TNT Bioremediation

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TNT Bioremediation
Dr. Joseph Hughes Rice University
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Extent of Problem

• Munitions production and handling has resulted
  in the widespread contamination of soils
  andgroundwaters by know toxins
• 2,4,6-trinitrotoluene (TNT)
• octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine
  (HMX)
• hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
• and related synthesis by-products.
• 700,000 cubic yards of soil and 10 billion
  gallons of groundwater at active U.S. military
  installations alone.

Compiled by J. Spain, USAF, 1996
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Explosives Compounds
TNT
RDX
HMX
DNTs
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Process Selection and Cost
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TNT Bioremediation Systems

• Microbial
• Slurry reactors
• Composting
• Plant-based
• Aquatic plant lagoons (groundwater)
• Terrestrial plants (soils)

Remediation not based on contaminant
mineralization
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Background TNT Metabolism

• Studied intensely yet questions remain regarding
  pathways and fate of metabolites
• Nitro group is site of initial biochemical
  modification
• Nitro group (-NO2) is
• a strong electron withdrawing group
• a ring deactivator
• decreases potential for oxygenase attack and ring
  fission
• Reduction followed by oxidation a common strategy
  for microbial bioremediation

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Nitro Group Reactivity
Reduction
Oxidation
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Aryl Nitro Reduction
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Initial Nitroaromatic Transformation
n 1
Adapted from Reiger and Knackmuss (1995)
n 0
n 2
n 3
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TNT Reduction
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Strategy for Binding
Lenke, et al., Biodegradation of Nitroaromatic
Compounds and Explosives,
Eds. Spain, Knackmuss, Hughes
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Slurry System
1 Substrate Medium Inocula
200 to 300 per ton of soil treated
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Composting Summary

• Amendments
• Manure, alfalfa, hay, grass clippings, leaves,
  garden soil, sewage sludge, apples, potatoes, saw
  dust, molasses waste, horse feed
• Temperature
• 35C to 60C
• Time
• 30 to 100 days
• Distribution
• Extractable gt humic acid gt humin gt fulvic acid
• Cost
• 130 to 225 per ton

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Common Observations

• TNT disappears
• Oxidation to CO2 does not occur
• Mass balance obtained from HPLC/GC is low
• 14C-distribution includes bound and soluble
  fractions
• Rate of disappearance and extent of binding is
  highly variable
• Endpoints are controversial

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Recent Interests

• Identify products of transformation under
  fermentative conditions
• Determine enzymes involved and factors critical
  to their expression
• Characterize fate under anaerobic-aerobic
  conditions
• Evaluate mutagenicity of intermediates and
  products

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Selection of Clostridia
Reduction of nitro groups well-documented
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Model Organism C. acetobutylicum

• Extremely well studied
• factors that influence growth
• effects of growth conditions on metabolism
• enzymology and electron transfer processes
• genetics
• Displays rapid rates of TNT transformation and
  other common nitroaromatics

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Experimental Methods

• Introduce 14C-nitroaromatic to C. acetobutylicum
• Whole cells and cell extracts
• Compare metabolites to standard materials
• Isolate and characterize novel products
• HPLC, column chromatography, acetylation
• MS, 1H-NMR, FTIR

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TNT Transformation
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TNT Radiochromatogram
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TNT Results
Results from cell extracts matched cell cultures
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Product Isolation
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Spectroscopy
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ImplicationsofFindings
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Its the Hydroxylamines!

• Hydroxylamines, not amines, are the primary
  products of reduction
• Complicates assessment of nitroaromatic fate
• Raises concerns in the ability to achieve
  toxicity reduction

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Analytical

• Primary intermediates and products of microbial
  metabolism are
• Oxygen sensitive
• Unstable
• Not amenable to EI-MS
• May be too polar for extraction
• Require either immediate HPLC analysis or
  immediate derivatization

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Its the Hydroxylamines!

• Hydroxylamines, not amines, are the primary
  products of reduction
• Complicates assessment of nitroaromatic fate
• Raises concerns in the ability to achieve
  toxicity reduction

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Reactivity of Aryl-Hydroxylamines

• Many reactions possible
• Binding to NOMmay compete withfurther
  metabolism

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Its the Hydroxylamines!

