Biological Remediation of Explosive Residues: Shree Nath Singh Editor

Environmental Science
Shree Nath Singh Editor
Biological
Remediation of
Explosive Residues
Environmental Science and Engineering
Environmental Science
Series Editors
Rod Allan
Ulrich Förstner
Wim Salomons
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Shree Nath Singh
Editor
Biological Remediation
of Explosive Residues
123
Editor
Shree Nath Singh
Head & Area Coordinator
Plant Ecology and Environmental Science Division
CSIR-National Botanical Research Institute
Lucknow
Uttar Pradesh
India
ISSN 1431-6250
ISBN 978-3-319-01082-3 ISBN 978-3-319-01083-0 (eBook)
DOI 10.1007/978-3-319-01083-0
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Dedicated to parents in heavenly abode
Preface
Cyclic nitramine explosives RDX, HMX, and CL-20 are commonly synthesized as
most widespread conventional explosives. Their use in military munitions largely
for the protection of national boundaries and mining operations, has resulted in
widespread contamination of soil and water reservoirs. Residual explosives have
the potential to move into soils as well as surface and ground water and affect
various ecological and human receptors. Therefore, U.S. Environmental Protection
Agency (USEPA) has included seven nitro-substituted explosives including TNT
and RDX as priority pollutants. Labscale studies have revealed that TNT, RDX,
and HMX are toxic to a wide spectrum of organisms including bacteria, algae,
plants, earthworms, mammals, and humans. No doubt, traditional treatments of
ammunition wastes, like open detonation and burning, adsorption onto activated
carbon, photo-oxidation, etc., are not only costly, but also damaging the envi-
ronment. Therefore, scientists are interested to develop an alternative technology
based on microbes and plants which will be not only cost-effective, but also
environment friendly.
In view of above facts, the editor has made his sincere efforts to compile the
latest developments on biological remediation of explosive residues in an edited
volume which will serve as a ready reckoner to the scientists, policy makers,
teachers and students, and military personnel for the remedial measures to
decontaminate the explosive residues in soils and waters by microbes and plants,
alone or in combination.
In this endeavor, I would like to profusely thank all the contributors for their
prompt response and active participation by contributing a review article on
different aspects of biological degradation of explosive residues. I would also like
to acknowledge my Ph.D. students associated with me Ms. Shweta Mishra,
Mrs. Babita Kumari and Ms. Nitanshi Jauhri for their academic and technical
support. Besides, untiring service, provided by Mr. Dilip Chakraborty in preparing
the manuscript meticulously, is heartily acknowledged.
Lastly, I would also like to thank my family members: Mrs. Manorama Singh
(wife), Ragini (daughter), and her kids Antra and Avantika and Pritish (son) for
their inspiration, endurance, and moral support to me in this endeavor.
vii
Contents
Biodegradation of Nitrophenol Compounds . . . . . . . . . . . . . . . . . . . . 1

Nobutada Kimura, Wataru Kitagawa and Yoichi Kamagata
Microbial Degradation of 2,4,6-Trinitrotoluene In Vitro and

in Natural Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Harald Claus
Bioremediation of Nitroglycerin: State of the Science . . . . . . . . . . . . . 39

John Pichtel
Bioremediation of Nitroexplosive Waste Waters . . . . . . . . . . . . . . . . . 67

Pradnya Pralhad Kanekar, Seema Shreepad Sarnaik,
Premlata Sukhdev Dautpure, Vrushali Prashant Patil
and Sagar Pralhad Kanekar
Degradation of TNP, RDX, and CL-20 Explosives by Microbes. . . . . . 87

Baljinder Singh, Jagdeep Kaur and Kashmir Singh
Assessment of Bioremediation Strategies

for Explosives-Contaminated Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
O. Muter
Bacterial and Fungal Degradation of Nitroglycrine. . . . . . . . . . . . . . . 149

Divya Bhatia, Anita Grewal, Meenu Rathi and Deepak Kumar Malik
Bioremediation of Perchlorate Contaminated Environment. . . . . . . . . 163

Atreyi Ghosh, Kannan Pakshirajan and Pranab Kumar Ghosh
Bioremediation of Nitroaromatics (NACs)-Based Explosives:

Integrating ‘-Omics’ and Unmined Microbiome Richness . . . . . . . . . . 179
Debasree Kundu, Chinmay Hazra and Ambalal Chaudhari
ix
x Contents
Bioremediation of 2,4,6-Trinitrotoluene Explosive Residues. . . . . . . . . 201

Sikandar I. Mulla, Manjunatha P. Talwar and Harichandra Z. Ninnekar
Phytoremediation of Soil Contaminated with Explosive

Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Katarzyna Panz and Korneliusz Miksch
Stable Isotope Tools for Tracking In Situ Degradation Processes

of Military Energetic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Anat Bernstein, Faina Gelman and Zeev Ronen
Biodegradation of Hexanitrohexaazaisowurtzitane (CL-20) . . . . . . . . . 285

Julius Pavlov and Mohammed Sidhoum
Pathways of 2,4,6-Trinitrotoluene Transformation

by Aerobic Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Ayrat M. Ziganshin and Robin Gerlach
In Situ Degradation and Remediation of Energetics TNT, RDX,

HMX, and CL-20 and a Byproduct NDMA in the Sub-Surface
Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Jim E. Szecsody, Steve Comfort, Herb L. Fredrickson, Robert E. Riley,
Fiona Crocker, Patrick Shea, Jim P. McKinley, Amy P. Gamerdinger,
Hardiljeet K. Boparai, Don C. Girvin, Jessa V. Moser, Karen Thompson,
Tom Resch, Brooks J. DeVary, Lisa Durkin and Andrew T. Breshears
Phytoremediation of TNT and RDX. . . . . . . . . . . . . . . . . . . . . . . . . . 371

Shree Nath Singh and Shweta Mishra
About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

Contributors
Anat Bernstein Volcani Center, Institute for Soil, Water and Environmental
Sciences, Agricultural Research Organization, 50250 Bet Dagan, Israel
Divya Bhatia Department of Biotechnology, University Institute of Engineering
and Technology, Kurukshetra University, Kurukshetra, Haryana, India
Hardiljeet K. Boparai School of Natural Resources, University of Nebraska,
Lincoln, NE 68583, USA
Andrew T. Breshears Pacific Northwest National Laboratory, Richland, WA
99352, USA
Ambalal Chaudhari School of Life Sciences, North Maharashtra University,
P.B. No. 80, Jalgaon 425001, India, e-mail: ambchasls@yahoo.com
Harald Claus Institute of Microbiology and Wine Research, Johannes Guten-
berg-University Mainz, Becherweg 15, 55099 Mainz, Germany, e-mail: hclaus
@uni-mainz.de
Steve Comfort School of Natural Resources, University of Nebraska, Lincoln,
NE 68583, USA
Fiona Crocker Environmental Laboratory at Waterways Experiment Station,
U.S. Army Engineer Research and Development Center, Vicksburg, MS 39180-
6199, USA
Premlata Sukhdev Dautpure Microbial Sciences Division, MACS-Agharkar
Research Institute, G.G. Agarkar Road, Pune 411004, India
Brooks J. DeVary Pacific Northwest National Laboratory, Richland, WA 99352,
USA
Lisa Durkin Pacific Northwest National Laboratory, Richland, WA 99352, USA
Herb L. Fredrickson Environmental Laboratory at Waterways Experiment
Station, U.S. Army Engineer Research and Development Center, Vicksburg, MS
39180-6199, USA
xi
xii Contributors
Amy P. Gamerdinger Pacific Northwest National Laboratory, Richland, WA

99352, USA
Faina Gelman Geological Survey of Israel, 30 Malkhey Israel St., 95501 Jeru-
salem, Israel
Robin Gerlach Center for Biofilm Engineering and Department of Chemical and
Biological Engineering, Montana State University, Bozeman, MT 59717, USA
Atreyi Ghosh Centre for the Environment, Indian Institute of Technology
Guwahati, Guwahati 781039, Assam, India
Pranab Kumar Ghosh Department of Civil Engineering, Indian Institute of
Technology Guwahati, Guwahati 781039, Assam, India
Don C. Girvin Pacific Northwest National Laboratory, Richland, WA 99352,
USA
Anita Grewal Department of Biotechnology, University Institute of Engineering
and Technology, Kurukshetra University, Kurukshetra, Haryana, India
Chinmay Hazra School of Life Sciences, North Maharashtra University, P.B.
No. 80, Jalgaon 425001, India
Yoichi Kamagata Bioproduction Research Institute, National Institute of
Advanced Industrial Science and Technology, Ibaraki, Tsukuba 305-8566, Japan
Pradnya Pralhad Kanekar Microbial Sciences Division, MACS-Agharkar
Research Institute, G.G. Agarkar Road, Pune 411004, India, e-mail: kanekarpp@
gmail.com
Sagar Pralhad Kanekar Microbial Sciences Division, MACS-Agharkar
Jagdeep Kaur Department of Biotechnology, Panjab University, Chandigarh
160014, India
Nobutada Kimura Bioproduction Research Institute, National Institute of
Advanced Industrial Science and Technology, Ibaraki, Tsukuba 305-8566, Japan,
e-mail: n-kimura@aist.go.jp
Wataru Kitagawa Bioproduction Research Institute, National Institute of
Advanced Industrial Science and Technology, Ibaraki, Tsukuba 305-8566, Japan
Debasree Kundu School of Life Sciences, North Maharashtra University, P.B.
No. 80, Jalgaon 425001, India
Deepak Kumar Malik Department of Biotechnology, University Institute of
Engineering and Technology, Kurukshetra University, Kurukshetra, Haryana,
India, e-mail: deepmolbio@rediffmail.com
Contributors xiii
Jim P. McKinley Pacific Northwest National Laboratory, Richland, WA 99352,

USA
Korneliusz Miksch Environmental Biotechnology Department, Silesian Uni-
versity of Technology, Akademicka 2A St., Gliwice, Poland
Shweta Mishra CSIR-National Botanical Research Institute, Rana Pratap Marg,
Lucknow 226001, India
Jessa V. Moser Pacific Northwest National Laboratory, Richland, WA 99352,
USA
Sikandar I. Mulla Department of Biochemistry, University of Karnataka,
Dharwad 580003, Karnataka, India
O. Muter Institute of Microbiology and Biotechnology, University of Latvia,
Kronvalda bulv. 4, Riga LV-1010, Latvia, e-mail: olga.muter@inbox.lv
Harichandra Z. Ninnekar Department of Biochemistry, University of Karna-
taka, Dharwad 580003, Karnataka, India, e-mail: hzninnekar@yahoo.com
Kannan Pakshirajan Department of Biotechnology, Indian Institute of Tech-
nology Guwahati, Guwahati 781039, Assam, India, e-mail: pakshi@iitg.ernet.in
Katarzyna Panz Environmental Biotechnology Department, Silesian University
of Technology, Akademicka 2A St., Gliwice, Poland, e-mail: katarzyna.panz@
polsl.pl
Vrushali Prashant Patil Microbial Sciences Division, MACS-Agharkar
Julius Pavlov Center for Environmental Systems, Stevens Institute of Technol-
ogy, Hoboken, NJ, USA
John Pichtel Ball State University, Muncie, IN 47306, USA, e-mail:
jpichtel@bsu.edu
Meenu Rathi Department of Botany, University College, Kurukshetra Univer-
sity, Kurukshetra, Haryana, India
Tom Resch Pacific Northwest National Laboratory, Richland, WA 99352, USA
Robert E. Riley Pacific Northwest National Laboratory, Richland, WA 99352,
USA
Zeev Ronen Department of Environmental Hydrology and Microbiology, Zuc-
kerberg Institute for Water Research, Ben-Gurion University of the Negev, 84990
Sede Boqer Campus, Israel, e-mail: zeevrone@bgu.ac.il
Seema Shreepad Sarnaik Microbial Sciences Division, MACS-Agharkar
xiv Contributors
Patrick Shea School of Natural Resources, University of Nebraska, Lincoln, NE

68583, USA
Mohammed Sidhoum Sustainability Division, Birdsall Services Group, Eaton-
town, NJ, USA, e-mail: msidhoum@birdsall.com
Baljinder Singh Punjab Pollution Control Board, Patiala 147001, Punjab, India
Kashmir Singh Department of Biotechnology, Panjab University, Chandigarh
160014, India, e-mail: kashmirbio@pu.ac.in
S. N. Singh CSIR-National Botanical Research Institute, Rana Pratap Marg,
Lucknow 226001, India, e-mail: drsn06@gmail.com
Jim E. Szecsody Pacific Northwest National Laboratory, Richland, WA 99352,
USA, e-mail: jim.szecsody@pnnl.gov
Manjunatha P. Talwar Department of Biochemistry, University of Karnataka,
Dharwad, Karnataka 580003, India
Karen Thompson Environmental Laboratory at Waterways Experiment Station,
U.S. Army Engineer Research and Development Center, Vicksburg, MS 39180-
6199, USA
Ayrat M. Ziganshin Department of Microbiology, Kazan (Volga Region) Fed-
eral University, ul. Kremlyovskaya 18, Kazan, Tatarstan, Russia 420008, e-mail:
a.ziganshin06@fulbrightmail.org
Biodegradation of Nitrophenol
Compounds
Nobutada Kimura, Wataru Kitagawa and Yoichi Kamagata
1 Introduction
Nitrophenol compounds have been used in a number of ways in medicines,

explosives, and pesticides (Munnecke 1976). Due to the intense yellow color of the
phenolate anion and pH reactivity [p-nitrophenol (PNP) is colorless at pH5.6 and
the corresponding phenolate anion has a maximum yellow color at pH 7.6],
nitrophenols are often used directly in titrations as indicators. Nitrophenols are
also used in monitoring the enzyme activity, such as ß-galactosidase activity
through detection of nitrophenol moiety and concomitant formation of the yellow
color. Picric acid, also called 2,4,6-trinitrophenol, has been extensively used as a
military explosive. In addition picric acid has been also used as a yellow dye, as an
antiseptic and in the synthesis of chloropicrin, or nitrotrichloromethane, CCl3NO2,
a powerful insecticide. A wide use of nitrophenol compounds and their subsequent
release leads to environmental pollution. The US Environmental Protection
Agency (USEPA) adds several mononitrophenols, dinitrophenols, and 2,4,6-trin-
itrophenol (picric acid) to its Emergency Planning and Community Right-to-Know
Act (EPCRA) list of hazardous and toxic chemicals (EPA 1995). Due to this
potential toxicity and persistence in the environment, to know the fate of the
compounds and to establish the technologies for rapid remediation detoxification
of these compounds are necessary. This chapter has focused on the microbial
degradation of three isomers of nitrophenol.
N. Kimura (&) W. Kitagawa Y. Kamagata

Bioproduction Research Institute, National Institute of Advanced Industrial Science
and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
e-mail: n-kimura@aist.go.jp
S. N. Singh (ed.), Biological Remediation of Explosive Residues, 1

Environmental Science and Engineering, DOI: 10.1007/978-3-319-01083-0_1,
2 N. Kimura et al.
2 Biodegradation of Nitroaromatic Compounds

by Microorganisms
Bacteria have evolved a variety of aerobic strategies for the removal of the nitro-
group during conversion of the nitroaromatic compouds to central metabolites
(Fig. 1).
Microbial degradation of nitrophenol compounds by microbial enzymes has
been reported by several workers (Sudhakar et al. 1978; Hess et al. 1990; Dickel
and Knackmuss 1991; Spain and Gibson 1991; Ecker et al. 1992; Groenewegen
et al. 1992; Lenke et al. 1992; Nishino and Spain 1993; Rhys-Williams et al. 1993;
Haigler et al. 1994; Jain et al. 1994; Nadeau and Spain 1995; Nishino and Spain
1995; Meulenberg et al. 1996; Rajan et al. 1996; Schafer et al. 1996, 1997, 1999;
Michan et al. 1997; Kadiyala and Spain 1998; Spiess et al. 1998; Behrend and
Heesche-Wagner 1999; Ebert et al. 1999; Katsivela et al. 1999; Rieger et al. 1999;
Zhao and Ward 1999; Bhushan et al. 2000; Kimura et al. 2000; Shinozaki et al.
2002; Kuda et al. 2011; Kristanti et al. 2012).
Different enzymes involved in biodegradation of various nitrophenol com-
pounds have been listed as below:
1. Monooxygenase: 2-Nitrophenol,4-Nitrophenol, 4-Nitroanisole
2. Dioxygenase: Nitrobenzene, 2-Nitrotoluene,3-Nitrobenzoate, 1,3-Dinitroben-
zene, 2,6-Dinitrophenol
3. Reductase: 2,4-Dinitrophenol, 2,4,6-Trinitrophenol
4. Mutase: Nitrobenzene,3-Nitrophenol,2-Chloro-5-nitrophenol, 4-Chloronitroben-
zene
5. Hydroxylaminolyase: 4-Nitrotoluene, 4-Nitrobenzoate, 3-Nitrophenol
The most widely studied approach involves the initial oxidative removal of the
nitro group as nitrite in a reaction catalyzed by a monooxygenase enzyme.
Some bacteria eliminate a nitro group following initial dioxygenation to a
dihydroxy intermediate. Aerobic bacteria attack 2,4-dinitrophenol (24DNP) and
Fig. 1 Nitroaromatic
compounds
Biodegradation of Nitrophenol Compounds 3
2,4,6-trinitrophenol through formation of a hydride-Meisenheimer complex before

elimination of the first nitro group as nitrite. A complete reduction of the nitro
group to the amine does not appear to be a mechanism that is widely used by
aerobic bacteria for productive metabolism. In one mechanism, the hydroxylamino
compound is attacked by an enzyme hydroxylaminolyase, resulting in the
production of the corresponding catechol and elimination of ammonia.
Bacteria, which were able to use nitrophenol compounds as a sole carbon or
nitrogen source were isolated by enrichment technique from environmental sam-
ples. Although a few studies on the microbial ecology and molecular evolution
were performed, they are ubiquitous at very low numbers in the contaminated soil
(Hanne et al. 1993; Kimura et al. 2000).
2.1 Nitrophenol
2.1.1 p-Nitrophenol
PNP is very important compound as a basic material for medicines, dyes, and
explosives. This compound is used on a large scale in the synthesis of the aspirin
substitute acetaminophen and in the manufacture of pesticides, such as parathion
and methylparathion (Spain and Gibson 1991; Zylstra et al. 2000). In the envi-
ronment, such pesticides are hydrolyzed and transformed to 4-NP (Munnecke and
Hsieh 1974, 1976; Sharmila et al. 1989). The toxicology of 4-NP has been studied
and reviewed by the Agency for Toxic Substances and Disease Registry (1992).
PNP irritates the eyes, skin, and respiratory tract leading to inflammation of these
parts. PNP has a delayed interaction with blood and forms methaemoglobin, which
is responsible for methemoglobinemia, potentially causing cyanosis, confusion,
and unconsciousness.
Microbial degradation of PNP has been described by several bacteria including
Arthrobacter, Bacillus, Flavobacterium, Moraxella, and Pseudomonas (Raymond
and Alexander 1971; Nelson 1982; Heitkamp et al. 1990; Spain and Gibson 1991).
At an early stage of the research, Simpson and Evans (1953) reported that Pseu-
domonas bacteria converted PNP into hydroquinone in association with the pro-
duction of NO2- (Simpson and Evans 1953). Until now, several 4-NP-degrading
bacteria have been isolated, and their degradation pathways have been also
studied. As shown in Fig. 2, the two major initial degradation pathways of 4-NP
have been characterized. The degradation pathway, in which 4-NP is converted to
maleylacetate via hydroquinone (hydroquinone pathway) (Fig. 2, top), was pref-
erentially found in gram-negative bacteria, such as Burkholderia spp. and
Moraxella spp. (Spain and Gibson 1991; Prakash et al. 1996). The degradation
pathway, in which 4-NP is converted via 4-nitrocathechol (4-NCA) and hydrox-
yquinol (hydroxyquinol pathway) (Fig. 2, bottom), was preferentially found in
gram-positive bacteria, such as Bacillus spp. and Arthrobacter spp. (Jain et al.
1994; Kadiyala and Spain 1998). Besides, anaerobic degradation of PNP has been
4 N. Kimura et al.
Fig. 2 Proposed pathway for PNP degradation by bacteria
reported. PNP was reductively converted into p-Aminophenol by Desulfovibrio

gigas and Clostridium pasteurianum (Gorontzy et al. 1993). Complete minerali-
zation of p-nitrophenol to CH4 and CO2 was observed after extended incubation
period in the anaerobic sewage sludge diluted to 10 % in a mineral salts medium
(Boyd et al. 1983).
Several studies of genetic information related to 4-NP degradation have been
carried out. In association with the hydroquinone pathway, 4-NP degradation genes
were cloned from Pseudomonas sp. strain ENV2030 and Pseudomonas putida JS444
(Zylstra et al. 2000). Similarly, in the case of hydroxyquinol pathway, 4-NP cata-
bolic genes were cloned from Rhodococcus opacus strain SAO101, Arthrobacter sp.
strain JS443, and Rhodococcus sp. strain PN1 (Kitagawa et al. 2004; Perry and
Zylstra 2007; Takeo et al. 2008; Yamamoto et al. 2011). A gram-positive 4-NP
degrader,R. opacus strain SAO101, was isolated from a sub-tropical island in Japan,
which was able to utilize PNP as a sole carbon source (Kimura and Urushigawa
2001). As shown in Fig. 3, the degradation pathway of strain SAO101 followed
(hydroxyquinol pathway) in which 4-NP is converted via 4-nitrocathechol (4-NCA)
and hydroxyquinol. To obtain genetic information, a novel 4-NP degradation gene
cluster from a gram-positive bacterium, Rhodococcus opacus SAO101, was iden-
tified and characterized (Kitagawa et al. 2004). The deduced amino acid sequences
of npcB, npcA, and npcC showed similarity with phenol 2-hydroxylase component B
(reductase, PheA2) of Geobacillus thermoglucosidasius A7 (32 %), 2,4,6-trichlo-
rophenol monooxygenase (TcpA) of Ralstonia eutropha JMP134 (44 %), and
hydroxyquinol 1,2-dioxygenase (ORF2) of Arthrobacter sp. strain BA-5-17 (76 %).
Fig. 3 Metabolic pathway for degradation of p-Nitrophenol by Rhodococcus opacus strain

