Bioremediation of TNT: What happens when a pollutant just disappears

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Bioremediation of TNT What happens when a
pollutant just disappears
J. B. Hughes, Ph.D., P.E. Professor and
Chair Civil and Environmental Engineering George
R. Brown School of Engineering Rice
University Houston, Texas
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Acknowledgments

• HSRC S/SW
• DTRA
• SERDP
• Texas Advanced Technology Program
• Ron Spanggord - SRI International
• L. Wolfe and S. McCutcheon - E.P.A. Athens
• Jim Spain - U.S.A.F. Armstrong Labs
• Debbie Roberts - University of Houston

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Research Group Collaborators

• Research Group
• Dr. R. Bhadra
• Dr. C. Y. Wang
• Dr. C. Zhang
• Dr. K. Yesland
• F. Ahmad
• M. Vanderford
• T. Khan
• A. Richardson
• J. Lauritzen
• L. Pucik

• Collaborators
• Dr M. Saunders
• Dr. J. Shanks
• Dr. F. Rudolph
• Dr. G. Bennett
• Dr. M. Tadros
• Dr. E. Lykissa

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Why Study TNT?

• Contamination present at numerous sites
• Fate poorly understood
• Alternative remediation being tested

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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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Mineralization of Aromatics
O2
O2
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Common Observations inTreatment Systems

• 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 is highly variable

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Nitroaromatic Metabolism Background

• Area of intense study for a decade
• Nitro group (-NO2) is
• a strong electron withdrawing group
• a ring deactivator
• decreases potential for oxygenase attack and ring
  fission
• Nitro group is prone to reduction

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Nitro Group Reactivity
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Established Metabolites
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Example of Disappearance
Bruhns-Nagel, et al., Biodegradation
of Nitroaromatic Compounds and Explosives, Eds.
Spain, Knackmuss, Hughes
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Objectives

• Identify novel intermediates and products of TNT
  metabolism by bacteria and plants
• Determine analytical methods for monitoring of
  intermediates and products
• Assess potential for toxicity reduction
• Examine metabolic pathways (enzymes, regulation,
  diversity)
• Technology transfer

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Organism Selection

• Clostriduim
• acetobutyicum and thermoautorophicum
• Reasons
• Representative
• Highly characterized
• Non-pathogenic
• Demonstrated nitro-reductase activity

• Myriophyllum and C. roseus
• Native, axenic, tissue cultures
• Reasons
• Practical
• Some characterization available
• Plant vs. bacteria testing

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TNT Transformation by Clostridia
TNT
5 minutes
15 minutes
35 minutes
65 minutes
100 minutes
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Transformation Pathway
?
(?)
Bamberger Rearrangement
Reduction
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Radiochromatogram
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Native Myriophyllum
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Plant Products
OxidationProducts!
BoundResidue
Intitial reduction may be at 2 position instead
of 4 position
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Mass Balance
Data taken at t 12 days
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ImplicationsofFindings
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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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Analytical - cont.

• Plant metabolites exist inside and outside of the
  plant
• Extracellular products are polar oxidation
  products and amino-dinitrotoluenes amenable to
  HPLC or GC analysis
• Intracellular products are high molecular weight
  conjugates and bound residues
• Without 14C, it is effectively impossible to
  track intracellular fraction

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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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Ferredoxin-Hydrogenase Couple
4Fe4S Hydrogenase
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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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Arylhydroxylamine Chemistry
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Nitrosoarene Chemistry
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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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Transient Mutagenicity
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Snail Mortality
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TNT Ring Fission?Hope is Alive!

• Bacteria
• 4-Amino-6-hydroxylamino-3-methyl-2-nitrophenol
• Plants
• 2-amino-4,6,dinitrobezoate
• 2,4-dinitro-6-hydroxy-benzyl alcohol
• 2,-N-acetoxyamino-4,6-dinitrobenzaldehyde
• 4,-N-acetoxyamino-2,6-dinitrobenzaldehde
• 2,4-dinitro-6-hydroxytoluene
• 2,6-dinitro-4-hydroxytoluene

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Objectives

• Identify novel intermediates and products of TNT
  metabolism by bacteria and plants
• Determine analytical methods for monitoring of
  intermediates and products
• Assess potential for toxicity reduction
• Examine metabolic pathways (enzymes, regulation,
  diversity)
• Technology transfer