• Hydroxylamines, not amines, are the primary
  products of reduction
• Complicates assessment of nitroaromatic fate
• Raises concerns in the ability to achieve
  toxicity reduction

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TNT Mutagenicity
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How Does This ImpactBioremediation ofTNT
ContaminatedSoil?
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Answers

• If we understand what enzyme(s) are responsible
  for hydroxylamine formation we can design for
  their expression
• If we understand hydroxylamine reactions, should
  be able to delineate fate processes and toxicity
  potential

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Suspected Role of Hydrogenase in TNT Reduction

• TNT reduction by cultures previously unexposed to
  nitroaromatic compounds suggests role of
  constitutive enzyme
• Rapid TNT reduction is observed only during
  acidogenic growth phase when H2 production occurs
  through hydrogenase activity

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The Hydrogenase Enzyme

• Reversibly oxidizes hydrogen
• Commonly found in a variety of microbial species
• Fe-only hydrogenase typically associated with
  hydrogen production in anaerobic bacteria

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Fermentation by C. acetobutylicum
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Fermentation by C. acetobutylicum
Acidogenesis
Solventogenesis
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TNT Transformation and Growth Stage
late solventogenic
mid-solventogenic
Concentration, mM
early acidogenic
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Suspected Role of Hydrogenase in TNT Reduction

• TNT reduction by cultures previously unexposed to
  nitroaromatic compounds suggests role of
  constitutive enzyme
• Rapid TNT reduction is observed only during
  acidogenic growth phase when H2 production occurs
  through hydrogenase activity
• CO and O2, know inhibitors of hydrogenase, slow
  or cease TNT reduction rates
• CODH from C. thermoaceticum, an enzyme similar in
  structure to hydrogenase, has been purified using
  TNT reduction activity as the selection basis
• Isolated hydrogenase catalyzed reduction via
  hydroxylamino-intermediates

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Metabolic Engineering

• Increase expression of enzymes through known
  over-expression promoter with cloned gene of
  interest (hyd A)
• Inhibit expression through cloned antisense RNA
  segments of gene of interest (hyd A)
• Examine resulting effects
• TNT reduction rates
• Correlate TNT transformation with hydrogenase
  activity

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pHTB Development Scheme
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Over-Expression Plasmid Map
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Antisense Plasmid Development Scheme
ori II
pSOS84
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Antisense Plasmid Maps
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Rates of TNT Reduction by strain type
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TNT Reduction vs. Hydrogenase Activity
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Why Enzyme Confirmation is Useful

• Hydrogenase enzymes commonly found in many
  microbial species (20 purified)
• Probable extrapolation to hydrogenases reductive
  capability for other nitroaromatic contaminants
• Antibody to be developed as an in situ probe for
  hydrogenase prevalence in a given microbial
  consortium

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Fate of Aryl-Hydroxylamines

• Many reactions possible
• Binding to NOMmay compete withfurther
  metabolism

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Anaerobic Sorption of 4HADNT onto IHSS Peat Humic
Acid
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Reactivity of Aryl Nitroso-Group

• Many reactions known
• Difficult to synthesize
• Reaction withthiols is central to binding

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Nitroso-Binding to Humic Acids
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Binding Results

• The nitroso-thiol reaction appears to be in large
  part responsible for the reaction
• Hydroxylamines showed no appreciable reactivity
  towards humic acids
• Exposure to oxygen resulted in binding to IHSS
  humics or protein (separate tests)
• The nitroso-forms of metabolites react with thiol
  nucleophiles(such as 1-thioglycerol)
• Nitrosobenzene shows rapid reactivity towards all
  IHSS humic acids
• increasing proteinaceous content of IHSS humic
  acids increased binding capacity
• selective thiol derivatizationof IHSS humic acids
  reduced binding and promoted azoxy formation
• The extent of the anaerobic bioremediation phase
  is likely to play a key role in the possible
  binding of nitroso intermediates