Bioremediation of Explosive
Contaminants
Matt Mahler
Introduction
 In the late 19th century many nitramine compounds
were created through the process of nitration.
 During WW I and WW II their application for
industrial and military purposes was investigated.
 By 1945 an estimated 1.2 million tons of soil
surrounding production plants had been
contaminated.
(Lewes et.al, 2004)
Introduction Cont’d
 Enter the environment through wastewater from
production plants.
 Many bioremediation techniques are currently
being investigated.
 Compounds Discussed: TNT, RDX, HMX and CL-20
(Lewes et.al, 2004)
Bioremediation of RDX
Readily degradable in a
variety of environments.
Aerobic and Anaerobic Mechanisms
Two-Electron Reductive Pathway and
Denitration
(Crocker et. al, 2006)
RDX - Two Electron Reductive
Pathway
Mechanism
Bacteria Invlolved
Mechanism I
Enterobacteria, E. Coli
Mechanism II or
McCormick's
Clostridium Acetylbutilecum
Pathway
Mechanism III
Aspirgillus Niger
Intermediates
Mono, Di and Tri-nitroso-RDX
MNX, hydroxyl amino, 1,3,5triamino-1,3,5-triazine
MNX
End Products
Ethanol and Formaldehyde
Ethanol and Formaldehyde
Ammonium, Nitrous Oxide,
Formaldehyde
RDX - Two Electron Reductive
Pathway Cont’d
Special Notes
Mechanism II: No Ring Cleavage
Mechanism III: Uses Oxireductase
Still Disagreement Among Researchers
RDX - Denitration
Believed to be the most common
method of RDX Degradation.
 Occurs aerobically and anaerobically
RDX - Denitration Cont’d
Pathway
Bacteria Invlolved
Intermediates
End Products
Aerobic
Rhodococcus rhodochrous, Williamsia and
Gordonia
-
NDAB, nitrous oxide,
ammonium, formaldehyde
and carbon dioxide
Anaerobic
K. pneumonia, C. bifermentans
MNX
Water, Nitrous Oxide and
Formaldehyde
RDX - Denitration Cont’d
Special Notes
Aerobic:
NDAB is Readily Degraded by Many Organisms
In the anaerobic denitration process two electrons
are added prior to ring cleavage.
Bioremediation of HMX
Most methods that degrade RDX have
also been shown to degrade HMX.
Also involves aerobic and anaerobic
processes.
Bioremediation of HMX
Aerobic
Methylobacterium Cometabolize HMX with
Carbon Dioxide
Anaerobic
Some process as RDX , however McCormick’s
Pathway not aplicable.
Bioremediation of CL-20
Recently Developed Nitramine
20% More Powerful Than HMX
Degradation Mechanisms Similar To
Those of RDX and HMX.
Bioremediation of CL-20
Anaerobic Degradation
Cloistridium Utilizes CL-20 for Cell Growth
Catalyzed by Dehydrogenase
End Products: Acetic Acid, Glyoxal, Nitrous
Oxide and Nitrogen Dioxide
Bioremediation of CL-20
Aerobic Degradation
While possible, CL-20 most occur in high
concentrations for process to take place.
In environments that support fungal
growth, white-rot fungi is responsible for
aerbic mineralization of CL-20.
Enhancing Anaerobic
Nitramine Treatment
Anaerobic Treatment of HMX, RDX and
TNT
Current Limiting Step in Process is the
Availability of Substrate.
Historically Starch Has Been Used
Enhancing Anaerobic
Nitramine Treatment
Anaerobic Treatment of HMX, RDX and
TNT
Recent Research Introduced Propylene
Glycol and Ethanol to Cultures
Consumption of these Molecules Produces
Hydrogen Gas
Enhancing Anaerobic
Nitramine Treatment
Enhancing Anaerobic
Nitramine Treatment
Conclusion
Addition of Propylene Glycol and Ethanol
Did Increase Rate of Degradation
Not Necessary for TNT and RDX
Degradation.
Enhancing Nitramine
Treatment
Enhancing Treatment CL-20
Added Sucrose, Pyruvate, Yeast, Acetate,
Glucose and Starch to act as Carbon
Sources
Enhancing Ntramine
Treatment
Conclusions
The addition of these substrates does
increase the rate of CL-20 Degradation.
Process is independent of microbial cell
growth.
Over half randomly selected microbes
could degrade CL-20.
Aerobic Degradation of CL-20
Researchers investigated the use of P.
Chrysosporium for its use as a CL-20
degrading molecule.
At the end of the 8 day experiment
concentrations of CL-20 were virtually
non-existant.
Growth of fungi was observed.
Case Study
Louisiana Army Ammunition Plant
Currently disposes of waste through
dumping and incineration.
Experiment performed to analyze the
potential use of land farming and soil slurry
as potential methods of waste treatment
Case Study
Results
Soil Slurry showed 99% removal of TNT
and near complete removal of HMX and
RDX at the end of the 182 day experiment
Land Farming showed 82% removal of
TNT and little to know RDX and HMX
removal after same amount of time.
Conclusion
The production of nitramines is only
expected to increase.
More efficient and economical
degradation mechanisms must be
found.
Conclusion
Mo le cu le
N am e o f D e g ra da t io n Me th od
B ac t e r ia I n v ol v ed
RDX
Two -El e c tro n R ed u ctive Pat h way
En te rob a ct e ira , E . C o li , C lo st rid iu m
A c e t yl b u t ile cu m , As pi rg illu s N ige r
RDX
An a e r ob ic D e n itra tion
K . pn e u m oni a , C. b ifer m ent a ns
RDX
A e ro b ic D e n it ra t io n
Rhod o co cc u s rho d o c h ro u s, W illia m si a an d
Gor d onia
HMX
A e ro b ic D e g rada t ion
M e thyloba c teri u m
HMX
An a e r ob ic D e n itra tion
K . pn e u m oni a , C. b ifer m ent a ns
CL - 20
A e ro b ic D e g rada t ion
P. C hr y s os p o riu m
CL - 20
An a e r ob ic De g rad a tio n
Clostrid ium
QUESTIONS?