Insitu Immobilization of Uranium VI by Dissimilatory Metal Reducing Bacteria DMRBs

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In-situ Immobilization of Uranium (VI) by
Dissimilatory Metal Reducing Bacteria (DMRBs)

• Gaurav Saini
• Winter 2004
• ENVE 541

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Overview

• Uranium- General Aspects
• Uranium Chemistry
• Radioactivity Contamination
• Management Strategies
• Dissimilatory Metal Reducing Bacteria (DMRB)
• Shewanella putrefaciens
• Geobacter metallireducens
• Reduction Mechanism
• Current Research
• Conclusions

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Uranium- General Aspects

• Discovery 1789, Germany
• Heaviest element 4 ppm in Earths Crust
• Use Nuclear industry, Research Institutions.
• Nuclear Weapons Cold War
• DOE 18 facilities 91 disposal sites
• Contamination Soil and Water
• Hanford, INEEL Complex, Oak Ridge, Savannah River
  etc.
• Suspected carcinogen, teratogen mutagen.
• Remediation Required.

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Uranium Chemistry U92

• Actinide Representative
• Isotopes 22 Total
• 3 Natural 238U, 235U 234U
• 238U92 Most Abundant (99.275)
• Half Life 4.5 billion years
• Electronic Configuration Rn.5f3.6d1.7s2
• Oxidation States 2345 and 6
• Stable 4 6 only
• U(VI) soluble and reactive
• Common Compounds oxides, fluorides, nitrates,
  chlorides, acetates etc.

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Uranium Chemistry (contd..)

• U(VI) Most Common form U3O82
• UO22 Fluorescent compound.
• U(IV) Stable form
• Compounds hydroxides, hydrated fluorides,
  phosphates
• Low solubility compounds

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Radioactivity

• Discovery 1896, Becquerel
• Process Fission
• Discovery 1939
• Products Kr Cs a, ß and ? radiations etc.
• Fissile Matter 235U and 234U.
• Enrichment Enhancing isotopic concentration
• Nuclear Reactors lt 10
• Weapons and sp. Reactors gt90
• Waste Generation
• Low level large quantity
• High level small quantity (relative!!)

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Radioactive Contamination

• First 50 years of Misuse (!!)
• Legacy of Cold War
• DOE sites heavily contaminated
• Use 113 facilities
• R D Weapon Testing
• Contamination at gt 50 facilities
• 1.7 Trillion gallons groundwater
• 40 million cubic meters soil.
• Post Cold War DOE
• Remediation Decontamination

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DOE Facilities across United States
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Hanford Disasters

• Plutonium for first WMD, WW II
• 1 million gallon waste lost to soil
• 100,000 curie strength
• Total disposal 440,000,000,000 gallons.
• Current Status Superfund Site
• Remediation efforts
• Workers unaware until 1986.

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Waste Volumes at DOE sites
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Management Strategies-I

• Storage
• Traditional SAFE
• Fire Drill Cascaded underground tanks
• Cribs, Pits
• Lost to sub-surface
• Current
• Double lined tanks
• Yucca Mountain Repository

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Waste Management at Hanford
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Management Strategies-II

• Treatment Remediation
• Treatment removal of contaminant
• Remediation Clean Up
• Traditional Method Ex-situ
• Pump--Treat
• Ground Water pumping from wells
• Soil excavation
• Cleaned matter back packing or reinjection
• Costly
• Labor intensive
• Subsurface Disturbance
• Incomplete Removal

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Ex-Situ Technique
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Management Strategies-III

• In-situ Technique
• Sending chemicals or bugs in.
• Low cost
• Better removal
• No disturbance to subsurface.
• Bioremediation Use of microbial cultures
• Contaminant energy and carbon source
• Transformations and Reduction
• Organics CO2 others.
• Metals Less mobile stable form

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In-Situ Technique
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Dissimilatory Metal Reducing Bacteria

• Metal reduction metabolism coupling
• Dissimilatory metabolism
• Energy derivation from metal reduction
• Electron Donor
• H2 (mostly)
• Organics
• Electron Acceptor
• Metals (mostly)
• Reduction to less mobile, stable forms

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DMRB (contd)