SAO101
This combination of degradative genes mixture converted 4-NP to hydroxyquinol

and 4-nitrocatechol (4-NCA) to hydroxyquinol. Furthermore, the crude cell extract
of E. coli containing pETnpcC converted hydroxyquinol to maleylacetate. These
results suggest that npcB and npcA encode the two-component 4-NP/4-NCA
monooxygenase while npcC encodes hydroxyquinol 1,2-dioxygenase.
Many aromatic degradation genes are known to be encoded on plasmid DNAs
(Worsey et al. 1978; Don and Pemberton 1981; van der Meer et al. 1991; Prakash
et al. 1996; Romine et al. 1999; Chauhan et al. 2000; Vedler et al. 2000). In
particular, several Rhodococcus strains harbor aromatic degradation genes on large
linear plasmids (Dabrock et al. 1994; Kosono et al. 1997; Shimizu et al. 2001).
Three strains of 4-NP degrading bacteria, i.e., Arthrobacter protophomiae RJK100
(Chauhan et al. 2000), B. cepacia RJK200 (Prakash et al. 1996), and Arthrobacter
aurescens TW17 (Hanne et al. 1993), have been reported to harbor plasmids
involved in 4-NP degradation. The pulsed-field gel electrophoresis (PFGE) anal-
ysis revealed that strain SAO101 had three large linear plasmids, designated
pWK301 (1,100 kb), pWK302 (1,000 kb), and pWK303 (700 kb) (Kitagawa et al.
2004; Kimura et al. 2006). By Southern hybridization analysis, a unique positive
hybridization signal was observed at the position of origin of electrophoresis (data
not shown). This result indicated that the npc genes were encoded on chromosomal
DNA.
6 N. Kimura et al.
The authors also isolated PNP-degrading bacteria by aerobic batch enrichment

from a laboratory-scale PNP-degradation reactor using a mineral salt medium
containing a low and high PNP concentration (Shinozaki et al. 2002). They iso-
lated two bacteria species, Pseudomonas sp. YTK17 and Rhodococcus opacus
YTK32, that utilize PNP as their sole carbon source of carbon and energy. These
strains exhibited differences in PNP degradation activity in relation to PNP con-
centration. Strain YTK17 showed a high level of degradation following pre-
exposure to a low PNP concentration, whereas strain YTK32 required a relatively
high PNP concentration for degradation to occur. These results indicated that
phylogenetically and physiologically different types of PNP-degradation bacteria
co-existed in a reactor.
2.1.2 2,4-Dinitrophenol
2,4-Dinitrophenol (2,4-DNP) is well known as an ‘‘uncoupler’’ compound

(Hanstein 1976). 2,4-DNP can cross membranes in its protonated form, acting as a
H+ carrier and dissipate the electrochemical gradient across cell membranes, thus
uncoupling the oxidative phosphorylation pathway without blocking oxygen
consumption (Alberts et al. 1989). The ability of 2,4-DNP to function as a
respiratory uncoupler causes toxicity to microorganisms (Bruhn et al. 1987), and
recommends restricting its concentration in the natural waters (Keith and Telliard
1979).
2,4-DNP-degrading strains, which have ability to utilize 2,4-DNP as a sole
carbon source, mainly belong to high GC gram-positive bacteria with one
exception, Janthino bacterium which used 2,4-DNP as a nitrogen source (Lenke
et al. 1992; Rhys-Williams et al. 1993; Blasco et al. 1999). Based on the sys-
tematic characterization of microorganisms that degrade 2,4-DNP, the authors
proposed that the isolated 2,4-DNP-degrading strains could be classified as two
different kinds of bacteria, Rhodococcus, Nocardia, and Nocardioides strains
based on phylogenetic and phenotypic properties (Kimura et al. 2000).
Generally, microbial degradation in reactors has been used as a tool for the
treatment of 2,4-DNP contaminated wastewater (Hess et al. 1990; Xing et al. 1995,
1999; Gisi et al. 1997). However, it takes a long time for microorganisms to
degrade 2,4-DNP in reactors due to the toxicity of the compound (Takahara 1980).
Knowledge about the microbial community in the 2,4-DNP digesting reactor is
useful for establishing the operational conditions needed to degrade this compound
efficiently. The microbial community of a 2,4-dinitrophenol digesting reactor was
investigated using molecular biological techniques based on 16S rDNA gene
sequences. A PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the
bacterial community in the reactor showed that species of Rhodococcus, Nocar-
dioides, and Nitrospira species were dominant in the reactor. The isolation and
phylogenetic analysis of 2,4-DNP-degrading bacteria from the reactor revealed
that isolated bacterial strains were found of two types having different 16S rDNA
sequences. One type of these strains was identified as relative of Rhodococcus
Hydride-Meisenheimer
complex
-
(H -Picric Acid)
Fig. 4 Proposed degradation pathway for picric acid and 2,4-Dinitrophenol by Rhodococcus
strains
koreensis, while an other type was a relative of Nocardioides simplex FJ21-A

(Kimura et al. 2003).
Although degradation of mononitroaromatic compounds and some dinitroaro-
matic compounds, such as 2,6-DNP, is initiated by oxygenation by a monooxy-
genase or dioxygenase (Zeyer JaK 1984; Spain and Gibson 1991; Ecker et al.
1992; Jain et al. 1994; Meulenberg et al. 1996; Schenzle et al. 1997), the analysis
of metabolization of trinitroaromatic compounds revealed that trinitroaromatic
compounds, such as 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenol (picric
acid), are reduced at an initial step of bacterial degradation, which is readily
susceptible to an initiation of reductive rather than oxidative attack in the presence
of electron withdrawing nitro groups as substituents (Fig. 4) (Lenke et al. 1992;
Vorbeck et al. 1998; Behrend and Heesche-Wagner 1999; Rieger et al. 1999). In
2,4-DNP degradation strains, the reductive pathway which converted 2,4-DNP
with concomitant liberation of stoichiometric amounts of nitrate and 4,6-dinitro-
hexanoatewas as found in Rhodococcus erythropolis HL24-1 and HL24-2 (Lenke
et al. 1992).
2.1.3 2,4,6-Trinitrophenol (Picric Acid)
2,4,6-Trinitrophenol (TNP) is used to be a main source of the military explosives.

Picramic acid (2-Amino-4, 6-dinitrophenol), which is produced by the reduction of
8 N. Kimura et al.
picric acid, is used in the manufacturing of azo dyes. Historical use of this
explosive by military and industries results soil and groundwater contamination.
The Department of Defense (DoD) estimates that more than thousand of sites are
contaminated with explosives and TNP (Walsh 1993; Thorne 1995). The EPA’s
Toxic Release Inventory (TRI) indicates that approximately 7.4 million pounds of
TNP was released to the environment in the United States from 1988 to 2002
during industrial activities (TRI 2002).
TNP-degrading strains, which have the ability to utilize TNP as a sole carbon
source, generally belong to high GC gram-positive bacteria (Lenke and Knackmuss
1992; Behrend and Heesche-Wagner 1999; Ebert et al. 1999; Walters et al. 2001).
The initial degradation step of TNP is very unique. F420H2-dependent reductive
attack on the aromatic ring creates H--Picric acid (the Hydride-Meisenheimer
complex) of picric acid which is further converted into 2,4-DNP through the release
of nitro group catalyzed by the F420-dependent reductase (NpdI) enzyme (Fig. 4).
Hydride-Meisenheimer complex of 2,4-dinitrophenol (H--2,4-DNP) was identified
as an intermediate of picric acid degradation by Nocardioides sp. strain CB 22-2
(Alberts et al. 1989; Ebert et al. 2001). This is feasible by assuming that nitrite is
released either from H--2,4-DNP or a tautomer of the protonated H--2,4-DNP.
Following nitrite release and hydrolytic ring cleavage, two metabolites were pro-
posed 4,6-dinitrohexanoate (Lenke and Knackmuss 1996) and 3-nitroadipate
Hydride-Meisenheimer complex
(H- -TNT)
Fig. 5 Proposed degradation pathway for Trinitrotoluene (TNT) by Rhodococcus erythropolis

strain HL PM-1 and Pseudomonas sp. strainJLR21. HADNT Hydroxylamino dinitrotoluene,
ADNT Amino dinitrotoluene, DANT Diamino dinitrotoluene
(Blasco et al. 1999). Several of the npd genes of R. (opacus) erythropolis HL PM-1,
showing sequence similarities to enzymes responsible for the b-oxidation of fatty
acids (Russ et al. 2000), might be involved in the oxidation of 4,6-dinitrohexanoate.
TNP degradation capacity of R. (opacus) erythropolis HL PM-1 is inducible by
2,4-DNP (Russ et al. 2000; Walters et al. 2001) but not by picric acid. TNP-
degrading strain R. erythropolis D3213 was shown to have a TNP degradation
pathway. This strain D3213 degrade TNP and 2,4-DNP constitutively. These results
suggested that strain HL PM-1 and D3213 had different abilities to degrade TNP.
Strain HL PM-1 possessed reductive enzyme systems, which catalyze ring
hydrogenation, i.e., the addition of a hydride ion to the aromatic ring of
2,4,6-Trinitrotoluene (TNT) (Fig. 5). The hydride-Meisenheimer complex thus
formed (H--TNT) was further converted to a yellow metabolite, which by elec-
trospray mass and nuclear magnetic resonance spectral analyses, was established
as the protonated dihydride- Meisenheimer complex of TNT (2H--TNT) (Vorbeck
et al. 1998).
3 Conclusion
In this chapter, current knowledge on the microbial degradation of nitrophenols

was reviewed focusing on the genetic and biochemical information of nitrophenol
degrading bacteria. The knowledge of microbial degradation of nitrophenols may
serve as a basis for the use of bioremediation systems for the removal of nitro-
phenol. The future direction of research in this area of nitropehnol degradation is
to make the appropriate bioremediation strategy for a nitrophenol-contaminated
site. To make strategy for bioremediation of nitrophenols, the fate of the targeted
pollutant in the environment must be well understood. Second, information on the
toxicity of intermediates produced during the microbial degradation of nitrophe-
nols is crucial for selecting the appropriate degradation pathway to be stimulated at
the remediation site. To understand the potential fate of intermediates of nitro-
phenol degradation, the complete pathway for nitrophenol degradation needs to be
elucidated and the metabolites of nitrophenol degradation have to be identified.
Third, information on the biodegradability of nitrophenols by natural microbes in
the environments must be obtained. In the literature, knowledge on the natural
consorcia for microbial degradation of nitrophenols in the environment has been
not presented.
In addition, only a few reports of gene sequence are known from selected
catabolic pathways and microorganisms. The sequence information in the database
needs to be increased to include genes for all of the possible pathways for nitro-
phenol degradation, by different microorganisms. The information of the different
gene sequences for nitrophenol degradation is useful for understanding the evo-
lution of pathways to solve question how two different pathways were evolved in
PNP-degrading strains. Furthermore, it is an important question to be answered
whether bacteria, that degrade nitropheols, have evolved mechanisms for
10 N. Kimura et al.
protection against these compounds. PNP and TNP are known to be toxic to
microorganisms. Knowledge of the protection mechanism for nitrophenols can be
used for the remediation of the environment so that the microorganisms can
degrade the nitrophenol compounds efficiently and effectively.
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pp 145–160
Microbial Degradation of 2,4,6-
Trinitrotoluene In Vitro and in Natural
Environments
Harald Claus
1 Introduction
2,4,6-Trinitrotoluene (TNT) is a nitroaromatic explosive that was released into soil

and water ecosystems mainly due to its massive use during the two world wars. As
a result, many sites used for TNT production have become seriously contaminated
with the explosive and related compounds (Fuller et al. 2004; Lewis et al. 2004).
Typical contaminated sites may contain up to 10 g/kg TNT in soils and up to
100 mg/l in water. TNT and its metabolites exhibit a high toxic and mutagenic
potential on both prokaryotes and eukaryotes (Spanggord et al. 1995; Honeycutt
et al. 1996; Lachance et al. 1999; Maeda et al. 2006). Consequently, there is an
urgent need to clean up contaminated sites to ensure environmental quality and
safety. It has been estimated that nearly 3,200 sites in Germany require environ-
mental restoration (Preuß and Eitelberg 1999). Various physical/chemical proce-
dures for remediation of TNT contaminated soils have been established, but all are
very cost-intensive. Carbon adsorption has often been used for removal of nitro-
aromatics from contaminated ground- and surface-waters (Wujcik et al. 1992).
Unfortunately, the matrix is expensive and the spent carbon still constitutes a
problematic waste (Schmidt et al. 1998).
Biological based remediation is an economical and ecological compatible
approach to detoxify areas contaminated with xenobiotics (Alvarez and Illman
2005; Crawford et al. 2005; Singh and Tripathi 2006). However, TNT is resistant
to oxidative microbial degradation and only low mineralization rates have been
sporadically reported with bacterial consortia and several white-rot fungi. The
reason is the presence of three electron withdrawing nitro-groups in TNT which
introduce steric constraints and confer a high electron deficiency to the aromatic
ring (Heiss and Knackmus 2002). Instead of oxidation, many bacteria catalyse the
H. Claus (&)
Institute of Microbiology and Wine Research, Johannes Gutenberg-University Mainz,
Becherweg 15 55099 Mainz, Germany
e-mail: hclaus@uni-mainz.de

16 H. Claus
reduction of one or two nitro-groups of TNT to monoaminodinitrotoluenes

(ADNT) and diaminonitrotoluenes (DANT). Another pathway is mediated by
addition of one or two hydride ions to the aromatic ring, resulting in the formation
of Meisenheimer-complexes (adducts of aromatic nitro-compounds with a nucle-
ophile) often accompanied by release of nitrite. The electron transfer is catalyzed
by different types of cytoplasmatic nitroreductases (Pak et al. 2000; Kim and Song
2005). Reactive nitroso- and hydroxylamino-intermediates can further react to
condensated azoxy-dimers and acetyl-derivates of TNT. Under strictly anaerobic
conditions, ADNT is further reduced to 2,4,6-triaminotoluene (TAT) which is
highly reactive and can polymerize or irreversibly bind to the organic soil matrix
(Thiele et al. 2002).
The reductive reactions are the basis of several treatment processes for the
bioremediation of TNT-contaminated soils (Breitung 1996; Lenke et al. 1998,
2000; Fuller et al. 2004; Lewis et al. 2004). However, there is a lack of biological
strategies to clean up contaminated water ecosystems. Some promising microbi-
ological approaches for detoxification of aquatic environments are addressed in
this chapter.
2 2,4,6-Trinitrotoluene
2.1 Toxicity
Due to its high blasting power and relative security of handling, TNT is still one of
the most used military explosives. It has been estimated that around 1.000,000 kg
TNT is produced per year (Singh et al. 2012). Five hundred thousand US gallons
of water, contaminated with TNT and other nitroaromatic compounds, may be
released into the environment by one TNT-manufacturing plant in one day. In
USA, 15 million acres at over 2000 sites are suspected or known to be contami-
nated with military munitions (Montgomery et al. 2011). At some munitions
manufacturing and processing sites, the contamination can be as high as 200 g
TNT per 1 kg of soil (Symons and Bruce 2006).
TNT forms yellow crystals and has a water solubility of 130 mg/l. At con-
taminated sites, it exists as a fine dust, flakes or crystallized chunks. Its hetero-
geneous distribution in soil restricts mobility, microbial degradation and also its
analysis.
In several studies, it has been demonstrated that TNT and most of its degra-
dation products are toxic to fish (Osmon and Klausmeier 1972), rats and mice
(Ashby et al. 1985) as well as to algae and aquatic plants (Sunahara et al. 1999).
For microorganisms, such as yeasts, actinomycetes and Gram-positive bacteria,
TNT is toxic at concentrations of ca. 50 mg/l (Klausmeier et al. 1973). Also
precursors and metabolites of TNT are classified as very toxic, carcinogenic and
mutagenic (Schneider et al. 2000; Haarck et al. 2001). Ribeiro et al. (2012)
Microbial Degradation of 2,4,6-Trinitrotoluene 17
reported that the toxic potential of effluents generated by TNT production (yellow
and red waters), produced from a plant located in Brazil was extremely high to all
test organisms (Daphnia similis, Danio rerio, Escherichia coli, Pseudomonas
putida and Pseudokircheneriella subcaptata).
Numerous symptoms of poisoning in humans following inhalation or dermal
absorption of mononitrotoluene, dinitrotoluene, and TNT are observed a few days
after exposure: headache, loss of appetite, dizziness, nausea, insomnia, numbness
of various parts of the skin and diarrhea. Strong changes in the hemogram are the
result of exposure. A particularly striking symptom is cyanosis, a bluish-red dis-
coloration of lips, fingernails and skin due to oxygen deficiency. That is caused by
reduced metabolites of TNT which are blamed for increased methemoglobin
formation and hemolysis. The metabolites of TNT cause liver damaging effects
(Koss et al. 1989).
2.2 Microbial Degradation of TNT
The degradation of TNT by microorganisms has been extensively studied for many
years and the results have been compiled in numerous reviews (Fritsche et al.
2000; Hawari et al. 2000; Lenke et al. 2000; Spain et al. 2000; Esteve-Núñez et al.
2001; Rodgers and Bunce 2001; Rosser et al. 2001; van Aken and Agathos 2001;
Heiss and Knackmus 2002; Fuller et al. 2004; Lewis et al. 2004; Schrader and
Hess 2004; Zhao et al. 2004; Stenuit and Agathos 2010; Rylott et al. 2011). Some
basic reactions are listed in Table 1.
2.2.1 TNT Degradation by Bacteria
There is one major problem with microbial TNT degradation: the three symmet-
rically arranged nitro-groups induce a high electron deficiency at the aromatic ring.
An oxidative degradation and the use of TNT as a source of carbon and energy is
extremely unlikely. Thus, the term degradation in this context means transfor-
mation or destruction of TNT, but not mineralization, i.e., use as the sole growth
substrate for a microorganism.
The initial metabolites in the biotransformation of TNT are hydroxylamino-
dinitrotoluenes (HADNTs) aminodinitrotoluenes (ADNTs), diaminomononitro-
toluenes (DANTs) and tetranitroazoxytoluenes (AZTs) (Hawari et al. 2000; Oh
et al. 2000). Because of the electron deficiency of the ring p system, the initial
degradation of TNT by microorganisms is characterized by reductive reactions
(Vorbeck et al. 1998). The nitro-moieties of TNT (-NO2) can be successively
reduced to nitroso (-NO), hydroxylamino (-NHOH) and finally amino (-NH2)
groups (Fig. 1). By strictly anaerobic bacteria, such as Clostridium sp., Desulf-
ovibrio sp. and archaebacteria as Methanococcus sp., TNT is completely reduced
18 H. Claus
Table 1 Degradation of TNT by microorganisms

Mechanism Products*
Primary enzymatic reactions
Stepwise reduction of nitro-groups of the aromatic ring by Nitrosodinitrotoluene
nitroreductases Hydroxyldinitrotoluene
Aminodinitrotoluene
Diaminodinitrotoluene
Hydride addition to the aromatic ring by flavoproteins of the Monohydride–
old yellow enzyme family (OYE) Dihydride-
Protonated Dihydride-
Meisenheimer complex
Secondary abiotic reactions
Condensation of NODNT/ HADNT Tetranitroazoxytoluene
Secondary
Diarylamines
Condensation of HADNT/2H- TNT.H+
Covalent binding to cell compounds Protein adducts
Secondary enzymatic reactions
Oxidation of the methyl-group
Acetylation of an amino-group
Oxidation of reduced metabolites (ADNT, DANT) by fungal Polymers
exoenzymes and coupling to organic soil components Humic acids adducts
*Different isoforms are formed depending on the microorganism and enzyme specificity
CH3 CH3 CH3 CH3

O2N NO2 O2N NO2 O2N NO2 O2N NO2
NO2 NO NHOH NH2
2,4,6-TNT 4-Nitroso- 4-Hydroxylamino- 4-Aminodinitrotoluene

dinitrotoluene dinitrotoluene
Fig. 1 Microbial transformation of TNT by reduction of nitro-groups
to 2,4,6-triaminotoluene (Boopathy and Kulpa 1994; Regan and Crawford 1994;

Crawford 1995; Ederer et al. 1997).
These reduced TNT compounds present the primary products of the microbial
metabolism. However, depending upon the reaction conditions (e.g., pH), they can
be further converted by biotic and abiotic mechanisms to azo-, azoxy-, hydrazone-,
and phenol-acetyl derivatives (Hawari et al. 2000). The intermolecular conden-
sation of partially reduced derivatives leads to formation of azoxytetranitrotolu-
enes (Haidour and Ramos 1996).
The second important pathway is the direct reduction of the aromatic ring by
addition of hydride-ions with the formation of monohydride-, and dihydride-
Meisenheimer complexes (Fig. 2). As both pathways (nitro-reduction and aromatic
Fig. 2 Microbial
transformation of TNT by
hydride addition to the
aromatic ring
2,4,6-TNT Monohydride-
ring-reduction) may co-exist in the same cell, condensation reactions between

dihydride-Meisenheimer complexes and hydroxylaminodinitrotoluene can lead to
the formation of diarylamines with concomitant liberation of nitrite (Wittich et al.
2009).
TNT enters the bacterial cell most probably by passive diffusion across the cell
barriers. In contrast, multi-drug efflux pumps are induced in Pseudomonas putida
KT2440 in the presence of TNT, suggesting the importance of active extrusion
systems in maintaining low intracellular TNT concentration to overcome toxicity
(as reviewed by Stenuit and Agathos 2010). As a result of TNT degradation,
different amounts of monomeric transformation products (ADNT, DANT) have
been found extracellular (Claus et al. 2007a, b). Conclusively, active efflux sys-
tems may also exist for these compounds.
2.2.2 TNT Degradation by Fungi
The enzymatic conversions mentioned above do not imply ring opening (Hawari
et al. 2000). This is the reason why TNT is transformed by bacteria, but usually not
mineralized. However, white-rot fungi and the litter degrading fungus Phanero-
chaete chrysosporium as well as Stropharia species have been shown to mineralize
TNT, at least in part, under aerobic conditions (Bumpus and Tatarko 1994; Fritsche
1998; Esteve-Núñez et al. 2001).
In a screening program, 91 fungal strains belonging to 32 genera of different
ecological and taxonomic groups (wood and litter decaying basidiomycetes, sap-
rophytic micromycetes) were tested for their ability to metabolize and mineralize
TNT (Scheibner et al. 1997b). All these strains metabolized TNT rapidly by
forming monoaminodinitrotoluenes (ADNT). Micromycetes produced higher
amounts of ADNT than did wood and litter decaying basidiomycetes. A significant
mineralization of (C14) TNT was only observed for certain wood and litter
decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and
Stropharia rugosa-annulata DSM11372 mineralized 42 and 36 %, respectively of
the initial added (C14) TNT to (C14) CO2 within 64 days. However, micromycetes
(deuteromycetes, ascomycetes, zygomycetes) were unable to mineralize (C14)
TNT significantly.
20 H. Claus
Responsible for this degradation is the ligninolytic enzyme system of white-rot

fungi which consists of a number of extracellular enzymes, especially peroxidases
(lignin peroxidase, Mn-dependent peroxidase) and oxido-reductases (laccases)
which catalyze the degradation of the wood constituent lignin (van Aken and
Agathos 2001). The metabolism starts again with reduction of the TNT nitro-
groups yielding 4-ADNT and 2-ADNT. Thereafter, various acylation reactions to
formylated and acetylated products may occur (Hawari et al. 1999). One of these
formylated products, 2-amino-4-formamido-6-nitrotoluene has been identified as a
substrate for lignin-peroxidase. The other products are broken down under lig-
ninolytic conditions as well (Hawari et al. 1999). The manganese-dependent
peroxidase is able to mineralize 4-ADNT directly, as shown for the lignin per-
oxidase negative fungus Nematoloma frowardii (Scheibner et al. 1997a). In
addition, an intracellular, cytochrome P-450 dependent enzyme system has been
identified which is involved in the mineralization of TNT in Bjerkandera adusta
(Eilers et al. 1999).
2.2.3 TNT as a Source of Nitrogen or Electron Acceptor
The vast majority of studies demonstrate that TNT can be exclusively transformed
in a co-metabolic manner (i.e., in the presence of a reduction equivalent donating
substrate), but not mineralized by an individual bacterial strain. Nevertheless, there
are some reports of at least partial mineralization of TNT by natural bacterial
consortia (Robertson and Jjemba 2005; Montgomery et al. 2011). Recently, an
amazing new strain VT11 of Acinetobacter sp. has been described which utilizes
TNT as sole growth substrate (Solyanikova et al. 2012).
In contrast, it is also established that various microorganisms can use TNT as a
source of nitrogen (Duque et al. 1993; Boopathy and Kulpa 1994) or as external
electron acceptor (Table 2). In the case of Pseudomonas sp. JLR 11, nitrite is
released from the aromatic ring and then further reduced to ammonium. Almost
85 % of the nitrogen of TNT can be incorporated into the cells as organic nitrogen
(Esteve-Núñez and Ramos 1998; Esteve-Núñez et al. 2000). As an intermediate of
nitrogen release, Meisenheimer-complexes have been identified (French et al.
1998; Heiss and Knackmus 2002). It has been proposed that the dihydride-com-
plex slowly rearomatizes with the concomitant release of nitrite. Another mech-
anism of N release from TNT involves its partial reduction to hydroxylamino
derivates and subsequent release of ammonium from the aromatic ring, probably
through an acid-catalyzed Bamberger-like rearrangement (Stenuit and Agathos
2010).
The yeast strain Geotrichum candidum AN-Z4 isolated from a polluted site is able
to transform TNT via the formation of unstable intermediate hydride-Meisenheimer-
complexes with their subsequent destruction and accumulation of nitrite and nitrate
(Ziganshin et al. 2010). Aeration of the medium promoted more profound destruc-
tion of this xenobiotic by the strain G. candidum AN-Z4 than static conditions.
Table 2 Microbial utilization of TNT as N-source or electron acceptor

