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TNT Bioremediation

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octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) ... Elution time, minutes. Absorbance. 5. 10. 15. 20. sample time. TNT Radiochromatogram. TNT Results ... – PowerPoint PPT presentation

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Title: TNT Bioremediation


1
TNT Bioremediation
Dr. Joseph Hughes Rice University
2
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
3
Explosives Compounds
TNT
RDX
HMX
DNTs
4
Process Selection and Cost
5
TNT Bioremediation Systems
  • Microbial
  • Slurry reactors
  • Composting
  • Plant-based
  • Aquatic plant lagoons (groundwater)
  • Terrestrial plants (soils)

Remediation not based on contaminant
mineralization
6
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

7
Nitro Group Reactivity
Reduction
Oxidation
8
Aryl Nitro Reduction
9
Initial Nitroaromatic Transformation
n 1
Adapted from Reiger and Knackmuss (1995)
n 0
n 2
n 3
10
TNT Reduction
11
Strategy for Binding
Lenke, et al., Biodegradation of Nitroaromatic
Compounds and Explosives,
Eds. Spain, Knackmuss, Hughes
12
Slurry System
1 Substrate Medium Inocula
200 to 300 per ton of soil treated
13
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

14
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

15
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

16
Selection of Clostridia
Reduction of nitro groups well-documented
17
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

18
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

19
TNT Transformation
20
TNT Radiochromatogram
21
TNT Results
Results from cell extracts matched cell cultures
22
Product Isolation
23
Spectroscopy
24
ImplicationsofFindings
25
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

26
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

27
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

28
Reactivity of Aryl-Hydroxylamines
  • Many reactions possible
  • Binding to NOMmay compete withfurther
    metabolism

29
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

30
TNT Mutagenicity
31
How Does This ImpactBioremediation ofTNT
ContaminatedSoil?
32
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

33
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

34
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

35
Fermentation by C. acetobutylicum
36
Fermentation by C. acetobutylicum
Acidogenesis
Solventogenesis
37
TNT Transformation and Growth Stage
late solventogenic
mid-solventogenic
Concentration, mM
early acidogenic
38
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

39
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

40
pHTB Development Scheme
41
Over-Expression Plasmid Map
42
Antisense Plasmid Development Scheme
ori II
pSOS84
43
Antisense Plasmid Maps
44
Rates of TNT Reduction by strain type
45
TNT Reduction vs. Hydrogenase Activity
46
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

47
Fate of Aryl-Hydroxylamines
  • Many reactions possible
  • Binding to NOMmay compete withfurther
    metabolism

48
Anaerobic Sorption of 4HADNT onto IHSS Peat Humic
Acid
49
Reactivity of Aryl Nitroso-Group
  • Many reactions known
  • Difficult to synthesize
  • Reaction withthiols is central to binding

50
Nitroso-Binding to Humic Acids
51
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
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