• Favor anoxia
• Spread Domain Bacteria
• Wide range
• Gram negative mostly
• Mainly Proteobacteria
• Some gram positives too
• Subsurface conditions favorable for Growth
• Natural presence sediments, aquatic env.
• Geobacter metallireducens and Shewanella
  putrefaciens extensively studied

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DMRB Phylogenetic Tree
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DMRB- Shewanella putrefaciens

• Formerly Pseudomonas strain 200 or Alteromonas
  putrefaciens
• Facultative and microaerophilic
• Isolated from Lake Oneida, NY.
• Gram negative, motile rods
• Heterotrophs
• E.D. H2, acetate, lactate, formate etc
• E.A. Nitrate, fumarate, thiosulfate, U(VI), Fe
  (III), Mn (IV), O2 etc.

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Shewanella putrefaciens

• Aerobic Growth
• Cytochrome plasma membrane
• No Ferric reductase activity
• Anaerobic Growth
• Cytochrome Outer membrane
• Ferric reductase activity of membrane
• Strains
• MR-1 renamed Shewanella oneidensis
• CN-32 extensive research currently

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Shewanella putrefaciens CN-32

• Isolated from Morrison formation, New Mexico.
• Morrison Uranium formation
• Natural occurrence subsurface
• Reduction of U(VI) to U(IV)
• Energy derived from reduction
• DOE funded studies

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Shewanella putrefaciens CN -32
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Geobacter metallireducens

• Isolated from sediment Potomac River, DC, 1987
• Degrade organics (PAH) with Fe (III) as electron
  acceptor. First bug
• Energy generation from metal reduction
• Obligate anaerobe
• Heterotroph
• Non-motile rod
• C-type cytochrome
• E.A. Mn(IV), U(VI), Fe(III), Nitrate, AQDS etc.
• E.D. Acetate, propionate, ethanol, benzoate etc.

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Geobacter metallireducens
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Reduction Mechanism

• U(VI) reduction vs. Fe(III) reduction
• U(VI) accumulation in Fe(III) rocks Bleached
  reduction spots
• DMRB DIRB
• Electron Transfer to Fe(III)
• Enzyme, electron shuttle, e.a., e.d.
• Cytochrome suspected shuttle enzyme.
• High reductase activity with high cytochrome
• Reductase activity at membrane
• Shuttle action uncertain
• U(VI) Scarcity of information

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Fe(III) Reduction by DMRB
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Reduction Inhibitors

• Nitrate Preferred electron acceptor
• Excess substrate No effect
• Mobilization of metals
• Oxygen growth inhibitor
• Competition
• No effect of Fe(III) co-reduction solution form
• Solid form reduction affected
• Calcium Ca-U-carbonate complex formation
• Pyrolusite Reoxidation to U(IV)

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Reduction Enhancement

• Humic Addition
• Electron shuttles
• Electron transfer to metal
• U(VI) Anthraquinone (AQDS)
• Chelating Agents
• Conversion to soluble form
• eg. NTA
• Bacterial Starvation
• Starvation adaptation
• Easy subsurface injection

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Additional DMRBs

• Desulfovibrio desulfuricans
• Geobacter sulfurreducens
• Shewanella algae BrY
• Shewnella oneidensis
• Desulfovibrio vulgaris

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PNNL Group

• Lead member Shewanella Federation.
• Biotechnology
• Physiology culturing
• Cell network modeling imaging
• NABIR projects
• Biotransformation
• Microbial ecology
• Current work Shewanella for subsurface
  remediation
• Lots of work on Shewanella putrefaciens

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Lovleys Group

• Extensive research on Geobacter
• Areas of interest
• Genome Geobacter
• Fuel cells DMRBs
• Bioremediation U(VI) and PAH
• Extremophile research
• Discovered Geobacter
• Dr. Lovley Group head
• 178 publications
• PI gt 10 projects
• Regular in Nature Science

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Conclusions

• DMRBs
• Potential for U(VI) reduction
• Scarcity of information
• Immobilization in long term ???
• Transport of reduced forms ???
• Unified approach Genome-Remediation-Transport-
  Long Term Stability

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Bioremediation Society