Mode of utilization Mechanisms Conditions
Nitro-groups of Pathway A
TNT as Reduction of TNT to HADNTs and 1 NAD(P) H+ required, pH \ 4.2
N-source subsequent release of ammonium
from the aromatic ring through
acid-catalyzed Bamberger-like
reaction
Pathway B
Condensation of HADNTs with 7 NAD(P) H+ required, 3 of them for
- +
[2H ]–TNT.H to form further conversion of NO2- to
secondary diarylamines with NH3 by nitrite reductase
concomitant release of nitrite
Nitro-groups of Pathway A Anaerobic/respirative
TNT as external Energy generation by utilization of
electron an electrochemical gradient
acceptors
Pathway B Anaerobic/fermentative
Reoxidation of reduced electron
carriers to maintain energy via
substrate level phosphorylation
Two possible pathways of TNT biodegradation were confirmed experimentally:

(1) via the destruction of the TNT-monohydride complex [(3-H-)-TNT] and (2) via
the destruction of one protonated TNT-dihydride complex [(3,5-2H-)-TNT.H+].
The best proven mechanism of N release from TNT involves the abiotic con-
densation of hydroxylamino-dinitroluene- (HADNT) isomers and protonated
dihydride-Meisenheimer-complexes to form secondary diarylamines with the
concomitant release of nitrite (Wittich et al. 2009). The nitrite is probably further
converted to ammonium by a nitrite reductase and finally assimilated via the
glutamine synthetase-glutamate synthase reaction (Caballero et al. 2005b). The
complete process requires seven reducing equivalents.
Some Pseudomonas strains use TNT as an alternative electron acceptor in the
respiratory chain forming ATP (Esteve-Núñez et al. 2000). Anaerobic Clostridiae
exploit TNT for reoxidation of reduced electron carriers to maintain the fermen-
tative metabolism (Stenuit and Agathos 2010).
2.2.4 Enzymes Involved in TNT Transformation
Initial TNT transformations are catalysed by nitroreductases which are related to

the old yellow enzyme (OYE) of yeast (French et al. 1998; Klausmeier et al. 2001;
Williams et al. 2004; Kim and Song 2005). They catalyse the pyridine nucleotide
dependent reduction of nitro-aromatic compounds. Nitroreductases are ubiquitous
in bacteria and higher organisms (fungi, plants, animals). They have recently
generated great interest from different points of view. Possible application areas
22 H. Claus
are not only restricted to bioremediation (Hannink et al. 2001), but also to specific
biocatalysis (Kadiyala et al. 2003) and cancer therapy (Denny 2002; Knox et al.
2003). Through their activity, they decisively determine the toxicity of nitroaro-
matic compounds (Homma-Takeda et al. 2002; Padda et al. 2003), although their
physiological relevance is still largely unknown. Nitroreductases may be classified
into two types.
Oxygen-insensitive Type I Nitroreductases
These are localized in the cytoplasm where they are constitutively expressed. They
are present as either monomeric or homodimeric flavin-mononucleotide (FMN)
containing proteins with a subunit size of approximately 25 kDa and use
NAD(P)H as an electron donor (Stenuit and Agathos 2010). They catalyze the
ubiquitous reduction of aromatic nitro-groups to amino-groups through two-
electron increments. The arising nitroso- and hydroxylamine-intermediates readily
undergo condensations reactions with themselves or other organic molecules
(proteins, humic acids) yielding polymeric products (Sarlauskas et al. 2004). Some
of these enzymes attack directly the aromatic ring by hydride-ion addition to TNT
and other nitroaromatics (Ramos et al. 2005). Type I nitroreductases have been
described in many Gram-negative bacteria, such as Escherichia coli (Whiteway
et al. 1998), Salmonella enterica (Nokhbeh et al. 2002), Enterobacter cloacae
(Haynes et al. 2002), Helicobacter pylori (Goodwin et al. 1998), Vibrio harveyi
(Lei et al. 1994), Vibrio fisherii (Riefler and Smets 2002), Rhodobacter capsulatus
(Blasco and Castillo 1993), Thermus thermophilus (Park et al. 1992), Pseudo-
monas pseudoalcaligenes (Sommerville et al. 1995), Pseudomonas putida
(Caballero et al. 2005a, b), Pseudomonas fluorescens (Pak et al. 2000), Seleno-
monas ruminatium (Anderson et al. 2002) and Klebsiella sp. (Shin and Song
2009).
Type II Hydride Transferases
They belong to the (b/a)8 barrel OYE family of flavoproteins (Stenuit and Agathos
2010). The enzymatic catalysis is characterized by a ping-pong reaction com-
prising two half-reactions. In the reductive part, the enzyme is reduced by
NAD(P)H to yield the enzyme-bound FMNH2. In the oxidative half-reaction,
FMNH2 is reoxidized by TNT in two competing pathways: (a) the ubiquitous
nitro-reduction of TNT and (b) the specific nucleophilic addition of hydride-ions to
TNT, leading to formation of mono- and dihydride-Meisenheimer-complexes.
Because of different redox-potentials, the reduction of the first nitro-group of
TNT occurs faster than that of the remaining groups. The reaction mostly starts at
the para-nitro group (Pak et al. 2000; Riefler and Smets 2002; Kim and Song 2005).
However, some bacterial strains produce mainly the ortho-derivate (2-ADNT)
which may be due to differences in substrate specificities of the degrading enzymes
(Oh et al. 2003; Maeda et al. 2006). In addition to nitro-group reduction,
nitroreductases of Enterobacter cloacae, E. coli, Pseudomonas fluorescens and
Pseudomonas putida catalyze reduction of TNT by hydride-additions to the
aromatic ring, yielding orange coloured hydride- and dihydride-Meisenheimer-
complexes under the release of nitrite (French et al. 1998; Khan et al. 2004;
Williams et al. 2004; Caballero et al. 2005b).
Besides the two major enzyme classes, an only iron hydrogenase from Clostridium
acetobutylicum has been found capable of reducing TNT to its dihydroxylamino-
derivate in a hydrogen depending manner (Symons and Bruce 2006).
Recent experiments indicated that the enzymes from Raoultella terrigena HB
prefer the nitro-group reduction pathway (Claus et al. 2007a, b) similar to its close
relative Klebsiella sp. (Kim and Song 2005). Apart from TNT, cells of R. terrigena
HB transformed dinitrotoluene and nitrobenzenes. The comparison of microbial
transformation of whole cells as opposed to a cell-free extract suggests that ni-
trophenolic compounds are substrates for the reducing enzymes, but they pre-
sumably do not pass the bacterial cell membrane and/or act as metabolic inhibitors.
In contrast, nitrobenzenes were as good substrates for whole cells and cell extracts.
Secondary transformations of TNT metabolites can be catalyzed by enzymes,
generally known as laccases (EC 1.10.3.2, para-benzenediol:dioxygen oxidore-
ductases). These are multi-copper proteins that use molecular oxygen to oxidize
various aromatic and non-aromatic compounds by a radical-catalyzed reaction
mechanism. The enzyme has been found in eukaryotes (fungi, higher plants,
insects) and more recently in many bacteria (Claus and Strong 2010). Numerous
articles have touted its diverse potential application in various biotechnological
processes. This is attributed to the enzyme’s broad-substrate spectrum, the use of
readily available oxygen as the final electron acceptor and apart from copper, no
requirement for co-factors or peroxide (Claus and Strong 2010).
TNT itself is not a substrate for these oxidative enzymes. However, after
conversion by nitroreductases, the reduced metabolites, such as aminodinitrotol-
uenes (ADNT), azoxy-compounds and diaminonitrotoluenes, can be efficiently
oxidized by laccase to polymeric products (Strong and Claus 2011). One approach
for the bioremediation of contaminated sites presents the immobilization of TNT
and its metabolites into the complex soil organic matter during composting or
during anaerobic and aerobic slurry treatment. The potential of laccases from
different white-rot fungi for immobilizing TNT degradation metabolites into the
humic matrix has been demonstrated by several research groups (Dawel et al.
1997; Thiele et al. 2002; Wang et al. 2002).
2.3 TNT Contamination in Germany
The total production of TNT, the main explosive of the last World Wars amounted
to about 800,000 tons in Germany. As a result of manufacture, accidents and
improper disassembling, TNT, precursors and by-products were heavily discharged
into soils and groundwater (Preuß 1996; Preuß and Eitelberg 1999). A study in 1996
listed 3,240 suspected locations, of which at least 750 might be contaminated with
explosives (Preuß 1996). It has been estimated that the contaminated area extends
to over 10,000 km2 (Preuß 1996). After the cessation of production and removal of
the relevant installations, most sites of the former weapon factories were converted
to residential or commercial areas (Schneider 1989). A notable example is the
24 H. Claus
terrain of a former TNT factory near Hamburg, where a nuclear power plant has
been installed. Due to high water consumption for the production process, explo-
sives plants were located in water-rich areas, i.e., important sources for drinking
water. After rainfalls, the nitroaromatics are still continuously leached out and
hence require expensive activated carbon filter systems to protect groundwater. An
ancient bomb factory from World War I (‘‘Espagit’’ at Hallschlag, Rhineland-
Palatinate) was not fully restored, but only secured in the core zone by a soil cover.
The leakage of water is collected by an underground ring pipeline and purified
through activated charcoal (Preuß and Eitelberg 1999).
2.4 Treatment of Contaminated Soils
TNT and some of its degradation products have a high persistence, toxicity and
mutagenicity (Spanggord et al. 1995; Honeycutt et al. 1996; Lachance et al. 1999).
For this reason, the fate of these compounds is of interest and remediation of
the contaminated sites is inevitable. Traditional remediation methods for
TNT-contaminated sites have been primarily conducted by physical–chemical
methods, including incineration, landfilling, thermal desorption and soil washing.
A study published in 1999 on the economics of various methods of soil remediation
suggested that biological soil remediation procedures in Germany are more favorable
for technical reasons vis-à-vis soil incineration or soil washing (Jansky and Neumann
1999). Furthermore, incineration of soil to get rid of explosives can result in the
exposure of workers to high levels of toxins (Symons and Bruce 2006).
In the past, various bioremediation technologies have been developed for soil
environments (Held et al. 1997; Daun et al. 1998; Drzyzga et al. 1999; Lenke et al.
2000; Fuller et al. 2004; Kröger et al. 2004; Lewis et al. 2004). Biological ex situ
methods rely upon the microbial community to treat contaminated media which
include soil slurry reactors, land farming and soil composting. Soil slurry is created
by transferring contaminated soil to a reactor, where aerated mixed with nutrients
the xenobiotics are degraded by indigenous microflora. Land farming involves
mixing of the contaminated soil with the surface layer of an uncontaminated soil
(0–30 cm depth) where added nutrients (fertilizers) and moisture synergistically
maximize indigenous microbial activity on nitroaromatic degradation. Soil com-
posting (static piles or windrows) is similar to land farming, but includes addition
of organic amendments, such as biosolids or green/animal manures and subsequent
mixing of contaminated soil with the amendments (Makris et al. 2010).
As a serious drawback, ex situ treatment may not be economically feasible for a
large-scale remediation of TNT-contaminated sites, as found in Europe and USA
(Makris et al. 2010). If we improve the biological methods for the in situ reme-
diation, it will be economical. Furthermore, the degradation of pollutants by
microorganisms is a very ecofriendly method, as the structure and biological
function of soils are not disturbed (Fritsche 1998). Various in situ bioremediation
methods have been developed and tested in the lab/field with relative success, such
as natural attenuation, biostimulation, bioaugmentation and phytoremediation.

Natural attenuation is the most simple and inexpensive bioremediation method that
relies upon the activity of the indigenous microbial community on the xenobiotics
which often takes long time (decades). Biostimulation of indigenous microor-
ganisms with the chemical amendments (N and P fertilizers) is another method
that relies on the adjustment of soil properties, such as nutrient content, pH, and
redox potential, resulting in enhanced microbial activity on the contaminant.
Bioaugmentation is defined as the addition of specific microorganisms (wild-type,
or genetically engineered) to the contaminated soil which have been previously
tested in the lab for their degradation ability. Phytoremediation is a low-cost and
environment-friendly bioremediation method which has shown a high promise for
use in TNT-contaminated soils (Makris et al. 2010; Rylott et al. 2011).
In experiments with radio-labeled TNT, it was repeatedly found that a major
part of TNT and its metabolites, independently of the individual process, quickly
and irreversibly bind to the soil matrix (Drzyga et al. 1998, 1999; Spain et al. 2000;
Weiss et al. 2004a). This could be demonstrated in an aerobic–aerobic soil slurry
process ([99 % bound residues), various composting variants (80 % bound resi-
dues), and by the use of fungi (86 % bound residues). The radioactivity was
determined in each case in the aqueous and methanolic-extracts as well as in the
organic fractions, i.e., fulvic acids, humic acids and humin.
Weiss et al. (2004b) studied the fate of (N15)-TNT in the course of an aerobic
bioremediation reactor process with Stropharia rugosoannulata. About 2 % of the
(N15) label was found as NO3- and NH4+, indicating simultaneous processes of
direct TNT denitration and reduction with cleavage of the amino groups. The
enrichment of NO2-/NO3- [up to 7.5 atom % of (N15)] suggested the formation of
Meisenheimer-complexes and denitration. The enrichment of N2O [38 % of the
(N15) label] demonstrated that both N atoms were generated from the labeled TNT
and indicated a novel formation process. The authors propose the generation of
N2O by cleavage from condensed azoxy-metabolites. In addition, 1.7 % of the
(N15) label was detected as biogenic amino-acids in the wheat straw containing the
fungus. Overall, 60 to 85 % of the applied (N15)-TNT was degraded and 52–64 %
was found as nonextractable residues in the soil matrix. Three percent was
detected as 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene.
Park et al. (2012) evaluated the toxicity of TNT contaminated soils after
passing through composting and slurry-phase bioreactor processes using the Sal-
monella mutagenicity assay. For composting, the percentage of mutagenicity
reductions of final composts in strain TA98 and TA100 with S9 metabolic acti-
vation were 90.3–93.7 % and 96.7–97.5 %, respectively. For slurry-phase biore-
actor processes, the percentage reductions of final residuals in strain TA98 and
TA100 with S9 metabolic activation were 95.0 and 99.1 % for anaerobic, 96.2 and
99.2 % for anaerobic/aerobic and 96.6 and 97.4 % for anaerobic treatment. It was
implied that slurry-phase treatment was a more effective process than composting
in reducing toxicity.
In order to biostimulate the capacity of bacteria to degrade TNT, Muter et al.
(2012) amended varying concentrations of nutrients consisting of inorganic salts,
26 H. Claus
plant extracts, and molasses to soil and liquid media. For the inoculum, they used a
consortium of bacteria which was isolated from explosives contaminated soils and
exhibited the ability to degrade TNT. Phylogenetically, the clones clustered into
seven different genera: Klebsiella, Raoultella, Serratia, Stenotrophomonas,
Pseudoxanthomonas, Achromobacter and Pseudomonas. The addition of the
consortium to a liquid environment along with 100 % nutrient amendment
decreased the amount of TNT (and its degradation products) by up to 90 % after
14 days incubation. When the total amount of TNT was less than 100 mg/l, the
concentration of TNT did not influence the amount of sugar consumed by the
bacterial consortium. In soil media, the TNT degradation process was dependent
on the concentration of nutrients added. At higher initial concentrations of TNT
(500 mg/kg), bioaugmentation (i.e., addition of a bacterial inoculum) had a sig-
nificant effect, especially when also nutrients were added to the soil.
Remedial measures are of increasing interest with plants (phytoremediation).
These methods are economical, but limited by the relatively low tolerance of
plants to TNT. In future, detoxification capacities might be enhanced using genetic
modifications (Makris et al. 2010; Rylott et al. 2011). In accordance with these
developments, Zhu et al. (2012) presented a system for TNT phytoremediation by
overexpressing the old yellow enzyme (OYE3) gene from Saccharomyces cere-
visiae. The resulting transgenic Arabidopsis plants demonstrated significantly
enhanced TNT tolerance and a strikingly higher capacity to remove TNT from the
media.
Various on-site biological methods of soil treatment were tested in Germany as
a part of feasibility studies. For example in Hallschlag, a two-stage reactor process
(soil suspension) and an anaerobic/aerobic composting process were tested, each
with 50 tons of soil (Schmitz 1995). Worldwide, there have been or will be also
off-site systems and highly developed in situ biological methods to clean up TNT-
contaminated soils. Time will show whether they pass the proof of sustainability
(Reinhard and Feldmann 1998; Thomas et al. 2001). For aqueous phases (without
solid matrix) such methods are unsuitable, even though the spread of contaminants
through groundwater and leachate is a particular hazard.
2.5 Treatment of Contaminated Waters
2.5.1 Microbial Entrapment
For contaminated soils, TNT biotransformation and immobilization to the organic

matrix represents an effective and low-cost procedure, if not to eliminate, but at
least to detoxify TNT and its metabolites. For aquifers, these procedures are not
applicable as long as TNT and its potential toxic transformation products remain in
the solution. So far, they have to be removed by expensive physico-chemical
adsorption till alternative microbiological procedures are not available.
In the following section the isolation and characterization of a TNT trans-

forming bacterial strain will be described which may be useful for this purpose
(Claus et al. 2007a, b). The microbiological and analytical methods presented
might concomitantly serve as a short practical outline for the study of microbial
TNT degradation.
Water and soil samples were collected from abandoned TNT production sites in
Germany (Hallschlag/Rhineland-Palatinate, Moschwig/Saxony-Anhalt). Microor-
ganisms were enriched in nutrient broth supplemented with TNT (10 mg/l). From
these cultures, single colonies were obtained on nutrient agar containing TNT
(10 mg/l) and further characterized. Bacterial identification was done by
sequencing of the 16S rDNA genes amplified by polymerase chain reaction (PCR).
By this procedure about 20 isolates of mainly Gram-negative bacteria were
obtained which were frequently isolated from contaminated soil in other studies
(Muter et al. 2012). About half of the isolates contained one or more plasmids
which is an indication of adaption to a stressful environment. The most efficient
isolate, R. terrigena strain HB, grew in the presence of 100 mg TNT/l, a con-
centration which is toxic to many microorganisms (Fuller and Manning 1997). It
grows well at temperatures between 4 and 40 °C with an optimum at 30 °C.
Growth at low temperatures is a hallmark of the genus Raoultella which is fac-
ultative anaerobe, having both a respiratory and a fermentative type of metabolism
(Drancourt et al. 2001).
For TNT degradation studies, the mineral-salt medium of Kalafut et al. (1998)
was used. Nitroaromatic compounds were added at concentrations between 40 and
400 lM to the mineral-salt medium before autoclaving. For (14C) studies, the
medium was spiked with uniformly ring labeled (14C)-TNT (33.3 kBq/ml). Glu-
cose concentrations were set to 0.3 and 3.0 % (w/v), respectively. The pH of the
culture medium was adjusted between 5.0 and 8.0 using a 200 mM Na phosphate
buffer. R. terrigena was precultured in Standard I nutrient-broth for 16 h at 30 °C
on a shaker before inoculation into mineral-salt media. These cultures were
incubated for 7 days under aerobic conditions on a rotary shaker at temperatures
between 10 °C and 37 °C. At regular intervals, aliquots were taken to determine
transformation products. At the end, cells and insoluble material were separated by
centrifugation at 40,0009g for 30 min. The bacterial cell mass was washed twice
with phosphate-buffered saline solution (pH 7.4), extracted with acetonitrile for
16 h at 30 °C and centrifuged as above. The resulting fractions (supernatant,
washings, acetonitrile extract) were analysed by HPLC and Radio-HPLC.
On minimal-salt agar supplemented with TNT (100 mg/l), R. terrigena strain
HB produced brownish pigments within and around the growth zone. Growth in
liquid media was determined by colony counts and optical density. After a lag time
of 24 h, the colony forming units (cfu) in the minimal-salt medium with TNT
increased rapidly and reached the same level as in the medium without TNT. The
significant increase of the optical density of cells grown in the presence of TNT
was thus not a result of higher cell densities, but obviously attributed to altered
spectroscopic properties of the cells and the culture media by accumulation of
coloured TNT metabolites, similar to those observed on solid agar-media.
28 H. Claus
Determination of the optical density is thus not an appropriate parameter for