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Relevant Publications

• Metabolism of Nitroaromatics and Explosive
  Compounds, Eds. J. Spain, H. Knackmuss, and J. B.
  Hughes, CRC Press, in press (summer 2000)
• Padda, R. S. C. Y. Wang, J. B. Hughes, and G. N.
  Bennett, Mutagenicity of Trinitrotoluene and its
  Metabolites Formed During Anaerobic Degradation
  by Clostridium acetobutylicum ATCC 824, accepted
  for publication in Environmental Toxicology and
  Chemistry, May, 2000.
• Huang, S., P. A. Lindahl, C. Wang, G. N. Benett,
  F. B. Rudolph, and J. B. Hughes,
  2,4,6-Trinitrotoluene (TNT) Reduction by Carbon
  Monoxide Deydrogenase from Clostridium
  thermoaceticum Product Identification, Kinetic
  Characterization, and Inhibitor Study, accepted
  for publication in Applied and Environmental
  Microbiology, January, 2000.
• Bhadra, R., D. G. Wayment, R. K. Williams, S. N.
  Barman, M. B. Stone, J. B. Hughes, and J. V.
  Shanks, Studies on Plant-Mediated Fate of the
  Explosives RDX and HMX, accepted for publication
  in Chemosphere, August, 1999.
• Tadros, M.G., A. Crawford, A. Mateo-Sullivan, C.
  Zhang, and J. B. Hughes Toxic Effects of
  Hydroxylamino Intermediates from Microbial
  Transformation of Trinitrotoluene and
  Dinitrotoluenes on Algae Selenastrum
  capricornutum, (200) Bulletin of Environmental
  Contamination and Toxicology, (64)579-585.
• Wang, C. Y., D. Zheng, and J. B. Hughes, (2000)
  Stability of Hydroxylamino and Amino
  Intermediates from Reduction of
  2,4,6-Trinitrotoluene, 2,4-Dinitrotoluene and
  2,6-Dinitrotoluene Biotechnology Letters,
  (22)15-19
• Wayment, D. G., R. Bhadra, J. Lauritzen, J. B.
  Hughes, and J. V. Shanks, (1999) A Transient
  Study of Conjugate Formation During TNT
  Metabolism by Axenic Plant Roots, Journal of
  Phytoremediation, (1)227-239.
• Bhadra, R., R. J. Spanggord, D. G. Wayment, J. B.
  Hughes, and J. V. Shanks, (1999)
  Characterization of Oxidation Products of TNT
  Metabolism in Aquatic Phytoremediation Systems of
  Myriophyllum aquaticum, Environmental Science
  and Technology, (33)3354-3361.

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Relevant Publications - cont.

• Hughes, J. B., C. Wang, and C. Zhang, (1999)
  Anaerobic Transformation of 2,4- and
  2,6-Dinitrotoluenes by Clostridium
  acetobutylicum A Pathway Through
  Dihydroxylamino-Intermediates, Environmental
  Science and Technology, (33)1065-1070.
• Bhadra, R., D. G. Wayment, J. B. Hughes, and J.
  V. Shanks, (1999) Confirmation of Conjugation
  Processes During TNT Metabolism by Axenic Plant
  Roots, Environmental Science and Technology,
  (33)446-452.
• Wang, C. and J. B. Hughes, (1998) Derivatization
  and Separation of 2,4,6-Trinitrotoluene Metabolic
  Products, Biotechnology Techniques,
  (12)839-842.
• Pucik, L. E. and J. B. Hughes, (1998)Fate of TNT
  and TNT-Transformation Products in Aerobic Mixed
  Cultures, Bioremediation Journal, (2)57-67.
• Hughes, J. B., C. Wang, K. Yesland, A.
  Richardson, R. Bhadra, G. Bennett, and F.
  Rudolph, (1998) Bamberger Rearrangement During
  TNT-Metabolism by Clostridium acetobutylicum,
  Environmental Science and Technology,
  (32)494-500.
• Hughes, J. B., C. Wang, R. Bhadra, A. Richardson,
  G. Bennett, and F. Rudolph, (1998) Reduction of
  2,4,6-Trinitrotoluene by Clostridium
  acetobutylicum through Hydroxylamino-Nitrotoluene
  Intermediates, Environmental Toxicology and
  Chemistry, (17)343-348.
• Wang, C. Y., R. Bhadra, and J. B. Hughes, (1997)
  The Rapid Separation of Reduction Products of
  2,4,6-Trinitrotoluene using TLC Biotechnology
  Techniques, (11)519-521.
• Tadros, M. G. and J. B. Hughes, (1997)
  Degradation of Polycyclic Aromatic Hydrocarbons
  (PAHs) by Indigenous Mixed and Pure Cultures
  Isolated from Coastal Sediments, Applied
  Biochemistry and Biotechnology, (63-65)865-870.

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Relevant Publications - cont.

• Vanderford, M., J. V. Shanks, and J. B. Hughes,
  (1997) Phytotransformation of Trinitrotoluene
  (TNT) and Distribution of Metabolic Products in
  Myriophyllum aquaticum, Biotechnology Letters,
  (19)277-280.
• Khan, T. A., R. Bhadra, and J. B. Hughes, (1997)
  Transformation of TNT and Related Nitroaromatic
  Compounds by Clostridium acetobutylicum, Journal
  of Industrial Microbiology and Biotechnology,
  (18)198-203.
• Hughes, J. B., J. V. Shanks, M. Vanderford, J.
  Lauritzen (1997), Transformation of TNT by
  Aquatic Plants and Plant Tissue Cultures,
  Environmental Science and Technology,
  (31)266-271.
• Pucik, L. E., and J. B. Hughes, (1996) Capillary
  Electrophoretic Separation of TNT and its
  Transformation Products, Journal of Capillary
  Electrophoresis, (3)209-215.