
500 μM Cd
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Cd Binding capacity of Cd-Resistant Bacteria
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0
12
3
45
6
7
89
10
S2500-1S2500-2S2500-3S2500-4S2500-5S2500-6S2500-7S2500-8S2500-9S2500-10
S2500-11
S2500-12
S2500-13
S2500-14
S2500-15
S2500-16
S2500-17
S2500-18
S2500-19
S2500-20
S2500-21
S2500-22
S2500-23
S2500-24
Isolates
Log
[Cd(
atom
)]/C
ell
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Analysis of S K-edge XANES spectrum of Klebsiella planticola Strain (Cd-1)
2472 eV
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Sharma, et al. 2000. Appl. Environ. Microbiol. 66: 3083–3087.
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Biotransformation
Heather AW and Tamar B. (2005) Current Opinion in Biotechnology. 16:261–268.
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Biotransformation
Fe(III), SO4-
Fe(II), H2S
e-
Electrons from organic C (lactate, acetate, ethanol) or H2
Metal reducing bacterium
Indirect reductive precipitation
U(VI), Cr(VI)Soluble, mobile
toxic
U(IV), Cr(III)Insoluble,immobileless toxic
Ox
Red Ox Red
Useful in above-ground and in situ treatments
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Direct reductive precipitation
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Wells at old Rifle mine inject acetate (vinegar) into the ground to stimulate the growth of
microbes already in the soil.
http://www.genomenewsnetwork.org/articles/12_03/geobacter.shtml
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BIOTECNational Synchrotron Research Center Dr. Richard T. Sayre, Plant Biology (Ohio State University)Dr. Sam Traina, Natural Resources (University of California at Merced )Members of My LabDepartment of Microbiology, Faculty of Science, Khon Kaen University