estimating bacterial growth on dependence of TNT.
The growth was coincident with the disappearance of TNT from the culture
media within 4 h incubation under optimum aerobic conditions (pH 7.0, 30 °C).
Already low nutrient concentrations (C0.05 % glucose) were sufficient to promote
growth and TNT removal by R. terrigena strain HB. The need for nutrient sup-
plementation and lack of (14CO2) production from ring-labelled TNT clearly
indicated a co-metabolic process. This was further confirmed by the effective TNT
transformation by resting cells.
In the culture supernatants, 2-ADNT and 4-ADNT were detected along with
small amounts of 2,4-DANT and tetranitroazoxy-compounds. In contrast to the
culture supernatant, the main transformation products found in the cell extracts
were azoxy-dimers. The radiochromatogram of the extract identified 3 peaks, two
of which could be assigned to TN-2,20 -azoxy and TN-4,40 -azoxy, respectively, in a
ratio of 1:10. The third peak corresponded to either TN-2,40 -azoxy or TN-20 ,4-
azoxy, or a mixture of these condensation products.
The (14C)-balance revealed that about 15 % of the initial radioactivity remained
in the culture supernatants, whereas up to 85 % was found in the cell pellet. Our
finding, that the main fraction of TNT metabolites is cell-associated, is deviated
from most other reports, where the main fraction of transformation products
remained in the supernatant in the form of ADNTs (Kalafut et al. 1998; Kim et al.
2002; Zhao et al. 2004). Similar to our study, a strain of Pseudomonas aeruginosa
MX accumulated 71 % of the initial (14C)-TNT in the cell pellet, leaving 21 % in
the supernatant. In the latter fraction, 2-ADNT was the main metabolite and TN-
2,20 -azoxy accumulated in the cells (Oh et al. 2003). As R. terrigena strain HB
grows rapidly at low temperatures and different redox conditions, it is a promising
candidate for the detoxification of TNT-contaminated waters under in situ con-
ditions. The metabolites associated with the cell fraction can be removed together
with the biomass, e.g. by filtration or flocculation (Fig. 3).
In order to optimize this process, we investigated the effects of culture condi-
tions on the TNT transformation in more details. Although TNT elimination was
observed at all incubation temperatures tested, pH 8.0 and 37 °C may regarded as
optimum with respect to the transformation velocity. Similar conditions have been
found for the biodegradation of TNT by Pseudomonas putida (Park et al. 2003).
TNT was completely eliminated at all concentrations tested, however, the amount
of glucose in the mineral salt media had a significant impact on the quantitative
and qualitative distribution of metabolites in the supernatants and cells. At low
glucose conditions (0.3 %), mainly ADNTs were detected along with the forma-
tion of smaller amounts of tetranitroazoxytoluenes. In contrast, at a tenfold higher
glucose concentration (3 % glucose), 2,4-DANT was the almost exclusively
detectable metabolite in the culture medium, accompanied by only minor amounts
of azoxy-dimers in the cell pellet. One explanation is that at high glucose con-
centrations, an excess of reduction equivalents is produced by aerobic metabolism.
As six electrons, provided by NAD(P)H, are needed for the complete reduction of
one nitro group in TNT as shown in Fig. 3 (Vorbeck et al. 1998; Heiss and
Cell-bound residues
TN-4,4´-azoxytoluene
Fig. 3 Model of TNT transformation and entrapment by R. terrigena HB. TNT enters the
bacterial cell by diffusion and is enzymatically reduced by nitroreductases. The products are
about 10–20 % ADNTs which are found extracellular in the solution. Another 80–90 % of the
initial TNT is converted to intra- or intermolecular coupling products which remain in the cell in
form of insoluble tetranitroazoxytoluenes or bound to proteins. In the course of TNT
transformation, R. terrigena HB forms brownish cells which can be removed from the solution
by sedimentation or filtration (Claus et al. 2006, 2007a, b)
Knackmus 2002), the surplus of NAD(P)H may be used for the reduction of a
further nitro-group. Farmore, high amounts of NAD(P)H will preclude the accu-
mulation of nitroso-dinitritoluenes, thus preventing azoxy-dimer formation
(Williams et al. 2004).
The efficiency of TNT removal under nearly in situ like conditions, was
demonstrated in experiments with water and soil samples originating from con-
taminated sites which contained a complex mixture of nitroorganic compounds.
Conclusively, these results have shown that R. terrigena strain HB eliminates
low and high TNT concentrations from water samples, but the efficiency of the
process is regulated by controlling temperature, nutrient and pH conditions. In
addition to TNT, the bacterium may be useful for the treatment of other nitroa-
romatic wastes as well.
2.5.2 Use of Immobilised Microorganisms
Another promising strategy to eliminate TNT from aquifers may be the use of
immobilized microorganisms in batch or continuously operating systems. As an
example, a Bacillus sp. YRE1 strain was isolated from red effluent and cells
immobilized on charcoal and polystyrene were checked for their ability to degrade
TNT by exposing them to different temperatures (Ullah et al. 2010). It was found
that both charcoal and polystyrene immobilized bacteria degraded TNT most
efficiently at 37 °C. Maximum percentage reduction in case of charcoal
30 H. Claus
immobilized Bacillus sp. YRE1 was calculated as 73.35 % at 37 °C, whereas,

polystyrene immobilized bacteria showed 70.58 % reduction. Bacillus sp. YRE1
immobilized on charcoal, showed maximum degradation at pH 7 with 93.81 %
reduction in TNT. Similarly, pH 5 was found to be optimum for the degradation of
TNT by polystyrene immobilized bacteria, with percentage reduction as high as
94 %. Charcoal immobilized cells showed increased transformation with 96 %
reduction in the presence of Tween 20, whereas, polystyrene immobilized cultures
caused 87.77 % reduction in TNT.
A combined process of immobilized microorganisms/biological filter to
degrade TNT in an aqueous solution was studied by Wang et al. (2010). The
results showed that the procedure could effectively degrade TNT to an extent that
it was not detected in the effluent of the system. GC/MS analysis identified 2-
amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene
and 2,4-diamino-6-nitrotoluene as the main anaerobic degradation products. Eth-
anol as the electron donor played a major role in the TNT biodegradation. Envi-
ronment Scan Electron Microscope analysis revealed that a large number of
globular microorganisms were successfully immobilized on the surface of the
carrier. Further analysis by Polymerase Chain Reaction (PCR)-Denaturing Gra-
dient Gel Electrophoresis (DGGE) demonstrated that a special bacterial commu-
nity for TNT degradation could be generated during the adaptation to the explosive
for 150 days. Nevertheless, one should be aware that toxic degradation products of
TNT may still remain in the contaminated water after treatment with immobilized
bacteria.
2.5.3 Application of Enzymes
TNT is no substrate for oxidoreductases, but small organic mediators have been
shown to increase the oxidative potential of laccase and allow the enzymatic attack
of molecules which are no natural laccase substrates (Claus and Strong 2010). The
presence of such a co-substrate may also facilitate the removal of recalcitrant TNT
metabolites from water environments. The addition of phenolic compounds
(200 mM ferulic acid and guaiacol) during the reductive transformation of TNT by
the fungus Trametes modesta prevented the accumulation of all major stable TNT
metabolites by at least 92 % (Nyanhongo et al. 2006). Acute toxicity tests of
individual TNT metabolites and in T. modesta cultures supplemented with 200 lM
TNT demonstrated that the biodegradation process leads to less toxic metabolites.
The presence of phenolics during the laccase reaction were very effective in
immobilizing the typical TNT metabolites (ADNTs and 2,2,6,6-azoxytetranitro-
toluene). When laccase from Trametes villosa was added to a solution containing
4-ADNT and TNT, only 30 % of the 4-ADNT and none of the TNT was trans-
formed. When the same experiment was done in the presence of catechol, 4-ADNT
was complete and up to 80 % of TNT was removed from the solution. This was
attained at close to a neutral pH which is beneficial for treatment of natural
environments (Wang et al. 2002).
2.6 Preventive Approaches to Minimize TNT Contamination
An innovative microbiological approach to reduce TNT contamination takes

advantage of the TNT-transforming Bacillus sp. strain SF, whose spores were
incorporated into an explosive formulation containing TNT and ammonium nitrate
(Nyanhongo et al. 2009). Upon addition of water to this new explosive mixture,
vegetative Bacillus cells grow out which immediately initiate TNT transformation
even after a 5-year storage of the bioexplosive at room temperature (Nyanhongo et al.
2009). The development of these self-cleaning explosive formulations opens new
perspectives for the application of specific TNT-transforming microorganisms, such
as spores of Clostridium bifermentans KMR-1 which can be used as a relatively stable
inoculant for TNT biodegradation (Sembries and Crawford 1997). The possibility to
lyophilize a Pseudomonas putida strain in the presence of cryoprotectants was also
investigated for the application of non-sporulating microorganisms into TNT-based
explosive formulations (Nyanhongo et al. 2009). However, the survival of P. putida
cells was limited in the bioexplosive formulation, underlining the need to optimize
the cryoprotective media and the lyophilization conditions (Nyanhongo et al. 2009).
This is a challenge since Pseudomonadaceae, a catabolically versatile and ecologi-
cally important group of bacteria, is also the most studied family for bacterial TNT
biodegradation. Recent studies on the microbial ecology of different TNT-polluted
soil samples using DGGE fingerprinting have also demonstrated the predominance of
members of Pseudomonadaceae in both long- and short-term contaminated sites
(George et al. 2008; Travis et al. 2008a, b). In addition, Gram-negative bacteria are
the best candidates for the microbial incorporation in self-cleaning explosive for-
mulations since they are more tolerant to TNT than Gram-positive bacteria (Fuller
and Manning 1997). In conclusion, the development of self-cleaning explosive
formulations using microbial catabolic capabilities has recently emerged as an
attractive strategy to prevent further TNT contamination (Stenuit and Agathos 2010).
A new environmentally benign synthesis route to manufacture military grade
TNT, which eliminates the production of red water, arising from the sulfiting
process for removing unsymmetrical trinitrotoluene isomers, was introduced by
Millar et al. (2011).
3 Conclusions
The metabolic capacities of microorganisms (bacteria, fungi) have been success-

fully exploited to clean up TNT contaminated soils by strategies generally referred
as bioattenuation or bioaugmentation. Comparable microbiological methods to
treat waters charged with nitroaomatics are scarce. The entrapment of TNT
metabolites within bacterial cells offers an opportunity to detoxify contaminated
waters after separation of the biomass, at least as long biotransformation and not
biomineralization is the state of art.
32 H. Claus
Alternative or additional strategies for cleaning contaminated aquifers and

waters may take advantage of immobilized microorganisms or oxidative enzymes
to enhance the formation of insoluble metabolites. Some innovative microbial
strategies are currently under development to minimize the danger of TNT con-
tamination during manufacturing process itself or by the design of innovative
explosive formulations.
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Bioremediation of Nitroglycerin:
State of the Science
John Pichtel
1 Introduction
Nitroglycerin [glycerol trinitrate, GTN, C3H5(NO3)3] was discovered in 1847 by

Asconio Sobrero, a student at the University of Turin (US Army 1984). A member
of the nitrate ester family of energetic compounds (Fig. 1), GTN was subsequently
applied at an industrial level by Alfred Nobel in the formulation of dynamite
(Bergengren 1960).
Nitroglycerin is a powerful impact-sensitive explosive. By virtue of this
quality, GTN is extensively used in the manufacture of dynamite, ignition car-
tridges, double- and triple-base smokeless powders, and rocket propellants, such as
those for intercontinental ballistic missile motors (Zhang et al. 1997; Pennington
et al. 2002). Additionally, since its introduction to medicine by Murrell in 1882,
nitroglycerin has been widely used as a vasodilator for the relief of angina pectoris
(Stayner et al. 1992; Podlipná et al. 2008; Saad et al. 2010).
Nitrate esters are relatively rare in nature (White and Snape 1993), however, it
was reported by Hall et al. (1992) that nitrate esters of long-chain alkenyl alcohols
may be produced by certain insects as sex pheromones. Methyl and ethyl nitrate in
sea water and air samples have been detected along Atlantic Ocean transects
(Chuck et al. 2002). Additionally, low concentrations of alkyl nitrates are gener-
ated in the atmosphere by the reaction of nitrogen oxides with hydrocarbons,
forming a component of photochemical smog (Day et al. 2003).
Nitroglycerin is considered a xenobiotic compound which is toxic to both
terrestrial and aquatic organisms including algae, vertebrates and invertebrates
(Burton et al. 1993; Halasz et al. 2010). At high levels it is known to be toxic to
microorganisms, fish, rats, and humans (Wendt et al. 1978; Urbanski 1965). At
concentrations ranging between 30 and 1,300 mg/kg, GTN has been reported to be
acutely toxic to mammalian species (Wendt et al. 1978). GTN can be readily
J. Pichtel (&)
Ball State University, Muncie, IN 47306, USA
e-mail: jpichtel@bsu.edu

40 J. Pichtel
Fig. 1 Chemical structure of several nitrate esters
absorbed through the skin and lungs, and excessive exposure has been linked to a
number of adverse health effects in humans. Chronic exposure causes severe
headaches, vomiting, decreased blood pressure, hallucinations, skin rashes and
methemoglobinemia. Acute exposure causes convulsions, cyanosis, circulatory
collapse, or death (Sittig 1991; Rom 1992). Exposure symptoms and mortality
have been widely reported among workers involved with explosives manufac-
turing (Stayner et al. 1992; Stucki 2004). The National Institute for Occupational
Safety and Health estimated that 8,000 workers involved in dynamite manufacture
were exposed to GTN via inhalation and dermal absorption (USDHEW 1978).
Twelve percent of 266 potentially exposed workers at Badger Army Ammunition
Plant (WI) suffered symptoms due to GTN exposure (Yost 2004). Hogstedt and
Axelson (1977) reported an association between ischemic heart disease and
cerebrovascular disease in studies of Swedish workers exposed to GTN.
Extensive manufacturing and use of GTN have resulted in its widespread
release and distribution in the biosphere. Data is sparse concerning the fate and
mobility of GTN in contaminated soils, sediments, and groundwater. Due to its
deleterious effects on public health and the environment as well as its explosive
nature, GTN contamination poses a significant risk that requires effective and
efficient remediation.
2 Properties and Uses of Nitroglycerin
Nitroglycerin is prepared by nitration of glycerin at approximately 25 °C. Glycerin

is added to a stirred mixture of 40 % nitric acid and 60 % sulfuric acid (Federoff
and Sheffield 1972) (Fig. 2). The resulting ester structure, C–O–NO, is chemically
distinct from nitro-organic compounds containing the C–NO2 group (White and
Snape 1993). The nitrate ester structure has important practical implications for
biodegradation.
Bioremediation of Nitroglycerin: State of the Science 41
Fig. 2 Nitration of glycerol

to produce nitroglycerin
Nitroglycerin is a liquid at standard temperature and pressure with a solubility

of 1.8 g/l at 20 °C (Verscheuren 1983; Pal and Ryon 1986). Its melting point is
13 °C (Fedoroff and Sheffield 1972) (Table 1). Nitroglycerin crystallizes in two
forms, a labile (unstable) form with a melting point of 2.8 °C and a stable form
with a melting point of 13.5 °C (Yost 2004). Its detonation velocity is 7,700 m/s
(Federoff and Sheffield 1972).
Dynamite is manufactured by absorbing GTN in a mixture of sawdust, starch,
and similar carbon-rich materials. Calcium carbonate is added to neutralize nitric
acid generated via spontaneous decomposition. Ethylene glycol dinitrate is added
as an antifreeze and oxidizers are also typically incorporated. Dynamites vary
markedly in composition and are identified by manufacturer and trade name
(Apache, Austin, Hercol, Toval, Trojan, etc.), strengths (20, 40 and 60 %),
application (ditching, quarrying, seismograph), additives (gelatin, ammonium
nitrate), and form (bulk, slurries, water gels). Three forms of dynamite are in
common use: straight dynamite, ammonia dynamite, and gelatin dynamite
(Table 2). Ammonia dynamite and straight dynamite contain ammonium nitrate
Table 1 Selected chemical Property Value

and physical properties of
nitroglycerin CAS number 55-63-0
Chemical formula C3H5(NO3)3
Molecular weight 227.09
Detonation velocity 7700 m/s
Density 1.59 g/cm3
Vapor pressure 2.6 9 10-4 mm Hg at 20 °C
Solubility in water 1.8 g/l
Heat of detonation 218 °C
Melting point 2.8 °C (labile form)
13.5 °C (stable form)
Boiling point 50–60 °C (decomposes)
log Kow 1.62–2.77
Henry’s law constant 2.71 9 10-7 bar m3 mol-1
Sources Rosenblatt et al. (1991), Windholz (1979), Pal and Ryon
(1986), Verscheuren (1983), Fedoroff and Sheffield (1972), Yost
(2004), Sunahara et al. (2009), Spanggord et al. (1980)
42 J. Pichtel
Table 2 Chemical and physical properties of selected forms of dynamite

Straight dynamite Ammonia dynamite Gelatin dynamite
Density, g/ml 1.3 0.8–1.2 1.3–1.6
Chemical composition 20–60 % GTN 12–22 % GTN 20–91 % GTN
23–60 % NaNO3 15–57 % NaNO3 40–60 % NaNO3
12–50 % NH4NO3 Gelatinized in nitrocellulose
Detonation velocity, m/s 3,600–6,000 2,700–4,600 4,000–7,400
Ft/s 11,800–19,700 8,900–15,000 13,000–24,000
Sensitivity High High High
Sources Meyer (2004), US Army (1984)
and sodium nitrate, respectively, as oxidizers; gelatin dynamite contains 1 %

nitrocellulose that serves to thicken the nitroglycerin and provides the dynamite
with additional brisance (shattering power) on detonation (Meyer 2004; Pichtel
2011).
Nitroglycerin is widely used for the production of propellants including
smokeless powder, which are divided into three classes based on presence of
additive compounds (Table 3). Single-base propellants contain nitrocellulose (NC)
as the primary energetic compound. Double-base propellants contain NC com-
bined with an organic nitrate, such as GTN. Cordite is a double-based propellant
containing 30–40 % GTN and petroleum jelly as a stabilizer. Triple-base pro-
pellants include GTN and NC mixed with nitroguanidine (NQ) (Juhasz and Naidu
2008; Walsh et al. 2008). Other additives may be included to adjust burn rate.
Also, binders or plasticizers are added to facilitate loading the propellant into the
shell. Additional compounds can be included to enhance propellant stability during
storage (Walsh et al. 1993).
Nitroglycerin is also used as a principal ingredient of ignition cartridges in
military projectiles, rockets, missiles and small arms ammunition. A common
ignition cartridge is composed of 57.75 % NC, 40 % GTN, 1.5 % potassium
nitrate and 0.75 % diphenylamine (Pennington et al. 2002). Rocket propellant
produced at Badger Army Ammunition Plant contained 50 % NC, 35 % GTN,
10 % diethylphthalate, 2 % 2-nitrodiphenylamine and various lead fillers (USD-
HEW 1978).
3 Environmental Contamination by Nitroglycerin
Nitroglycerin has been detected in soil and groundwater at numerous locations

worldwide (Table 4). Contamination is a result of routine military training exer-
cises and armed conflict. However, GTN has also entered the biosphere due to
improper management and disposal from both commercial and military activities.
Live-fire and demolition ranges at US and Canadian military bases have been
assessed for contamination by energetic compounds (i.e., explosives and
Table 3 Chemical composition of double-base smokeless powder

Component Structure Amount Function
name by
weight
(%)
Nitroglycerin 4–40 Energetic: raises the energy
content; improves physical
properties; reduces
hygroscopicity
Rosin (abietic 0–4 Deterrents: coats the exterior of

acid) the propellant granules to
reduce the initial burning rate
on the surface and reduce
initial flame temperature and
ignitability
Diphenylamine 0–1 Stabilizer: prevents nitrocellulose

and nitroglycerine from
decomposing by neutralizing
nitric and nitrous acids
Ethyl centralite 0–1 Plasticizer: reduces the need for

volatile solvents
Nitrocellulose Variable Energetic
propellants) (Jenkins et al. 1998, 2007, 2008; Thiboutot et al. 1998, 2004a, b;
Ampleman et al. 2000, 2004; Walsh et al. 2001, 2004; Hewitt et al. 2004;
Pennington et al. 2005, 2006a, b; Martel et al. 2009). Affected sites include
antitank rocket, rifle grenade, demolition, tank firing, mortar, artillery and C-130
gunship ranges. Nitroglycerin is released to soil primarily at firing positions of
anti-tank rocket ranges due to the use of double-based propellant in M72 rockets
(Pennington et al. 2002, 2006a, b; USACHPPM 2002; Thiboutot et al. 2004a, b).
Residues have been deposited at distances up to 100 m in front of the muzzle
(Pennington et al. 2006a). The major deposition of residue, however, is behind the
firing line due to back blast. Studies conducted at anti-tank rocket firing points at
Yakima Training Center (WA) (Pennington et al. 2002, 2006b), Fort Bliss (TX)
(USACHPPM 2002), CFB Gagetown (Thiboutot et al. 2004a, b), CFB Valcartier
(Jenkins et al. 2004), and CFB Petawawa (Brochu et al. 2008) indicate highest
GTN concentrations behind the firing line due to back blast of shoulder-fired
rockets.
44 J. Pichtel
Table 4 Nitroglycerin contamination in surface soils at selected US and Canadian military

installations
Facility Maximum Source or Media Reference
concentration
(lg/kg)
Camp Edwards, MA 69.6 n/a Clausen et al. (2010)
Fort Lewis, WA 344 105 mm Howitzer Jenkins et al. (2001)
Fort Lewis, WA 936,000 Jenkins et al. (2001)
Massachusetts Military 130,000 Gun firing position Pennington et al. (2001)
Reservation, MA
Yakima Training Facility, WA 13,000 Anti-tank (LAW Pennington et al. (2001)
rockets)
Yakima Training Facility, WA 17,000 120 mm tank Pennington et al. (2001)
Yakima Training Facility, WA 25,700 155 mm Howitzer Pennington et al. (2001)
firing position
Yakima Training Facility, WA 246 81 mm mortar Pennington et al. (2001)
firing position
Yakima Training Facility, WA 1,340 Claymore mine Pennington et al. (2001)
Fort Bliss-Dona Ana Range, 1,060 155 mm Howitzer Pennington et al. (2001)
NM range
Fort Bliss-Dona Ana Range, 1,370 Artillery range Pennington et al. (2001)
NM
Fort Bliss-Dona Ana Range, 1,160 Anti-tank range Pennington et al. (2001)
NM
Fort Bliss-Dona Ana Range, 20,000 Crater of 155 mm Pennington et al. (2001)
NM Howitzer
CFB Shilo, Manitoba, Canada 18.1 Gun Propellants Pennington et al. (2001)
CFB Shilo, Manitoba, Canada 408 MILAN missile* Pennington et al. (2001)
Fort Drum, NY 0.5 lg/m2 60 mm mortar Jenkins et al. (2002)
Camp Ethan Allen, VT 1,593 lg/m2 81 mm mortar Jenkins et al. (2002)
Wellington Anti-tank Rocket 110,000 Anti-tank Pennington et al. (2002),
Range (2005)
CFB Valcartier 788,000 5–15 m behind Jenkins et al. (2004)
firing line
CFB Valcartier 339,000 15–25 m behind Jenkins et al. (2004)
firing line
CFB Petawawa 2,400,000 0–10 m behind Brochu et al. (2008)
firing line
CFB Petawawa 250,000 Anti-tank range Brochu et al. (2008)
CFB Petawawa 2,240,000 Anti-tank range Pennington et al.
(2006a)
Cold Lake Air Weapons Range, 950,000 Anti-tank range Pennington et al.
Alberta, Canada (2006a)
Cold Lake Air Weapons Range, 1,500,000 10 m behind firing Pennington et al.
Alberta, Canada point (2006a)
CFB Gagetown, New 4,700,000 Firing line, anti- Pennington et al.
Brunswick, Canada tank range (2006b)
CFB Gagetown, New 6,560,000 Firing line, anti- Thiboutot et al.
Brunswick, Canada tank range (2004a, b)
(continued)
Table 4 (continued)
Facility Maximum Source or Media Reference
concentration
(lg/kg)
WATC Wainwright, British 4,453,000 Soil Pennington et al.
Columbia, Canada (2006a)
Schofield Barracks, HI 14,000,000 0–10 m behind Jenkins et al. (2007),
firing point Hewitt et al. (2004)
*
MILAN Missile d’Infanterie Leger Antichar, anti-tank missile
Nitroglycerin also occurs in the target areas as a result of incomplete detonation

of ordnance (i.e., low-order detonation). Concerns have arisen that such residues
may pose a threat for GTN to migrate through the soil profile and contaminate
groundwater and surrounding environments, resulting in adverse environmental
and health impacts. Accidental detonation is also a concern (Pennington et al.
2006a).
During manufacturing operations, soils have become enriched in GTN from
propellant and explosives machining, casting and curing, improper storage prac-
tices and improper disposal of contaminated wastewaters (Pugh 1982; Best et al.
1999). For example, GTN has been detected in soil at an abandoned nitroglycerin
manufacturing plant in Somerset West, South Africa (Marshall and White 2001).
For decades, the US military used unlined evaporation/percolation lagoons for
disposal of wastewaters from manufacturing, demilitarization, and load, assemble,
and pack (LAP) operations. Many propellant and explosive formulations have
subsequently accumulated at the surfaces of lagoons. Additionally the destruction
of energetic mixtures was historically accomplished via open burning, detonation
or incineration techniques. These practices can leave unburned energetic residues
on the soil surface (Walsh et al. 2010). As more stringent environmental regula-
tions are enacted at state and federal levels, these techniques are no longer con-
sidered feasible.
4 Behavior of GTN in the Environment
When GTN is released into soil and the subsurface, it becomes mobile in part due
to its moderate aqueous solubility and low partition coefficient values (e.g., log
Kow = 1.62 and log Koc = 2.77) (Spanggord et al. 1980; Sunahara et al. 2009).
Since its specific gravity is 1.59, GTN behaves as a DNAPL when comes in
contact with water.
A number of mechanisms may be involved in the natural attenuation of GTN in
soil, aquifers and groundwater. These include adsorption to solid media (e.g.,
colloidal clay, Fe and Al oxides, humic compounds, microbial biomass), gradual
dissolution into the aqueous phase, advection, hydrodynamic dispersion, and
46 J. Pichtel
diffusion, as well as hydrolysis and biological degradation (Wiedemeier et al.

1999; Husserl 2011).
5 Remediation Technologies for GTN
A range of chemical and physical technologies are available for remediation of

soils, sediments and groundwater contaminated with energetic materials. Because
of concerns about the toxicity of GTN as well as its energetic properties, efforts
have been underway to develop safe and cost-effective methods for treating GTN-
enriched media. However, due to its impact sensitivity, solubility and behavior as a
DNAPL in subsurface environments, remediation of GTN-contaminated soil and
groundwater is plagued with a host of practical difficulties. In one of the most
straightforward procedures, soil is excavated, sieved to remove large items such as
buried drums, tree stumps, etc., and fed into a mobile high-temperature incinerator.
Excavation may, however, inadvertently release contaminated airborne particles.
Additionally, GTN is an impact-sensitive explosive, hence its excavation is
potentially dangerous. Lastly, hazardous waste incinerators bear substantial
operational costs.
Contaminated groundwater can be subjected to so-called pump and treat
technology, where GTN present in extracted water is adsorbed on activated carbon
and/or exposed to rigorous chemical reaction. For example, GTN can be reduced
using inorganic reagents (e.g., sodium sulfite) and alkaline hydrolysis (Meng et al.
1995). Other physico-chemical methods include oxidation using potassium per-
manganate or ozone (Smith et al. 1983), reduction on elemental Fe (Oh et al. 2004)
and pyrite (Oh et al. 2008), sorption using nano-structured silica-based materials
(Saad et al. 2010), and microwave digestion (Halasz et al. 2010). Unfortunately,
pump and treat is a slow process which is often costly. There are additional
concerns that drilling into GTN-enriched soil or groundwater could result in
detonation (Jones and Lee 1997).
The above methods have been found to reduce GTN concentrations in waste-
waters; however, several decomposition products have been generated that are
unsafe to public health and the environment. Reacting GTN with alkalis or strong
acids forms glycidol and several glycidylnitrite and nitrate by-products (Kaplan
et al. 1982). Likewise, GTN treated with sodium sulfite may yield undesirable
nitrite and nitrate compounds (Accashian et al. 2000). Hence, secondary treat-
ments may be needed to eliminate toxic products.
These physico-chemical techniques require the use of hazardous reagents and
carry substantial operating costs, plus the need to remove nitrogenous by-products.
As a result of these practical complications, natural in situ approaches to GTN
treatment and removal are appealing both to industry and regulatory officials.
Preference is for safe, cost-effective, and environmentally-friendly methods which
ensure complete GTN transformation to innocuous end-products.
6 Bioremediation of GTN
6.1 Biochemistry and Microbiology of GTN Transformations
Biological remediation techniques (bioremediation) are among the most effective

and widespread clean up methods for soils contaminated by energetic materials
(Scalzo et al. 1998; Husserl 2011). The most commonly employed techniques
include bioslurry, composting, land-farming and phytoremediation. Primary
benefits include their relatively low cost, fewer negative impacts to the local
environment, and (in the case of phytoremediation) greater acceptability due to its
aesthetic appeal. Drawbacks to these technologies are that they are slower
than several physico-chemical technologies with less certainty of complete
contaminant removal. In addition, relevant microbial populations may be incapable
of decomposing some of the complex and/or toxic molecular structures of
contaminants.
The microbially-catalyzed reactions involved in GTN degradation have been
examined in detail. Laboratory studies have demonstrated microbial degradation
of GTN under both aerobic and anaerobic conditions (Meng et al. 1995; White
et al. 1996; Bhaumik et al. 1997; Christodoulatos et al. 1997; Marshall and White
2001) with and without additional nitrogen or carbon sources. However, most of
these studies have involved test media other than natural soils—biodegradation has
been evaluated in laboratory culture media, digester sludge, and wastewater
treatment systems (Smith et al. 1983; White et al. 1996; Bhaumik et al. 1997;
Blehert et al. 1997; Christodoulatos et al. 1997; Accashian et al. 1998; 2000;
Marshall and White 2001). Little is known regarding GTN degradation in soil and
groundwater.
All recent work is in agreement as regards a single denitration pathway under
both aerobic and anaerobic conditions in which GTN is used as a nitrogen source.
Biodegradation occurs via successive denitrations to glycerol dinitrates (GDNs)
and glycerol mononitrates (GMNs). As depicted in Fig. 3, GTN metabolism
produces glycerol 1,2- and 1,3-dinitrate (1,2-GDN and 1,3-GDN), and glycerol 1-
and 2-mononitrate (1-GMN and 2-GMN). The removal of the last nitro group to
obtain glycerol is consistently the slowest and most difficult step (Wendt et al.
1978; Marshall and White 2001). Once glycerol is formed, it is biologically
transformed to CO2 and H2O.
The identical denitration reactions apparently occur in mammalian systems
(Spain et al. 2000). Furthermore, during abiotic GTN reduction utilizing elemental
Fe, a similar chain of intermediates and final products was observed; however,
nitro groups were eventually reduced to ammonia (Oh et al. 2004; Husserl 2011).
The recalcitrance of nitrate esters to biotransformation has been observed by
various researchers (US Army 1989; Meng et al. 1995; Binks et al. 1996; Ramos
et al. 1996). Resistance to microbial attack is likely the result of both the toxicity
and xenobiotic nature of these molecules. The tendency of GTN to partition to
organic matter, deduced from log Kow values ranging from 1.62 (Sunahara et al.
48 J. Pichtel
Fig. 3 Transformation of nitroglycerin showing successive steps in denitration (DNG dinitro-

glycerin, MNG mononitroglycerin) (Wendt et al. 1978)
2009) to 2.77 (Spanggord et al. 1980), may explain its reported toxicity (Ducrocq
et al. 1989; Meng et al. 1995). Microbial cells experienced significant inhibition or
death when exposed to a medium containing organic compounds with log
Kow = 1–5 (Heipieper et al. 1994). This is a result of partitioning into the lipid
bilayer of the microbial cell membrane which causes leakage. Disruption of
membrane potential may occur along with loss of proteins and lipids and ulti-
mately, cell death (Simkins and Alexander 1984). It is, therefore, likely that GTN
inhibits microbial activity through membrane solvent toxicity.
Microbe-mediated denitration of GTN to 1,2-GDN (log Kow = 6.02), 1,3-GDN
(log KOW = 5.05) and GMNs (log KOW = 1.46) (Leo et al. 1971; Williams and
Bruce 2002) may further promote cellular toxicity as per the membrane leakage
theory (Simkins and Alexander 1984). In addition, the biological metabolism of
nitrate esters is characterized as a reductive process which typically results in the
formation of nitrite (White and Snape 1993). Nitrite is known to be toxic to cells at
high concentrations via several mechanisms (Sijbesma et al. 1996). Such data on
the toxicity of GTN and its metabolites pose challenges regarding microbial
capabilities for its decomposition. Earliest reports stated that the GTN molecule
was resistant to biological attack (Logan 1953; Rudolfs 1953; Smith and Dick-
inson 1972; ADPA 1975). Concentrations from 100 to 150 mg/l inhibited
microbial denitration reactions (US Army 1974, 1976). Toxic effects on mixed
microbial populations were noted at concentrations ranging from 600–900 mg/l
(US Army 1973, 1974; ADPA 1975). As late as 1989, a treatability study con-
ducted at the Badger Army Ammunition Plant (WI) revealed that the presence of
GTN in the influent feed caused toxic and/or inhibitory effects on microbial cul-
tures and resulted in system failure (US Army 1989).
Contemporary studies, however, indicate that GTN undergoes conversion by a
variety of microorganisms. Kozioroski and Kucharski (1972) described an acti-
vated sludge process for treatment of GTN manufacturing wastewater. Using
mixed cultures in laboratory-scale activated sewage systems GTN was found to
undergo biological modification—bacteria significantly reduced GTN concentra-
tions (US Army 1975). Thin layer chromatography and HPLC analysis of extracts
of mixed cultures showed partial conversion to GDNs (US Army 1974, 1975).
However, it was not determined whether the GTN molecule was supporting
microbial growth. Smets et al. (1995) calculated the thermodynamic feasibility of
biochemical GTN denitration assuming a sequential denitration pathway via the
dinitrate and mononitrate isomers, in which each denitration step is reductive and
mediated by a glutathione S-transferase (Ducrocq et al. 1989; Servent et al. 1991).
It was concluded that complete mineralization of GTN by bacterial cells under
both aerobic and anoxic conditions, without the addition of external carbon and
nitrogen sources, was thermodynamically feasible.
6.1.1 Bacterial Metabolism: Mixed Cultures
Wendt et al. (1978) examined GTN decomposition using activated sludge, in batch
and continuous bioreactors, treated with excess carbon sources under varying
cultural conditions. Biodegradation occurred via successive denitrations, with each
succeeding step proceeding at a slower rate (Fig. 3). A mixed culture established
in a two-stage bench-scale activated sludge system converted GTN to 1,3-GDN
and 1,2-GDN in roughly equivalent quantities. The final effluent was devoid of tri-,
di- and mononitrate esters. Several pure, but unidentified bacterial cultures were
isolated and subsequently grown in the batch culture to convert GTN to 1,3-GDN,
1,2-GDN and GMN. It is not known whether complete denitration had occurred
because residual dinitrate and mononitrate isomers were detected in finished
medium from both the batch and continuous bioreactors. Furthermore, no attempts
were made to determine glycerol concentrations in the spent medium. Reduction in
GTN concentration in control treatments was negligible without supplemental
carbon which suggested that GTN biotransformation was a co-metabolic process.
50 J. Pichtel
High destruction efficiencies of GTN were reported in a sequencing batch

reactor used to treat munitions wastewater from a propellant manufacturing facility
(Pesari and Grasso 1993). An influent concentration of 200 mg/l GTN was reduced
to below detection limits after \5 h of aeration. Sorption did not contribute
appreciably to GTN reduction. High concentrations of nitrate, a product of GTN
degradation, were treated in an anoxic process. It is not known whether complete
denitration of GTN was achieved, however, as detection of GDNs or GMNs was
not attempted. Co-metabolism was proposed as the mechanism of GTN transfor-
mation since GTN-acclimated cultures were not capable of using GTN as the sole
carbon source. Ethyl acetate occurring in the waste stream served as a satisfactory
substrate (Pesari and Grasso 1993). Batch reactors proved a viable treatment for
biological oxidation, nitrification, and denitrification of the wastewater in a single
unit.
GTN was completely degraded by anaerobic bacterial consortia in the presence
or absence of co-substrates (Christodoulatos et al. 1997). Anaerobic mineralization
was complete in 26 d with addition of 2,000 mg/l glucose as compared to 114 d
without a carbon source. Denitration converted GTN to 1,2-GDN and 1,3-GDN,
then to 1-GMN and 2-GMN. Significant regioselectivity of the enzymatic degra-
dation was observed with preferential action on the middle (C2) nitro group which
favored production of 1,3-GDN and 1-GMN. The by-products of anaerobic
nitration, i.e., nitrate and nitrite, were further reduced to N2 (Christodoulates et al.
1997). Specific removal rates of nitrate esters were low, but increased substantially
upon addition of glucose as a co-substrate. The rate of decomposition was
recorded as:
GTN [ 1; 2 GDN [ 1; 3 GDN [ 1 GMN [ 2 GMN
Following the above steps, the authors suggest that an available carbon source
is produced. However, it is possible that the original digester sludge contained
utilizable sources of carbon. The advantage of anaerobic mineralization of GTN is
a lower co-substrate requirement compared with aerobic methods (Bhaumik et al.
1997). Furthermore, anaerobic reactions substantially reduce quantities of nitrate
and nitrite, two undesirable products of dinitration.
6.1.2 Bacterial Metabolism: Pure Cultures
Several studies have examined GTN biodegradation under aerobic and anaerobic
conditions using individual bacterial and fungal species (Kaplan et al. 1982;
Ducrocq et al. 1989; Servent et al. 1992; White et al. 1996). Complete mineralization
of GTN was not reported in any of these studies, as a primary carbon source was
required in order to initiate denitration.
Pseudomonas putida and P. fluorescens isolated from GTN-contaminated soils
sequentially degraded toxic levels of GTN to GDN and GMN isomers, but could
not denitrate GMN (Blehert et al. 1997). Microbial isolates from soil and sediment,
which had been previously exposed to nitrate esters, were studied by Meng et al.
(1995). The most effective bacteria for transforming GTN were identified as
Bacillus thuringiensis/cereus and Enterobacter agglomerans. The biodegradation
pathway for both isolates was shown to be the sequential denitration as presented
in Fig. 3. Resting cells denitrated GTN with the formation of nitrite, indicating a
reductive denitration reaction.
Several bacteria capable of metabolizing GTN were isolated under aerobic and
nitrogen-limiting conditions from soil, river water, and activated sewage sludge
(White et al. 1996). An Agrobacterium radiobacter strain, isolated from sewage
sludge, denitrated GTN with the concomitant formation of 1,2-GDN and 1,3-GDN,
with significant regioselectivity for the production of the 1,3-GDN isomer. Both
GDN isomers were subsequently converted to 1-GMN and 2-GMN. This strain
was unable to denitrate the GMN isomers, resulting in their accumulation. No
other GTN derivative or metabolite was produced in significant quantities. These
cultures were also capable of metabolizing another nitrate ester, pentaerythritol-
tetranitrate (PETN), probably to its trinitrate and dinitrate analogs (White et al.
1996).
Sequencing batch and packed bed reactors were used to assess GTN degrada-
tion under aerobic and anaerobic conditions (Bhaumik et al. 1997). Using activated
sludge, anaerobic digester sludge and the white rot fungus Phanerochaete chry-
sosporium, GTN was denitrated to GDN and GMN isomers. The reactions were
apparently identical in both mixed bacterial and P. chrysosporium cultures. The
rate of conversion was, however, slower for the latter. The denitration pathways
were the same in aerobic and anaerobic environments (Fig. 3). In the presence of
co-substrates, both aerobic and anaerobic microorganisms showed marked regi-
oselectivity at the first and second denitration steps with preferred scission of the
central nitrate ester group. This favors generation of 1,3-GDN and 1-GMN
(Fig. 3). Significantly higher rates of denitration were measured in the packed bed
reactor as compared to batch systems for all microbial types tested. In packed bed
reactors, it is possible for GTN and its intermediates to completely decompose,
given sufficient retention time to allow for destruction of the more recalcitrant di-
and mononitrates (Bhaumik et al. 1997).
In the first report of complete denitration of GTN used as a primary growth
substrate by a bacterial culture under aerobic conditions, GTN metabolism was
studied from aeration tank sludge previously exposed to GTN (Accashian et al.
1998). Aerobic enrichment cultures removed GTN rapidly. The denitration of all
glycerol nitrate esters was concurrent, and 1,2-GDN and 2-GMN were the primary
isomers observed. Nitrite comprised the major fraction (69–100 %) of released
nitrogen, with lesser quantities of nitrate detected. Accumulation of nitrite implies
a reductive rather than a hydrolytic denitration mechanism. Reductive denitration
is congruent with published accounts of denitration of nitrate esters by bacterial
(Binks et al. 1996; White et al. 1996; Blehert et al. 1997; Snape et al. 1997) and
fungal (Servent et al. 1991, 1992) cultures. In contrast to Bhaumik et al. (1997)
and Christodoulatos et al. (1997), the kinetics of GTN biotransformation were
52 J. Pichtel
10-fold faster than reported for complete GTN denitration under anaerobic con-
ditions (Accashian et al. 1998).
Bacteria were isolated from the soil samples collected from a wash water soak
away at a closed nitroglycerin manufacturing facility (Marshall and White 2001).
The isolates, identified as P. putida, Arthrobacter species, Klebsiella species and
Rhodococcus species exploited GTN as its sole nitrogen source and removed nitro
groups sequentially. The Arthrobacter strain removed only the first nitro group; the
Klebsiella strain demonstrated a preference for removal of the central nitro group
from GTN, while the other five strains showed no regioselectivity. For those
strains which removed a second nitro group from 1,2-GDN, a preference for
removal of the end nitro group was demonstrated, thus producing 2-GMN. After a
long lag period, Rhodococcus species was capable of removing the final nitro
group from GMN and thus achieved complete biodegradation of GTN. It is
unknown, however, if glycerol was the final product or if a different nitrated
intermediate accumulated. The authors claim this was the first report of a single
bacterial species that could rapidly and completely denitrate GTN without addition
of a supplemental N source.
6.1.3 Fungal Metabolism
Transformation of GTN by Geotrichum candidum and P. chrysosporium has been

reported by various workers (Ducrocq et al. 1989, 1990; Servent et al. 1991, 1992;
Zhang et al. 1997). G. candidum was able to denitrate GTN to 1-GMN and 2-GMN
(Ducrocq et al. 1989), while P. chrysosporium denitrated GTN only to 1,2-GDN
and 2-GMN (Servent et al. 1991, 1992). However, evidence of complete GTN
denitration was not observed with either culture (Ducrocq et al. 1989, 1990;
Servent et al. 1991, 1992). An organism identified as Penicillium corylophilum
Dierckx was isolated from a double base propellant which contained nitrocellu-
lose, GTN, 2,4-dinitrotoluene, diphenylamine, and di-n-butyl phthalate. The
organism was able to partially degrade nitrocellulose with xylan as a supplemental
carbon source (Sharma et al. 1995) and to completely denitrate GTN in buffered
medium with glucose and ammonium nitrate (Zhang et al. 1997). GTN was
transformed stepwise to DNG (48 h) and MNG (168 h). Complete denitration of
GMN was achieved in 336 h. The presence of ammonium nitrate is apparently
necessary for the production of the enzymes responsible for denitration of GMN.
During the metabolism of GTN, P. chrysosporium produced nitrite which was
converted to nitrate. EPR spectra also suggested the formation of nitric oxide
which was present as an (Fe2+)–heme–NO complex (Servent et al. 1991). Analysis
of the subcellular location of the enzyme activities suggested the presence of GST
activity in the cytosol, together with both cytosolic and microsomal cytochrome-
P450-like enzymes (Servent et al. 1992).
6.1.4 Transformation via Enzymes
In recent years, the bacterial and fungal enzymes capable of cleaving nitrate esters
have been characterized in detail (Blehert et al. 1997; Snape et al. 1997; Williams
and Bruce 2002; Marshall et al. 2004). The conversion of GTN to GDN has been
shown to involve a/b barrel oxidoreductase flavoproteins (French et al. 1996;
Blehert et al. 1997; Snape et al. 1997) which are members of the Old Yellow
Enzyme (OYE) family (Stott et al. 1993). These oxidoreductase flavoproteins are a
familiar assemblage of enzymes. Therefore, it may be expected that their ability to
biodegrade GTN may occur widely in the biosphere (Marshall and White 2001).
The distinct physiological role of OYEs is as yet unknown; however, based on the
broad substrate specificity of several members of this family, it is suggested that
they do not have a single physiological substrate in vivo (Fitzpatrick et al. 2003). It
is hypothesized that this family of enzymes is involved in general stress response,
helping to maintain the redox state of the cell (Husserl 2011).
Five different enzymes capable of denitration have been studied and some of
their crystal structures have been determined (Blehert et al. 1997, 1999; Snape
et al. 1997; Williams and Bruce 2002; Fitzpatrick et al. 2003; Marshall et al. 2004;
Williams et al. 2004). These enzymes are all oxidoreductases which use NADPH
as a reducing agent, removing nitro groups from GTN and releasing nitrite
(Blehert et al. 1997; French et al. 1998; Husserl 2011). Several reports state that
these enzymes are regioselective and therefore produce different ratios of meta-
bolic intermediates. In most cases, both DNG isomers are produced, although
selectivity for either C1 or C2 has been observed (Blehert et al. 1997; Snape et al.
1997; Husserl 2011). A number of flavoproteins capable of excising the first nitro
group from GTN have been identified and characterized (Marshall et al. 2004).
Enzymes include pentaerythriol tetranitratereductase (Onr) from Enterobacter
clocae (French et al. 1996), NerA from A. radiobacter (Snape et al. 1997; Marshall
et al. 2004), YqjM from Bacillus subtilis (Fitzpatrick et al. 2003), and reductases
XenA and XenB from P. putida and P. fluorescens (Blehert et al. 1997).
Meng et al. (1995) examined a potential GTN treatment strategy involving
bacterial enzymes. Resting cells of B. thuringiensis/cereus and E. agglomerans
denitrated GTN with the formation of nitrite (Meng et al. 1995). Denitration
activities were expressed constitutively in both isolates, and GTN was not required
for enzyme induction. Dialysis of cell extracts did not affect denitration which
demonstrates that dissociable and depletable co-factors are not required. In long-
term studies with excess cell extract, the isolates had the ability to completely
convert GTN to glycerol; however, continuous addition of cell extracts was nec-
essary (Meng et al. 1995). Husserl (2011) identified an OYE homolog in Arth-
robacter sp. strain JBH1 which denitrates GTN and is capable of selectively
producing 1-MNG. A glycerol kinase homolog transformed 1-GMN into 1-nitro-3-
phosphoglycerol which could be later introduced into a broader metabolic pathway
where the last nitro group is removed. In the overall process, GTN is converted to
CO2 and biomass and some of the nitrite released is incorporated into biomass.
54 J. Pichtel
To be practical for treatment of GTN-affected media, denitration enzyme(s) must

have simple co-substrate requirements, since few NAD(P)H- or ATP-requiring
enzymes are industrially useful (Meng et al. 1995). Tan-Walker (1987) proposed
that hydrolytic activities were responsible for GTN denitration. A hydrolytic path-
way has the advantage of simple co-substrate requirements which facilitate the
development of an enzymatic strategy for transforming GTN-containing wastes.
6.2 Bioremediation of GTN
6.2.1 Bioslurries, Packed Bed Reactors, and Soil Columns
Bioslurry or activated sludge treatment of GTN applies the principles of operation

of conventional municipal wastewater treatment. Contaminated liquid effluent, soil
or sediment is transferred from the affected site to a reactor (man-made or natural,
e.g., lagoon) and water is added as necessary. Influent water may be pre-treated to
remove excess concentrations of Fe or other potentially interfering elements.
Solution pH should be adjusted to the circumneutral range in order to optimize the
availability of nutrients (e.g., P and trace elements). The system can be amended
with macronutrients (N, P, K), micronutrients (Cu, Co, Mn, Zn) and other sup-
plements, such as surfactants, to promote microbial activity and GTN degradation.
In some cases, microbial inocula may be added. The slurry is then mixed con-
tinuously to promote aeration, suspension of particles, and breaking up of
agglomerations (Fig. 4). A portion of the sludge, presumably enriched in
GTN-degrading organisms, may be recycled into the aeration compartment.
Fig. 4 Schematic diagram of a bioslurry reactor. Source: Fundamentals of site remediation, 2nd
ed. J. Pichtel 2007. Rowman & Littlefield Pub
During soil slurry incubations, no biodegradation of triple (M3 IAIEl) and double
base (NOSIH-AA2) propellants was observed by Adrian (1996). The GTN com-
ponent of both propellants was degraded in both experimental and sterile control
bottles in more than one week, suggesting an abiotic mechanism as being
responsible for degradation. When an additional electron donor was added to the
reaction medium, 62 % of nitroguanidine was biodegraded under methanogenic
conditions. Adrian (1996) has concluded that biological treatment processes have
only a limited role in disposing of production grade propellants.
Using slurry reactors, Yost (2004) determined the fate of GTN in a surface soil
and an aquifer soil spiked with GTN. GTN degradation was examined under
aerobic and anaerobic conditions at three pH levels. Degradation was independent
of soil carbon content and supplemental carbon inputs. Radiolabeled 14C-GTN
studies indicated persistence of unidentified GTN constituents (Yost 2004), pre-
sumably GDN and/or GMN. Nitroglycerin remained in solution at pH 6 under
aerobic conditions in both soils. This may be a cause for concern as regards
persistence of GTN in contaminated soils of acidic regions in the Pacific Coastal,
Atlantic and Southeastern states.
A trickling filter (packed bed or fixed film reactor) consists of a bed of coarse
materials such as stones or plastic media, over which contaminated water is slowly
applied. Trickling filters are commonly employed by municipalities for treating
wastewater for BOD removal. A typical design consists of a bed of stones placed
from 1 to 3 m deep in a large diameter basin (Fig. 5). The stones provide sub-
stantial surface area for microbial attachment as they metabolize the organic
contaminants. The flow from the filter is passed through a sedimentation basin
(secondary clarifier or final clarifier) to allow solids to settle out (Pichtel 2007).
Reactor columns packed with 1/6-inch plastic flakes resulted in high rates of
GTN decomposition (Bhaumik et al. 1997). Anaerobic reactors resulted in higher
conversion efficiency and at lower co-substrate requirements than did aerobic sys-
tems. These are, therefore, preferable in field applications to aerobic systems which
Fig. 5 Trickling filter

system for treatment of
contaminated water.
Source: Fundamentals of site
remediation, 2nd ed.
J. Pichtel 2007. Rowman &
Littlefield Pub
56 J. Pichtel
have substantial co-substrate requirements and greater capital and operating costs
due to requirements for aeration (Bhaumik et al. 1997). The authors suggest that
performance of packed bed reactors can be improved by optimizing co-substrate
concentration and retention time. BOD is usually present in sufficient quantities
and addition of co-substrates would not be required in commercial applications of
the system. Using columns packed with soil Clausen et al. (2010) observed a
decrease in GTN concentrations indicating biologically mediated degradation.
However, initial GTN concentrations were low (1 mg/l) and no mass balances or
biodegradation indicators were used. Therefore, it could not be concluded whether
GTN biodegradation rates in porous soil are sufficiently high for bioremediation to
be considered a viable treatment option. Using columns packed with field soil,
Asbaghi and Pichtel (2012) found that GTN was degraded significantly (p \ 0.05)
more rapidly in non-autoclaved compared to autoclaved soils.
Using porous soil column systems, Husserl (2011) found that Arthrobacter
JBH1 was capable of growing on GTN at pH values as low as 5.1 and at GTN
concentrations as high as 1.2 mM (Husserl 2011). The author proposed that bio-
augmentation with this strain could result in complete mineralization in GTN-
contaminated soil and sediments without addition of other carbon sources. The
presence of other explosive contaminants including trinitrotoluene and 2,4-dini-
trotoluene lowered GTN degradation rates.
6.2.2 Composting
Many studies have used composting to treat soils contaminated with TNT, RDX
and HMX (Isbister et al. 1984; Lowe et al. 1989; Griest et al. 1990; Williams et al.
1992; Pennington and Brannon 2002). Methods of composting include static piles,
windrow, and in-vessel systems. During field-scale composting, contaminated soil
is excavated and transferred to an impervious surface equipped with means of
collecting drainage and runoff. Contaminated soil is mixed with bulking agents
such as wood shavings, straw, hay, etc., following which amendments such as
livestock manure, food waste, or municipal solid waste are added. In turned pile
systems, the mixture is aerated by agitating the solids to promote decomposition of
contaminants. In static pile systems, perforated tubing (typically PVC) is inserted
within the compost mass. Air can be either drawn in by vacuum, or blown out
(Pichtel 2007). During 45-day compost incubations using bench-scale reactors, no
biodegradation of triple (M3 IAIEl) and double base (NOSIH-AA2) propellants
was observed by Adrian (1996). The only discernible change to the propellant was
a slight discoloration of particle surfaces.
In our laboratories, we are investigating the ability of both aerobic thermophilic
composting and vermiculture (worm composting) using Lumbricus rubellus (red
wiggler fishing worms) in decomposing GTN in double base smokeless powder.
Preliminary data indicate that thermophilic composting is significantly more rapid
in decomposing GTN as compared with vermiculture. Furthermore, in many sit-

uations GTN appears to be toxic to worm culture even at very low concentrations.
6.2.3 Phytoremediation
Phytoremediation is defined as the engineered use of green plants to treat con-

tamination in soil, sediment, and groundwater. The method is considered an
environmentally conscious, cost-effective, and aesthetically appealing alternative
to traditional soil remediation techniques. Plants remove contaminants from soil
and water through various mechanisms; certain species take up contaminants and
store them in roots and/or shoots. Certain compounds, typically low molecular
weight organics, may be released through the stomata. Plants may be employed in
rhizofiltration where roots sorb and/or precipitate contaminants. Plants capable of
using enzymes to uptake and subsequently transform contaminants into innocuous
compounds, are used in phytodegradation. In rhizodegradation, plants assist soil
microorganisms by providing a habitat—the rhizosphere—which creates favorable
conditions for solubilization and decomposition of contaminants (USEPA 1999).
Rhizospheric Degradation of Nitroglycerin
The rhizosphere is the biologically and chemically active zone directly adjacent to
the root where soil microorganisms are strongly influenced by the presence of root
exudates (Brady and Weil 2009). The rhizosphere has been documented to contain
a high diversity of microbial types as well as number of microorganisms compared
to non-vegetated (i.e., ‘‘bulk’’) soil. Many of the microorganisms and enzymes
associated with decomposition of nitroesters occur in the rhizosphere.
Yellow nutsedge (Cyperus escalantus) and common rush (Juncus effuses) took
up GTN from hydroponic culture and incorporated GTN into biomass. Although
yellow foxtail (Setaria glacula) did not accumulate GTN, the authors proposed
that this plant transformed GTN through enzymatic processes (Reifler and Medina
2006). Flax seed (Linimumus itatissimum) cell cultures accumulated and trans-
formed GTN in wastewater within a 24-day period into 1,2-DNG and 1,3-DNG
(Podlipna et al. 2008). Perennial ryegrass (Lolium perenne) root exudates trans-
formed GTN into GDN in the rhizosphere, following which it was taken up into
roots and shoots (Rocheleau et al. 2011). Goel et al. (1997) demonstrated that
sugar beet (Beta vulgaris) cellular extracts degraded GTN to DNG and MNG
through a PETN reductase enzyme. To encourage nitroester decomposition in a
plant already known to be a viable phytoremediation species, French et al. (1999)
cultivated tobacco (Nicotiana tabacum) plants genetically modified to express the
PETN reductase enzyme gene. These plants degraded nitroesters at levels beyond
the capacity of wild varieties of tobacco.
58 J. Pichtel
160
NG (mg/kg) plant tissue

140
120
100
80
60
40
20
0
0 1 5 10
SP rate (%)
Oat Oat CB Sedge Sedge CB
Fig. 6 Uptake of GTN by oat (Avena sativa) and sedge (Carex vulpinoidea) CB = composted
biosolids
In our laboratories, we are examining the feasibility of rhizosphere-enhanced

phytoremediation for the removal of GTN as applied in commercial smokeless
powder (Asbaghi and Pichtel 2012). Double base smokeless powder was applied to
soil at rates as high as 5 % (w/w), and mesocosms were cultivated with oats
(Avena sativa) and sedge (Carex vulpinoidea). Microbial activity in the rhizo-
sphere was found to be a major contributor to GTN decomposition. Only modest
quantities of GTN removal could be accounted for by abiotic processes, such as
sorption. Nitroglycerin decomposition by photolytic processes was observed;
however, this effect is considered to be a minor contribution to GTN removal. Soil
bacterial numbers remained relatively constant regardless of the rate of smokeless
powder application. Plant uptake of GTN increased with dosage rate (Fig. 6), but,
the overall effect of plant uptake to GTN removal from soil was minimal.
Amendment of soils with composted biosolids imparted a positive effect on GTN
decomposition and/or removal from soil.
Plant Selection for Phytoremediation
Plant species are selected for phytoremediation projects based on site character-
istics and their ability to remove or decompose the contaminants present. Plants
must be capable of surviving on site by adapting to climatic factors and local soil
characteristics. Preferred plant attributes include ability to uptake or transform
contaminants, rapid growth, production of large quantities of biomass, ease of
maintenance, and overall hardiness in local conditions. Plants with dense, fibrous
root structures, such as grasses, are often preferred. Plants with nitrogen-fixing
capabilities, such as legumes, are desirable since they usually require less input of
fertilizers and are less likely to compete with soil microorganisms for nutrients
(USEPA 2001).
7 Conclusions
Review of the published literature reveals numerous gaps in our knowledge of

GTN biodecomposition. Various microbes are known to utilize GTN as a sole
nitrogen source, i.e., A. radiobacter, Enterobacter cloacae, P. putida, P. fluores-
cens, Klebsiella, Rhodococcus, G. candidum, P. chrysosporium, and possibly
others. This clearly indicates that the ability to biodegrade GTN is widespread in
the biosphere. Nitroglycerin degradation has been successful in the laboratory
using natural and inoculated organisms under both aerobic and anaerobic condi-
tions with and without the addition of supplemental carbon sources. Microbes were
isolated from laboratory culture media, digester sludge, wastewater from nitro-
glycerin manufacturing plants, contaminated ammunition facility soil and waste-
water lagoon soil. In only limited cases were field soils used as test media. Hence,
there is a need for more comprehensive study of GTN reactions in soil material.
This includes soil fractured as a result of detonations, whose surface properties
will differ markedly from that of natural soil (Douglas et al. 2009). Nitroglycerin
may persist longer in oxidized than in reduced environments. However, investi-
gation reveals that it will degrade to undetectable levels under both conditions.
Many studies have concentrated on the fate of nitroglycerin alone in solution
form. However, the contamination found on military installations is most likely not
GTN alone in soluble form, but GTN occurring within a complex propellant
formulation. Combined with propellant ingredients, GTN biotransformation
reactions may be altered, resulting in a more complex and persistent compound.
Given the toxic and recalcitrant nature of many energetic compounds, for example
TNT, RDX and HMX, their presence at sites being considered for cleanup by
bioremediation may lower GTN degradation rates, and could potentially result in
GTN recalcitrance. Research is needed to determine rates of GTN decomposition
in the presence of co-contaminants, in particular TNT, RDX, HMX and various
propellants (e.g., dinitrotoluene, nitroguanidine, nitrocellulose, perchlorate).
GDN and GMN may inadvertently bypass treatment in conventional biological
systems such as activated sludge. Since these intermediate biotransformation
metabolites are more water soluble than the parent compound, they can migrate
faster with a higher potential to reach groundwater. Being mutagenic (Ellis et al.
1978), they must be completely converted to glycerol if GTN is to be successfully
treated on the industrial scale. The performance of any GTN treatment method
must, therefore, be assessed according to its ability to completely degrade the
nascent metabolites. Since rates of conversion are reduced with each denitration
step, it is necessary that the construction of a biological treatment process be based
on rate of accumulation and disappearance of the mononitrate isomer (Bhaumik
et al. 1997). Furthermore, it is well established that nitrite, a product of GTN
denitration, is toxic to cells at high concentrations through a variety of mecha-
nisms. Nitrite concentrations must be monitored to ensure minimal impact on
microbial populations.
60 J. Pichtel
If an enzyme system which denitrates GTN to yield glycerol and nitrate could
be identified and developed at the industry scale, issues regarding its toxicity,
recalcitrance, and explosive capabilities may be lessened. Microbiological treat-
ment of toxic compounds such as GTN suffers from fluctuation and instability
when microbial populations are subjected to shock loading. Shock loading is rather
common during routine treatment of wastes resulting from GTN manufacture,
because production processes are typically operated in a batch or semi-continuous
mode, with wastes being generated irregularly (Meng et al. 1995).
Little has been published on the optimal environmental (field) conditions for
organisms known to decompose energetic materials. It is essential to understand the
effect of soil pH, redox potential, nutrient levels, presence of interfering com-
pounds, etc., in order to optimize bioremediation of nitroglycerin-impacted soils.
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Bioremediation of Nitroexplosive
Waste Waters
Pradnya Pralhad Kanekar, Seema Shreepad Sarnaik,

Premlata Sukhdev Dautpure, Vrushali Prashant Patil
and Sagar Pralhad Kanekar
1 Introduction
Explosives, particularly nitro explosives, are synthesized globally and are high
energy materials, consisting of elements like carbon, hydrogen, oxygen, and
nitrogen. When subjected to any stimuli, they can undergo a very rapid, self-
propagating, and exothermic decomposition reactions, resulting in the formation of
more stable materials like CO2, H2O, and N2 with the development of sudden high
pressure.
Among modern explosives, many are polynitroaromatic compounds. In the
early 20th century, scientists had developed more than 60 highly explosive com-
pounds, like Glycerol trinitrate (GTN), Pentaerythritol tetra nitrate (PETN),
Trinitrotoluene (TNT), Royal Demolition Explosive/Research Department
Explosive (RDX, hexogen, cyclonite), High Melting Explosive (HMX, octogen)
etc. Recently developed nitroexplosives, such as Triaminotrinitrobenzene (TATB),
Diaminodinitroethylene (FOX-7) and CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,
8,10,12-hexaazaisowurtzitane) have been not studied in details, whereas among
most commonly used nitroexplosives like Trinitrotoluene (TNT), Royal Demoli-
tion Explosive (RDX) and High Melting Explosive (HMX), TNT and RDX have
been investigated in details for microbial degradation. Chemical structures of some
nitroexplosives are illustrated in Fig. 1.
P. P. Kanekar (&) S. S. Sarnaik P. S. Dautpure V. P. Patil S. P. Kanekar

Microbial Sciences Division, MACS-Agharkar Research Institute, G.G.Agarkar Road, Pune,
Maharashtra 411004, India
e-mail: kanekarpp@gmail.com

68 P. P. Kanekar et al.
Fig. 1 Chemical structures of some nitroexplosives TNT, RDX, HMX, TATB and FOX-7
2 Application of Nitroexplosives
The nitroaromatic and nitramine explosives are used in the military applications,
such as buster charges for artillery shells and component of solid fuel rocket
propellants to implode fissionable material in nuclear devices (Yinon 1990). Both
RDX and HMX are more energetic and stable than TNT which is used in both
conventional and nuclear weapons.
3 Toxicity of Nitroexplosives
Nitroaromatic compounds are pervasive pollutants whose toxicity is well docu-

mented. Toxicity of the nitroaromatics is manifested in symptoms as irritation of
the digestive tract, methaemoglobinaemia, disturbed heart function, kidney trou-
ble, dysfunction of the whole vascular system and severe jaundice. The vasodi-
lating, cardio-vascular and toxic effects of these compounds can be linked to the
formation of nitric oxide, a biologically active free radical species (Karmarkar
et al. 2000). In general, toxicity of explosives and other nitroaromatic compounds
is well described by Kanekar et al. (2003). From the laboratory studies, TNT, RDX
and HMX were found to be toxic to bacteria, algae, plants, earthworms, aquatic
invertebrates and animals, including mammals and humans (Aken et al. 2004b).
RDX, formerly used as a rat poison, is classified as a possible human carcinogen
(Class C), while HMX is listed as a contaminant of concern and classified as Class
D carcinogen by the EPA. The permissible limits of most commonly used
explosives in drinking water laid down by US EPA are described in Table 1.
Nitramine per se may not cause severe toxicity, but the metabolic intermediates
derived from the nitramine may be highly toxic. Therefore, it is important not only
Table 1 Permissible limits of nitroexplosives in drinking water

Explosive Permissible limit (mg/l) Reference
TNT 0.049 Alnaizy and Akgerman (1999)
RDX 0.002 Aken et al. (2004b)
HMX 0.04 Zoh and Stenstrom (2002)
Bioremediation of Nitroexplosive Waste Waters 69
to assess the environmental fate of nitramines, but also to determine the nature of
the products of biotransformation or biodegradation or both (McCormick et al.
1981), because interconversions between the two may involve the production of
the corresponding nitroso and hydroxyl amino derivatives which are known to be
more toxic than the parent molecules. Polycyclic nitroaromatic compounds are not
very toxic or carcinogenic, but can be activated not only by reduction of the nitro
group by intestinal microflora, but also by mammalian cyctochrome P-450 med-
iated by oxidation of the aromatic ring (Spain 1995). In humans, RDX affects
primarily the central nervous system as well as renal and gastrointestinal systems
(Ronen et al. 1998).
Since production of all nitroexplosives involves the nitration process using
concentrated nitric acid, the wastewater generated is highly acidic in nature and
contains very high concentration of nitrates along with traces of explosives and
other nitro compounds. Hence, contaminated wastewater shows diverse effects on
the living organisms. The nitroexplosives as well as their intermediate products are
very toxic, showing symptoms of methaemoglobinaemia, kidney trouble, jaundice
etc. in humans. Therefore, it is necessary to remove these compounds from the
waste water.
4 Environmental Contamination by Nitroexplosives
Nitroexplosives are highly energetic materials, but essentially needed for the
security and defense of any nation, hence their production is unavoidable. Pro-
duction, use and recalcitrance of explosives over a long period, have led to their
environmental persistence which is a serious concern. The main sources of con-
tamination of land as well as marine and ground water sources due to explosives
are handling, military training, dumping of huge amounts of unexploded ordnance,
ordnance waste disposal by open burning/open detonation, land mines, commer-
cial use of explosives in propellants and mining, effluents from explosive manu-
facturing plants and now-a-days, their rampant use by terrorists (Karmarkar et al.
2000; Fournier et al. 2002; Adrian et al. 2003; Bhushan et al. 2004).
In order to protect the environment from pollution due to nitrates and residual
explosives, it is necessary to study the degradation of nitro explosives. Although a
majority of the nitroexplosives are sparingly soluble in water near neutrality, the
intermediate metabolites may have higher solubility than the final product. In
general, during production of nitroexplosives, nitration is carried out by using
nitric acid and hence, the pH of the wastewater generated is highly acidic.
Therefore, there are fair chances of solubility of nitroexplosives in highly acidic
wastewaters.
Contaminated groundwater, processed water from closed military bases and
production plants and weapon dismantling facilities inflate the expanse of the
treatment needs of explosives in contaminated soils. The concentrations of
explosives in contaminated soil are extremely heterogeneous, ranging from 0.7 to
74,000 mg/kg for RDX, from 0.7 to 5,700 mg/kg for HMX and from 0.08 to
87,000 mg/kg for TNT (Best et al. 2006). Since recreational waters are within
3 miles downstream from the plant, their concentrations exceeding health per-
missible limits have been detected in the groundwater used for the human con-
sumption (George et al. 2001). A single TNT manufacturing plant can generate
over 1.8 mega liters of waste waters per day (Halasz et al. 2002).
5 Different Processes for Treatment of Waste Waters
Rodgers and Bunce (2001) have described various methods used for remediation
of nitroaromatic explosives. These can be of three types viz. physical, chemical
and biological. Physical and chemical methods are neither cost-effective nor
environment-friendly. Therefore, biological methods employing microorganisms
and plants are preferred over physical and chemical methods.
5.1 Biological Methods
The biological degradation could be stated as breaking of organic matter present in

waste water by microorganisms to simpler forms which will not further decompose
any more. The degradation can be achieved by both aerobic and anaerobic
bacteria.
The biological methods are eco-friendly treatment methods which include the
use of natural occurring organisms like microorganisms, plants etc. Microbes are
known to have evolved diverse degradative pathways mediated by various
enzymes to mineralize specific nitro compounds. A few of them are able to use
nitroaromatic compounds as N and/or C and energy source. The high nitrogen
content of nitramines suggests that there are potential nitrogen sources for the
microorganisms. Hence, efforts were made to isolate microorganisms capable of
degrading nitroaromatic and nitramine pollutants (Binks et al. 1995; Rodgers and
Bunce 2001).
Microorganisms are known to degrade nitroaromatic compounds in both aer-
obic and anaerobic conditions. Under aerobic conditions, removal or effective
metabolism of nitro groups can be accomplished by four different strategies viz.
(a) some bacteria can reduce the aromatic ring of dinitro and trinitro compounds
by the addition of a hydride ion to form a hydride-meisenheimer complex which
subsequently rearomatizes with the elimination of nitrite; (b) monooxygenase
enzymes can add a single oxygen atom and eliminate the nitro group from ni-
trophenols; (c) dioxygenase enzymes can insert two hydroxyl groups into the
aromatic ring and precipitate the spontaneous elimination of the nitro group from a
variety of nitroaromatic compounds; (d) reduction of nitro group to the corre-
sponding hydroxylamine is the initial reaction in the effective metabolism of
nitrobenzene, 4-nitrotoluene, and 4-nitrobenzoate. The hydroxylamines undergo

enzyme-catalyzed rearrangements to hydroxylated compounds that are substrates
for ring-fission reactions. In anaerobic mechanism, anaerobic microorganisms
reduce the nitro group via nitroso and hydroxylamino intermediates to the corre-
sponding amines (Spain 1995).
6 Aerobic Degradation of Nitroexplosives
The first investigation into the possibility of degrading nitro compounds by bio-
logical methods was carried out by Erikson (1941) wherein nitro compounds, like
nitrobenzene, picric acid and trinitro resorcinol, were found to be used as the
nutrients by some actinomycetes. This observation was later confirmed by Moore
(1949) and Rogovskaya (1951). Simpson and Evans (1953) reported degradation
of nitrophenols by Pseudomonas sp., while Jensen and Gundersen (1955) found
nitrophenols to be degraded by Corynebacterium.
Bacterial degradation and fungal transformations of TNT have been described
by a number of researchers. Osman and Klausmeier (1972) reported degradation of
TNT using sewage effluent, pond water, soil suspension etc. and also by a pure
culture of Pseudomonas aeruginosa in presence of glucose. Won et al. (1974)
studied metabolic decomposition of a-2,4,6-Trinitrotoluene by Pseudomonas sp.
Traxler et al. (1975) demonstrated ring cleavage by Pseudomonas sp. during
degradation of a-TNT. McCormick et al. (1976) have reported reduction of a-TNT
by hydrogen in presence of enzyme preparations from the anaerobic bacterium
Veillonella alkalescens. However, Parrish (1977) reported fungal transformation of
a-TNT. Kanekar and Godbole (1983, 1984) have also extensively studied bio-
degradation of a-TNT. Binks et al. (1995) isolated a number of microbes that were
able to degrade nitroaromatic and nitramine pollutants.
Biological degradation of cyclic nitramines under aerobic conditions is scantly
reported. Aerobic degradation studies, using RDX as a nitrogen source, led to the
isolation of 3 Rhodococcus strains and a strain of Stenotrophotromonas malto-
philia (Binks et al. 1995). Another aerobic Rhodococcus sp., strain DN22, isolated
from the soil of a site of munitions manufacture and storage, was found to grow
exponentially in minimal medium containing RDX as the sole nitrogen source.
Resting cells, grown on RDX, showed the highest degradative activity, compared
to cells grown on alternative nitrogen sources. This indicated that the RDX deg-
radation system was inducible (Coleman et al. 2002). Toze and Zappia (1999)
developed microcosms to determine the ability of microorganisms to degrade
munition compounds, such as TNT, dinitrotoluenes, nitrotoluenes and RDX from
the wastewater. They observed 76 % removal of TNT and 94 % removal of RDX
within 45 days of incubation.
Aerobic biodegradation of HMX has been described by Spanggord et al. (1983).
Morganella morganii, Providencia rettgeri and Citrobacter frundii belonging to
the family Enterobacteriaceae were isolated by Kitts et al. (1994) from the
explosive contaminated soils to study their ability to transform HMX. Reardon

(1992, 1994) carried out work on immobilized cell bioreactors for biodegradation
of 2,4-dinitrotoluene. Pinar et al. (1997) demonstrated use of Klebsiella oxytoca
isolate 15 for the removal of nitrates from industrial wastewaters generated during
production of dinitroethylene glycol. The culture was isolated from the soil of
nitration factory and was able to tolerate nitrates at the concentration of 0.5–1.0 M.
Boopathy et al. (1998) reported the metabolism of TNB, RDX and HMX by sulfate
reducing bacterial consortium of Desulfovibrio spp., where bacteria used explo-
sives as a sole source of nitrogen.
Fungi can degrade organic compounds using several enzymes, such as perox-
idases that are known to catalyze a number of free radical reactions. The elec-
tronegative -NO2 group in RDX readily accepts a free electron to form an anion
radical (Spain et al. 2000). White rot fungus Phanerochaete chrysosporium
degraded RDX in liquid cultures, generating CO2 and N2O with traces of MNX
(mononitroso derivative of RDX) after 60 days (Sheremata and Hawari 2000).
Fournier et al. (2004a) employed a sequential treatment of bacteria, followed by
fungus for nitramine degradation. Subsequent incubation of the soil with the
fungus Phanerochaete chrysosporium led to the removal of NDAB with the lib-
eration of N2O. In cultures with the fungus alone, NDAB was degraded to release
N2O. The production of 14CO2 increased from 30 to 76 % in this process. Deg-
radation of HMX by Phanerochaete chrysosporium was also studied by Fournier
et al. (2004b). Fungal isolates, like Acromonium, Penicillium, Rhodotorula and
Bullera, were studied by Bhatt et al. (2006) for degradation of both RDX and
HMX.
Dautpure (2007) isolated Providencia rettgeri MCM B-437 from the soil col-
lected from the sites exposed to HMX, using enrichment technique and found it
capable of removing appreciable amounts of HMX from a synthetic medium.
Optimization of environmental parameters revealed that P. rettgeri showed better
degradation of HMX in a synthetic medium (C:N ratio 65:1) completely devoid of
any nitrogen source other than HMX. Besides, P. rettgeri showed better degra-
dation efficiency (75 % removal of HMX in 6 days) than the earlier report by Kitts
et al. (1994). However, an inhibition of HMX degradation in presence of RDX had
been a general observation. In spite, P. rettgeri could degrade HMX in the pres-
ence of RDX. There is an advantage with MCM B-437, as it has a potential for the
remediation of contaminated site with HMX and RDX together. The detection of
nitramine product (m/z 331) during HMX degradation by P. rettgeri indicates a
ring cleavage route for HMX degradation.
Patil et al. (2011) isolated microorganisms from the soil contaminated with
diaminodinitroethylene (FOX-7) containing waste water from High Energy
Materials Research Laboratory (HEMRL), Pune. Out of 8 microbial cultures, four
isolates (3 actinomycetes and 1 bacterium) showed 20–40 % removal of FOX-7 at
the initial concentration of 500 mg/l incorporated in Davis Mingioli synthetic
(DMS) medium within 96 h at ambient temperature (28 ± 2 °C) in an orbital
shaker. Based on morphological characteristics, three isolates of actinomycetes
Fig. 2 FOX-7 utilizing

strain of Streptomyces sp.
showing spores in chains
after 48 h of incubation on
GYP medium at ambient
temperature
were tentatively identified as Streptomyces sp. (Fig. 2) and one isolate as Micro-
coccus sp.
Biodegradation of Triaminotrinitrobenzene (TATB) has been also reported
(Anonymous 2010–2011). Microbial cultures were isolated from the soil samples
collected from premises of TATB production unit at HEMRL, Pune. Five isolates
(2 bacteria and 3 actinomycetes) could remove TATB and nitrate in the range
11–37 and 11–48 %, respectively from Davis Mingioli’s synthetic (DMS) medium
supplemented with 0.05 % peptone and TATB at the initial concentration of
100 mg/l. The bacterial isolates were identified as Enterobacter cloacae complex,
Escherichia harmanii and Streptomyces sp. as an actinomycete. These isolates
could use TATB as a source of nitrogen in the presence of acetate as a carbon
source and could remove TATB in the range of 20–30 %.
7 Metabolic Pathways of Biodegradation
Biodegradation pathways for a few of the nitroexplosives have been described in

the literature. Davis et al. (1997) reported the degradation of TNT and TNB by
Pseudomonas vesicularis isolated from soil and detected the major metabolites as
dinitrobenzene, nitrobenzene, nitroaniline etc. Kroger et al. (2004) studied the
biological reduction of TNT as a part of mineralization through formation of
aminodinitrotoluene (ADNT) and diaminonitrotoluene (DANT). Degradation of
the aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB) by Methylobacterium sp.
strain JS178 was reported by Fournier et al. (2005). NDAB is a ring cleavage
metabolite that accumulates during aerobic degradation of RDX by Rhodococcus
sp. However, this product is also formed during alkaline hydrolysis of either RDX
or HMX and photolysis of RDX. Mineralization of RDX by strains of aerobic
bacteria Gordonia and Williamsia sp. was reported by Thompson et al. (2005).
Tront and Hughes (2005) reported oxidative microbial degradation of 2,4,6-tri-
nitrotoluene (TNT) with the detection of 3-methyl-4,6-dinirocatechol as an
intermediate using 14C TNT and its mineralization to CO2. Sherburne et al. (2005)
demonstrated the cleavage of triazine ring of RDX through formation of nitrous
oxide by Acetobacterium paludosum under anaerobic conditions. A complex
nature of reductive transformation of TNT by Cellulomonas sp. strain ES6 in the
presence/absence of ferrihydrite and anthraquinone-2, 6-disulfonate was demon-
strated by Borch et al. (2005). Crocker et al. (2006) have reviewed the biodeg-
radation pathways for cyclic nitramine explosives RDX, HMX and CL-20.
Bacterial pathways for degradation of nitroaromatic compounds have been
reported by Symons and Bruce (2006). Dautpure (2007) has also proposed a
pathway of degradation of HMX by Providencia rettgeri. The studies indicated
direct ring cleavage pathway based on the metabolites detected by LC–MS.
The metabolism of nitroaromatic compounds using chemotaxis of Ralstonia sp.
SJ 98 was studied in details by Samanta et al. (2000) and Pandey et al. (2002). The
enzyme system, having a role in the degradation of DNB with the formation of
intermediate metabolites, was demonstrated by Dey (2002). The intermediate
metabolites were formed during degradation of o-nitrobenzoate by Arthrobacter
protophormiae RKJ100 (2003). Jain et al. (2004) studied the transformation of
2,4,6-trinitrotoluene by a marine yeast isolate Yarrowia lipolytica NCIM 3589.
Ningthoujam (2005) isolated Brevibacterium linens strain from the garden soil
which was capable of degrading P-nitrophenol at the concentration of 300 mg/l in
presence of yeast extract. Transformation of different nitroaromatic compounds
viz. o-nitroaniline, m-nitrotoluene, 2,4,6-trinitrotoluene and o-nitrophenol by
Acinetobacter juinii AB under aerobic conditions was reported by Soojhawon
et al. (2005). They observed an induction of bacterial oxidase systems, such as
cytochrome P450, aminopyrine N-demethylase, acetanilide hydroxylase and glu-
tathione-s-transferase. Biodegradation of nitrophenol and other nitroaromatic
compounds by Arthrobacter protopharmiae RKJ100 was worked out in details
(Pandey et al. 2003; Labana et al. 2005a, b).
8 Detoxifying Enzymes Involved in Biodegradation
Kanekar et al. (2003) have reviewed the biodegradation of nitroexplosives by both

aerobic as well as anaerobic microbes involving removal or productive metabolism
of nitro groups. However, only a few reports are available on the enzymes
involved in degradation of nitroexplosives. They have stated that aerobic degra-
dation involves presence of enzymes, such as monooxygenases and/or dioxygen-
ases which can add one/two oxygen atoms and eliminate the nitro group from a
number of nitroaromatic compounds. A reduction of nitro compounds by a hydride
ion forms a hydride-Meisenheimer complex which subsequently rearomatizes with
the elimination of nitrite or reduction of the nitro group to the corresponding
hydroxylamine. The enzyme systems, having a role in the degradation of DNB
with the formation of intermediate metabolites, was also demonstrated by Dey
(2001). Anaerobic degradation involves a reduction of nitro group via nitroso and
hydroxylamino intermediates to the corresponding amines by the anaerobic

microorganisms (Spain 1995).
Degradation of nitroexplosives by the enzymes, like nitroreductases, manga-
nese peroxidases, laccases etc., are reported by various workers (Binks et al. 1995;
Spain 1995; Sheremata and Hawari 2000; Rodgers and Bunce 2001). Under aer-
obic conditions, cytochrome P450 enzyme is known to accept polynitro com-
pounds as electron acceptors. Degradation of nitro explosives by the reductive
pathway could be due to non-specific nitroreductase enzymes present in both
aerobic and anaerobic organisms (Rodgers and Bunce 2001). Since nitroaromatic
compounds containing only nitro groups are not the direct substrates for lignin
degrading enzymes (e.g., lignin peroxidase and/or manganese peroxidase), these
enzymes reduce the aromatic nitro group to an amine, resulting in the degradation
of nitroaromatic compounds (Sheremata and Hawari 2000). Some of the enzymes,
like nitroreductases, manganese peroxidase, laccases etc., are also reported to be
involved in the degradation of nitroaromatic compounds (Rodgers and Bunce
2001; Coleman et al. 2002).
Biodegradation of nitroaromatic compounds generally involves reduction of
nitro functional groups or some times cleavage by the enzymes. Biotransformation
of RDX by NAD(P)H-nitro oxidoreductase was studied by Bhushan et al. (2002)
under anaerobic conditions at pH 7 and at 30 °C. They also reported formation of
hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine
as the intermediate metabolites detected by LC/MS analysis with the formation of
nitrous oxide, formaldehyde and ammonium ions as end products. Kutty and
Bennett (2005) have given an account of biochemical characterization of TNT
transforming oxygen-insensitive nitroreductases NitA (Mol. Wt. 31 kDa) and NitB
(Mol. Wt. 23 kDa) by Clostridium acetobutylicum ATCC 824.
Involvement of cytochrome P450 in the metabolism of nitro explosives, like
RDX and TNT is reported by several workers. Bhushan et al. (2003) studied the
cleavage of RDX using rabbit liver cytochrome P450 and found a similar pattern
of metabolism as observed in biotransformation by Rhodococcus sp. strain DN22.
This provides a strong evidence that a cytochrome P450 enzyme is the key enzyme
responsible for RDX biotransformation by Rhodococcus sp. strain DN22. Bhushan
et al. (2005) studied the biotransformation of CL-20 by a membrane-associated
and NADH-dependent dehydrogenase enzyme from Clostridium sp. EDB2. Torre
et al. (2006) reported the involvement of cytochrome P450 in the metabolism of
TNT in a marine organism i.e., Anguilla anguilla. Multiple enzymes, which attack
TNT, were reported to be present in Escherichia coli (Gonzalez-Perez et al. 2007).
Nitrate reductases are ubiquitous enzymes in diverse groups of microorganisms
and their physiological role is to reduce nitrate to nitrite via a two-electron transfer.
Enzymes, such as nitroreductases (types I and II), hydrolases and hydrogenases, are
implicated in the transformation of cyclic nitramines (Kitts et al. 2000; Hawari et al.
2001). Many of the facultative bacteria of Enterobacteriaceae family and some
strictly anaerobic bacteria including Providentia rettgeri degrading HMX are
reported to possess these enzyme systems (Dautpure 2007). Coleman et al. (2002)
studied the biodegradation of RDX by Rhodococcus strain DN22 which was found
to possess plasmid-borne cytochrome P450 enzyme system.
Stenuit and Agathos (2011) have given an overview on the recent advances in
the genetics and biochemistry of biodegradation of nitroexplosives with special
reference to promising enzymatic families, viz. the Old Yellow Enzyme (OYE)
family, the class VI cytochrome P450 system, and the family of nitronate mon-
ooxygenases or nitroalkane oxidases which help in evaluating and optimizing the
performance of a bioremediation process. In their review on microbial remediation
of explosives, Singh et al. (2012) mentioned about biodegradation and biotrans-
formation pathways of some explosives. They have discussed the detoxifying
enzymes, metabolism and molecular basis of degradation of the toxic compounds.
This information will be useful for developing economically feasible methods for
bioremediation of sites contaminated with toxic organic compounds.
9 Anaerobic Degradation of Nitroexplosives
Most of the studies on degradation of cyclic nitramines are focussed on RDX,

while only a few have targetted HMX. But considering the structural similarity of
the two explosives, degradation of RDX can provide as a guideline for the deg-
radation of HMX. Successful degradation of RDX is consistently reported under
both aerobic and anaerobic conditions (Light et al. 1997; Speital et al. 2001; Beller
2002). The addition of biodegradable organic carbon and phosphorus under
anaerobic condition accelerated the rate of degradation of RDX. The half-lives for
the degradation of RDX under anaerobic and microaerobic conditions were
approximately 60 days which was decreased to 40 days with addition of organic
carbon and phosphorus. Supporting studies for the anaerobic degradation are
provided by the significant biodegradation of RDX in Pantex soil slurries under
nitrate reducing conditions, but not under aerobic, sulfate- reducing, or metha-
nogenic conditions (Light et al. 1997). Pennington et al. (2001) observed that in
field conditions, HMX persisted in the surface soil where oxygen is available, but
not detected in deep aquifers, where an anaerobic environment is expected to
prevail (Monteil-Rivera et al. 2003).
In situ degradation of two nitramine explosives was favoured by the nutrient
addition. Anaerobic incubation of a mixture of RDX and HMX in the marine
sediment demonstrated improved removal of HMX when supplemented with
carbon sources, such as glucose, acetate, or citrate (Zhao et al. 2004a). However,
Acetobacterium paludosum degraded RDX fastest under anaerobic conditions,
when auxiliary growth substrates (yeast extract ? fructose) and nitrogen sources
(ammonium) were not added. Degradation of RDX was faster under autotrophic
(H2-fed) than heterotrophic conditions. Thus, an absence of easily assimilated
nitrogen sources, such as ammonium, enhances RDX degradation (Sherburne et al.
2005).
Boopathy et al. (1998) reported that sulfate reducing Desulfovibrio spp. can use
nitramine explosives as sole source of nitrogen for growth and as a result, the
concentrations of TNB (1,3,5-trinitrobenzene), RDX and HMX in the culture
media dropped from initial concentration of 25 ppm to below detection limit
(\0.5 ppm) within 18 days of incubation with concomitant production of
ammonia. This indicated that sulfate-reducing bacteria may be useful in the
anaerobic treatment of explosives-contaminated soil. Using electron donors, such
as ethanol, propylene glycol or butyrate that produce H2, stimulated the anaerobic
biotransformation of HMX and biotic breakdown of HMX (Adrian et al. 2003).
Microcosms, amended with both Fe0 filings and municipal anaerobic sludge,
mineralized RDX faster than separate treatments, resulting in 51 % 14CO2
recovery after 77 days (Oh et al. 2001). Bhushan et al. (2004) demonstrated a
chemotaxis-mediated biodegradation of three cyclic nitramine explosives CL-20,
RDX, HMX where local population of Clostridium sp. strain EDB2 first initiated
biotransformation of nitramines with the release of NO2-. Biodegradation of HMX
using enrichment cultures developed from anaerobic digester sludge under various
electron-acceptor conditions, such as sulfate reducing, nitrate reducing, ferment-
ing, methanogenic, and mixed electron accepting conditions, exhibited fastest
removal of HMX (Boopathy 2001). Degradation of HMX under anaerobic con-
ditions is also reported by some other researchers (McCormick et al. 1984; Kitts
et al. 1994; Hawari et al. 2001; Zhao et al. 2004a, b, 2007; Bhatt et al. 2005).
Nishino and Spain (2001, 2002, 2004) studied the biodegradation of nitroaromatic
compounds, especially dinitrotoluene in the anaerobic conditions.
10 Bioremediation of Nitroexplosive Containing

Waste Waters
Microbial degradation of these nitroaromatic compounds and waste water gener-

ated in their manufacture was demonstrated by some workers (Kanekar and
Godbole 1983, 1984; Dey et al. 1986). However, reports on successful treatment
techniques for the contaminated waste water from nitramine manufacturing
sources are sparse. Bioremediation of HMX wastewater, using horizontal packed
bed bioreactor (HPBBR) and a soil isolate of yeast Pichia sydowiorum, was
reported by Kanekar et al. (2009). Singh et al. (2009) have reported biodegradation
of high explosive production effluent containing RDX and HMX by denitrifying
bacteria. Ahmad et al. (2007) carried out a bench-scale treatability study using an
organic mulch—a complex organic material populated with its own consortium of
microorganisms for the treatment of RDX- and HMX-contaminated groundwater
obtained from a plume. Kusßçu and Sponza (2011) described an application of Box-
Wilson experimental design method for the treatment of synthetic wastewater
containing 2,4-dinitrotoluene (2,4-DNTA) using a sequential anaerobic migrating
blanket reactor (AMBR)/aerobic completely stirred tank reactor (CSTR) system.
Freedman and Sutherland (1998) operated a sequential treatment plant of anoxic

filters, followed by aerobic filters and finally activated sludge reactor for treating
waste water discharges from Holston Army Ammunition Plant. Ronen et al. (1998)
described a biological process comprising of an anoxic stage and then followed by
an aerobic one, to treat real RDX-contaminated waste water from munitions
factory. This waste water contained nitramine and RDX together with high levels
of nitrate and organic solvents, such as cyclohexanone and acetone.
Using enrichment technique, Dautpure (2007) extensively studied a yeast iso-
late, Candida ishiwadae MCM Y-2, obtained from soil collected from sites
exposed to HMX. This was capable of reducing the COD and nitrate contents of
the HMX containing effluent. Hence, C. ishiwadae was proved to be a prospective
candidate for the treatment of effluent from HMX manufacture. The results of flask
culture and laboratory scale reactor studies can be used to set up a pilot scale
treatment plant for HMX effluent. Microbial treatment employing C. ishiwadae,
reduced the toxicity of the effluent as tested by the fish bioassay.
There are a few reports available on the biotreatment of high nitrate effluent.
A study of COD/N ratios of three different types of bioreactors including activated
sludge reactor, a biologically mediated activated carbon fluidized bed reactor and
an upflow immobilized cell reactor, used a high-strength nitrate waste water
(the nitrate-nitrogen was 1,200 mg/l i.e. 5,314 mg/l nitrate) (Chen et al. 1999).
In another report, Zala et al. (1999) have isolated denitrifiers which could reduce
nitrate from 1,200 to 100 mg/l in 48 h under aerobic conditions with fusel oil as a
carbon source. Up to 95–100 % nitrate removal was achieved on scale-up to 50 l
at COD: nitrate-nitrogen ratio of 3.45 with a retention time of 48 h.
Bioremediation of FOX-7 wastewater was studied by Patil et al. (2011) with
respect to environmental factors e.g., temperature, pH, incubation period, sup-
plementation with carbon and nitrogen source, initial concentration of waste water
pollutants, etc. Maximum removal of FOX-7 was observed at the initial concen-
tration of 400 mg/l (incubation period 96 h and supplementation of medium with
0.01 % peptone). Three isolates could use FOX-7 as a sole source of nitrogen in
presence of 0.1 % pyruvate and succinate as carbon sources and removed FOX-7 in
the range of 13–51 and 22–52 %, respectively. Thus, these microbial cultures
would be useful in carrying out biodegradation of FOX-7 on a larger scale. Sim-
ilarly, a lot of work has been done on the remediation of nitramine-contaminated
soil (Williams et al. 1992; Speitel et al. 2001; Monteil-Rivera et al. 2003; Fuller
et al. 2004; Hatzinger et al. 2004).
11 Phytoremediation
Exploration of plants for remediation is an emerging cost-effective and eco-

friendly approach. The strategies involving plants are commonly called phyto-
technologies which include phytoremediation. Phytotechnologies are defined as
the use of plants to remediate, treat, stabilize or control contaminates in soil or
water. Phytotechnologies and phytoremediation exploit the natural plant physio-

logical and biochmeical processes. Plants, mainly pondweed, arrowroot, coontail
and poplar, have been employed for remediating TNT contamination in a con-
structed wetland (Rodgers and Bunce 2001). The plant system could degrade
0.019 mg/l TNT per day (Rodgers and Bunce 2001). TNT removal rates increased
with increasing plant density and removal kinetics enhanced with increasing
temperature up to 34 °C (Medina et al. 2000). The rate of phytoremoval of TNT
was 30 mg/l from a hydroponic system by Stonewort (Algae nitella) and Parrot
feather (Myriophyllum aquaticum).
Several agricultural and indigenous terrestrial plants were examined for their
capacity to accumulate and degrade HMX. Only traces of mononitroso-HMX were
detected in contaminated soil extracts and leaf extracts. Mechanism for HMX
translocation and accumulation in the foliar tissue is mainly transpirational flux
and evaporation (Groom et al. 2002). These reports highlight the phytoremediation
potential of explosives. Besides, phytoremediation is more rugged than microbial
bioreactors with respect to physical conditions and changes in contaminant
loading.
Nitrate removal by plants was also studied by a few research workers. Best
et al. (2006) studied removal of nitrocompounds (explosives) from groundwater
using aquatic plants. Kanekar et al. (2003) have reviewed use of some aquatic and
terrestrial plant species for phytoremediation of nitroexplosives, such as TNT,
HMX etc.
In a study by Bhadra et al. (2001), M. aquaticum removed RDX from the
aqueous medium. RDX level was decreased by about 75 % in the presence of live
plants compared to 10 % in the presence of dead plant matter. RDX disappearance
in the presence of dead plant matter typically represents that fraction was sorbed
into biomass. However, HMX was not metabolized by M. aquaticum (Bhadra et al.
2001).
Plants may also metabolize nitramines. Aken et al. (2004a) highlighted trans-
formation of RDX by Populus deltoides x nigra DN34. The report suggested that
transformation of RDX by plant tissue cultures may occur through a three-step
process: (a) a light-independent reduction of RDX to MNX and DNX by intact
plant cells; (b) a plant/light- mediated breakdown of the heterocyclic ring of RDX,
MNX or DNX into CH2O and CH3OH; (c) a further light-independent minerali-
zation of the C1 metabolites by intact plant cells.
A number of reports have also revealed the possibility of plant-symbiotic
bacteria transforming nitramines. Aken et al. (2004b) demonstrated that a pink-
pigmented symbiotic bacterium Methylobacterium sp. strain BJ001, isolated from
poplar tissues, could mineralize HMX. From their work, it appears that this
symbiosis might be useful in phytoremediation of explosive-contaminated sites.
Groom et al. (2002) investigated the potential of agricultural and indigenous ter-
restrial plants to accumulate and degrade HMX. Wheat and ryegrass demonstrated
rapid growth in the presence of HMX. Their work speculates that the capacity to
survive and accumulate significant quantities of HMX identifies these plant species
as potential candidates for phytoremediation. However, the use of edible plants for
phytoremediation is not advisable on account of direct entry of the contaminant

into the food chain. Moreover, phytoremediation, like bioremediation, also suffers
from unpredictable climate variation. Although plants are effective remediators
due to their large amount of biomass, they are less efficient per unit of biomass
than bacteria. One major drawback is that many of the metabolic products of
phytoremediation remain unidentified, making it difficult to assess their long-term
fate and toxicity. Moreover accumulation of explosives in plants to levels sig-
nificantly above soil concentration is relevant to the assessment of both phyto-
remediation potential and environmental risks (Groom et al. 2002).
Phytoremediation seems to be a good complementary remediation approach
(Dautpure 2007). Terrestrial plant species, like Poplar and Glyricidia, were
explored for remediation of HMX wastewater. These plant species could appre-
ciably remove nitrate and HMX from microbially treated HMX wastewater. The
darkening and wilting of leaves was minimized at higher dilution of the effluent
(Fig. 3).
A strategy of microbial remediation, followed by phytoremediation, offers
better treatment technology for the effluent from a HMX manufacturing plant.
12 Future Perspective
The process of development of new explosives will continue to match their

demand for security and defense of nation. To protect the environment from
nitroexplosives, bioremediation processes are to be suitably designed. A search for
natural resources, like microbes and plants for degradation of newer explosives,
has become inevitable. Enzymatic degradation of these compounds can be further
investigated. Biosensors can be developed using enzymes involved in degradation
and the genes regulating their activities for detecting explosives from the con-
taminated soils.
Fig. 3 Effect of effluent on

Glyricidia showing darkening
of leaves and wilting of plant;
(a) 1:10 diluted effluent,
(b) 1:50 diluted effluent,
(c) control
13 Conclusions
Production of nitroexplosives is unavoidable and hence, developing methods for

bioremediation of toxic waste waters generated during their manufacture, is the
only choice left to mankind. Natural resources, like microorganisms and plants,
have been explored for their mechanisms of degradation and detoxification of the
nitroexplosives and bioremediation of waste water explosives. A search for new
organisms and development of new strategies for bioremediation of nitroexplo-
sives waste waters is an on-going process to meet the environmental challenges of
explosive contamination of soil and water.
Acknowledgments The authors thank the High Explosive (HE) factory, Pune and High Energy
Material Research Laboratory (HEMRL), Defense Research and Development Organization
(DRDO), Pune for providing nitroexplosive waste water samples and relevant information. Part
of the work on biodegradation of nitroexplosives was supported by Department of Biotechnology,
Govt. of India, New Delhi, Indo-US Science and Technology Forum (IUSSTF) Govt. of India and
US Govt. and HEMRL, Pune, The authors are thankful to authorities of MACS’ Agharkar
Research Institute, Pune for providing necessary facilities to carry out the work, compile the data
and present in the form of a book chapter.
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Degradation of TNP, RDX, and CL-20
Explosives by Microbes
Baljinder Singh, Jagdeep Kaur and Kashmir Singh
1 Introduction
Life on the planet earth is supported by the continuous cycling of elements. Due to
massive mobilization of natural resources and industrial synthesis of chemicals, a
number of environmental problems have arisen as a consequence of incorporation
of the synthesized molecules into ongoing biological cycles. The quality of life on
earth is linked inextricably to the overall quality of environment. The problem
associated with contaminated environment now assumes increasing prominence in
many countries. Contamination sources are mainly associated with their manu-
facture, use, loading, storage and disposal processes. The variety of materials and
processes, used in modern day industrial activities, cause different types of con-
taminants. The numbers of contaminants found to date are enormous and types of
mixtures are countless. Their unplanned intrusion into ecosystems affects flora and
fauna including human beings, thus exerts serious ecological problems. Now-a-
days, xenobiotic compounds, like explosives waste, are regarded as major envi-
ronmental contaminants around the world.
Explosives are materials with high nitrogen and oxygen contents on detonation
expand to create a shock wave which exerts high pressures on the surroundings,
causing an explosion and leaving toxic waste in the environment (Singh et al. 2012).
An explosive is a material, either a pure single substance or a mixture of substances,
which is capable of producing an explosion by its own energy (Davis 1972; Sickler
1992). The specific property of explosives depends on its components: the initiator,
the detonator, the booster charge, and the main charge. The initiator or primary
explosive consists of a small quantity of material that is very sensitive to heat, spark,
impact, or friction (Fig. 1). The secondary explosives are physical mixtures of one or
B. Singh
Punjab Pollution Control Board, Patiala 147001 Punjab, India
J. Kaur K. Singh (&)
Department of Biotechnology, Panjab University, Chandigarh 160014, India
e-mail: kashmirbio@pu.ac.in

88 B. Singh et al.
Explosives
Low Explosives or High Explosives

Propellants
e.g. Black powder,
Smokeless powder, Flash Primary Secondary Tertiary
powder e.g. Lead Azide, Lead e.g. RDX, PETN, e.g. TNT, TNP,
Styphnate, Mercury etc. GTN, dynamite, etc.
Fulminate, etc.
Fig. 1 General classification of explosives
more high explosives with various additives (use of mixtures provides greater
flexibility in explosive design and additives extend the range of performance). Low
explosives or propellants are combustible materials, which burn, but do not explode,
and function by producing gas which produces an explosion. High explosives are
detonated under the influence of shock of the explosion of a suitable primary
explosive. They do not function by burning, but can be ignited by a flame and in
small amount, generally burn tranquilly and can be extinguished easily. If heated to a
high temperature by external heat or by its own combustion, it sometimes explodes.
Unlike primary explosives, high explosives cannot be exploded readily by heat or by
shock and are generally more brisant and powerful. High explosives (e.g. cyclic
nitramines) produce more power because of their the higher density, bigger mole-
cules and more realizable energy packed into the same space through the formation
of covalent bonds between closer atoms (Sunahara et al. 2009).
Explosives are used primarily for military purposes, industries, mining and
agricultural activities. A large scale manufacturing testing, firing ranges and
destruction of ammunition stocks have created a number of environmental prob-
lems and increasing concern about their persistence in air, water and terrestrial
ecosystems (Spain 2000). Interactions between the chemicals and various com-
ponents of the environment determine the behaviour of an explosives waste.
Explosives, dumped in the sea, burned or detonated in remote areas, can travel
long distances from the contamination site by water flow and leaching into the soil.
Presently, a large number of sites across the globe are affected with explosives
contamination. These sites are potential or actual sources of human exposure to
explosives causing harmful health effects. Environmental contamination by
munitions constituents primarily occurs in soils at munitions manufacturing plants,
load and pack operations, firing ranges, and demilitarization areas (Jenkins et al.
2001). The ammunition producing plants were mostly located in forests, not only
to protect them from reconnaissance by the enemy but also to supply them with
larger amounts of water, which is necessary, for instance, for the production of
TNT (Steuckar et al. 1994).
Degradation of TNP, RDX, and CL-20 Explosives by Microbes 89
The increased awareness of the harmful effects of explosives has led to a

dramatic increase in research on various strategies that may be employed to clean
up the environment. The methods currently used for the remediation of contam-
inated sites are expensive and sometimes highly impractical and can result in the
formation of toxic products. The limitations faced by physical and chemical
treatment technologies, could be overcome with the help of microbes. With dis-
covery in the 1960s that many soil microorganisms are capable of metabolizing
these explosives waste, the use of biological processes to degrade hazardous
materials became a viable and acceptable possibility. Microbes, the oldest
inhabitants on earth, are versatile and also adaptive to the changing environment,
will provide a cost-effective measure to combat the present problems of contam-
ination. Microbes and their diverse metabolic enzymes are typically employed for
safe removal of environment contaminants, either through direct destruction or
indirectly through transformation of the contaminant to a safer intermediates
(Pieper and Reineke 2000; Farukawa 2003).
The main classes of important explosives are nitrate esters, which include
glycerol trinitrate (GTN; propane-1,2,3-triyl trinitrate) and pentaerythritol tetra-
nitrate (PETN; 2,2-bis[nitrooxymethyl]-propane-1,3-diyl dinitrate); nitroaromatics
like 2,4,6-trinitrophenol (TNP) and TNT and nitramines with hexahydro-1,3,5-
trinitro-1, 3,5-triazine, commonly known as Royal Demolition Explosive or
Research Department Explosive (RDX), and octahydro- 1,3,5,7-tetranitro-1,3,5,7-
tetrazocine (HMX) and 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-hexaazaisowurtzi-
tane (CL-20) (Singh et al. 2012). However, because of a higher stability and
detonation power, nitramines HMX and RDX are presently the most-widespread
conventional explosives (Singh et al. 2012). In the present chapter, we will dis-
cusses three important explosive wastes, TNP, RDX and CL-20.
2 TNP Toxicity
As an explosive waste, TNP is converted into salts, such as ammonium picrate

(ammonium 2,4,6-trinitrophenoxide), which are more shock sensitive than the parent
acid (ACGIH 1984). Exposure to TNP or its salts through inhalation of dust or through
skin contact can cause dermatitis, general weakness, muscle pain, anuria, followed by
polyuria, and temporary coma (Swartz 1944; Sunderman et al. 1945). Metabolites
isolated from TNP exposed person’s urine included N-acetylisopicramic acid,
picramic acid, N-acetylpicramic acid and unidentified components. Approximately,
60 % of the TNP was reported to be excreted unchanged (Wyman et al. 1992). TNP
intake at 100 mg/kg per day causes hemolytic anemia and testicular toxicity and
mortalities at higher dose of 500 mg/kg per day in a dose-finding study in young rats
(Takahashi et al. 2004). Goodfellow et al. (2007) evaluated acute toxicity of TNP
and picramic acid on commercially important aquatic species, rainbow trout
(Salmo gairdneri), and American oysters (Crassostrea virginica). They found that
concentrations ([0.001) in 96-h LC50 for rainbow trout and 144-h LC50 for American
90 B. Singh et al.
oysters caused lethal effects. The LD50 for TNP following oral dosing of male and
female rats was established as 290 and 200 mg/kg, respectively (Wyman et al. 1992).
They found that the primary depots (per gram tissue basis) were blood, spleen, kidney,
liver, lung, and testes after 24 h of oral administration of [14C] TNP (100 mg/kg).
Therefore, general recalcitrance (Lenke et al. 2000) and toxic properties of TNP pose
a threat to life (Aguirre et al. 1993).
3 Microbial Degradation of TNP
Microbes have the ability to use TNP as a sole nitrogen source under aerobic
conditions (Lenke and Knackmuss 1992; Behrend and Heesche-Wagner 1999).
Nitro groups, due to the strong electron-withdrawing properties on the aromatic
ring, are subjected to initial reductive transformation for biodegradation of TNP
(Rieger et al. 1999; Heiss and Knackmuss 2002; Singh et al. 2011). Due to the
presence of three nitro groups on phenol, it is difficult to degrade TNP at high
concentrations using microbes (Shen et al. 2009). Therefore, the screening of
microbes capable of degrading TNP is a critical step for formulating an effective
strategy for bioremediation of TNP. Gram positive bacteria, Rhodococcus (Rieger
et al. 1999), Nocardioides (Rajan et al. 1996; Behrend and Heesche-Wagner 1999)
and Bacillus (Singh et al. 2011) are important genera capable of degrading TNP.
Different pathways of TNP degradation have been studied under conditions not
typically found in the sub-surface environment (Fig. 2).
The occurrence of nitroaromatic compounds in the environment has selected
microorganisms that are able to utilize nitroaromatic compounds as carbon and/or
nitrogen sources for growth. Examples include degradation by bacteria (Table 1)
or fungi that may use picric acid as an energy source (Lenke and Knackmuss 1992;
Rieger and Knackmuss 1995; Gazdaru et al. 1996; Rajan et al. 1996; Rieger et al.
1999; Heiss et al. 2002; Takeo et al. 2003; Hofmann et al. 2004; Singh et al. 2011).
The predominant route of biological transformation of nitroaromatic compounds is
oxidation. However, the presence of three electron-withdrawing nitro-groups
around the ring prevents oxidation renders such compounds resistant to biodeg-
radation (Symons and Bruce 2006). Nitroaromatics with two or more nitro groups
are hydrogenated. The initial step of TNP biodegradation is a hydrogenation
reaction, yielding the hydride Meisenheimer complex of TNP (H--TNP) (Fig. 3).
A hydride ion, provided by NaBH4, reduces the aromatic nucleus to form a H--
TNP. TNP is hydrogenated to form Meisenheimer complex (hydride r-complex)
(Lenke and Knackmuss 1992). TNP possesses an electrophilic aromatic nucleus
due to the negative mesomeric effects of the nitro groups. In addition, nitrogen and
oxygen atoms are electronegative and therefore attract the p-electrons of the ring
(negative inductive effect).
Erikson (1941) was the first to observe a microbial attack on TNP by Micro-
monaspora strains. Gundersen and Jensen (1956) described the metabolism of
TNP by Corynebacterium simplex which was isolated from soil as a 4,6-dinitro-2-
Fig. 2 Biodegradation TNP +

pathway of picric acid. The
Rhodococcus opacus HLPM1
scheme is based on articles
cited in the text Hydride transferase II
TNP hydride Meisenheimer complex
Hydride transferase I
TNP dihydride Meisenheimer TNP dihydride

complex (aci form) Meisenheimer
complex (nitro form)
TNP dihydride denitrase
2,4-dinitrophenol hydride
2,4-dinitrophenol dihydride
Spontaneous
2,4-dinitrocyclohexanone
2,4-dinitrocyclohexanone
hydrolase
2,4-dinitrocyclohexanoate
Carbon dioxide
methylphenol-degrading organism. Tabak et al. (1964) observed a color change

from yellow to orange-red in enrichment cultures with TNP by phenol adapted
bacteria.
Rieger et al. (1999) showed that Rhodococcus (opacus) erythropolis HL PM-1
was able to use TNP as a sole source of carbon, nitrogen, and energy (Rieger et al.
1999). They proposed that the observed nitrite release occurs from the protonated
form of H--TNP, forming DNP (2, 4 dinitrophenol).
3.1 Hydrogenation of H2-TNP
The first enzymes of the initial TNP degradation were purified and characterized
from Nocardioides simplex FJ2-1A by Ebert et al. (1999). Nitroaromatics with two
or more nitro groups are hydrogenated. The initial step of TNP biodegradation is a
hydrogenation reaction, yielding the H--TNP. Further investigations have shown
Table 1 Microorganisms capable of degrading TNP
92
Microorgaism Standard Degradation pathway Conditions involved Percentage Degradation product Reference
concentration (concentration of transformation (metabolite)
of TNP substrate, ativity of
enzyme)
Bacillus cereus 1.3 mM Metabolism of TNP was H-TNP-synthesizing Degradation of TNP Hydride meisenheimer Singh et al.
strain PU accompanied by enzyme was complex (H-TNP) (2011)
transient accompanied by
accumulation of an stoichiometric
orange-red release of
metabolite, hydride 2.1 ± 0.15 mol
meisenheimer nitrite/mol TNP
complex (H-TNP), at 539 lmol/h g
complete reductive dry cell wt
removal of the nitro
group as nitrite
Nocardioides 5 to 6 mM TNP used as a sole H2-TNP-synthesizing 2.9 ± 0.1 mol of [H2]-Meisenheimer Behrend and
sp. Strain source of carbon, enzyme nitrite per mol complexes of TNP and Heesche-
CB 22-2 nitrogen, and of TNP 2,4-dinitrophenol (H2- Wagner
energy. DNP), as well as 2,4- (1999)
Transformation was dinitrophenol
accompanied by
stoichiometric
nitrite release
(continued)
B. Singh et al.
Table 1 (continued)
Microorgaism Standard Degradation pathway Conditions involved Percentage Degradation product Reference
concentration (concentration of transformation (metabolite)
of TNP substrate, ativity of
enzyme)
Nicardioides 1.76 mM TNP used as a sole Degradation of TNP Transient formation of 2,4- Rajan et al.
simplesx source of carbon, was dinitrophenol (1996)
(ATCC 6946) nitrogen, and accompanied by
energy. stoichiometric
Transformation was release of
accompanied by nitrite/mol TNP
stoichiometric
nitrite release
Rhodococcus Hydride-Meisenheimer Conversion of the aci- 2,4-DNCH (2,4- Hofmann et al.
opacus HL complex as a initial nitro form of 2H_- dinitrocyclohexanone). (2004)
PM-1 metabolite, npdH TNP to H--DNP by 4,6-DNH (4,6-
gene encode enzyme the nitrite- dinitrohexanoate)
tautomerase, eliminating enzyme,
catalyzing a proton HTI converts H--
shift between the DNP to 2,4-DNCH,
aci-nitro and the hydrolase
Degradation of TNP, RDX, and CL-20 Explosives by Microbes
nitro forms of the converting 2, 4-

dihydride DNCH to 4, 6-DNH
Meisenheimer
complex of TNP
Rhodococcus 0.5 mM TNP used as nitrogen 0.5 mM TNP and 2 mol of nitrite Hydride-Meisenheimer Lenke and
erythropolis source 10 mM succinate in from 1 mol of complex of TNP and Knackmuss
HL 24-2 mineral medium TNP (maximum 2,4,6- 1992
specific Trinitrocyclohexanone
activities of the as dead end metabolite
cells were
17–22, umol/
93
min/g of protein
94 B. Singh et al.
that H--TNP is further hydrogenated to produce dihydride Meisenheimer complex

(2H--TNP), as an intermediate of the pathway (Lenke and Knackmuss 1996; Ebert
et al. 2002; Heiss et al. 2002). TNP is hydrogenated by this mechanism forming a
Meisenheimer complex, hydride r-complex (Lenke and Knackmuss 1992). The
hydride transferring enzyme system [NADPH-dependent F420 reductase (NDFR)
and hydride transferase II (HTII)], isolated from N. simplex FJ2-1A, was able to
hydrogenate H--TNP in a subsequent step, generating the dihydride r-complex,
2H--TNP (Ebert et al. 2002). TNP possesses an electrophilic aromatic nucleus due
to the negative mesomeric effects of the nitro groups. Further, 2H--TNP exists in
two tautomeric forms, aci-nitro and nitro form (Hofmann et al. 2004). In addition,
nitrogen and oxygen atoms are electronegative and therefore, attract the p-electrons
of the ring (negative inductive effect). The electron density at the nitro groups
makes them easily reducible, which was observed in most cases during
TNT decomposition by one-electron transfer or two-electron transfer forming a
nitroso derivative, followed by two consecutive electron transfers, producing a
hydroxylamine and an aromatic amine (Esteve-Nunez et al. 2001). The electron
withdrawing effects of the nitro groups also facilitate a nucleophilic attack at the
aromatic ring. A hydride ion, e.g. provided by NaBH4, reduces the aromatic
nucleus to form a hydride Meisenheimer r-complex. The occurrence of TNP in the
environment has selected microorganisms that are able to utilize it as carbon and/or
nitrogen sources for their growth (Table 1).
3.2 Nitrite Elimination from 2H2-TNP
Ebert et al. (2002) described nitrite release from 2H--TNP by an enriched enzyme
from N. simplex FJ2-1A, which was not fully identified nor characterized. Behrend
and Heesche-Wagner (1999) had detected H--DNP formerly with resting cell
experiments by Nocardioides sp. CB 22-2 during the conversion of TNP.
The first step in TNP metabolism by R. erythropolis HL PM-1 is formation of
H--TNP (Fig. 3). Generation of Meisenheimer complexes of nitroaromatic com-
pounds is a well-known chemical reaction (Meisenheimer 1902; Severin et al.
1969). In contrast, there are only two examples of microbial conversion of ni-
troaromatic compounds to Meisenheimer complexes; both of these involve TNP
and TNT (Lenke and Knackmuss 1992; Vorbeck et al. 1994).
4 Factors Effecting Microbial Degradation of TNP
Based on a series of preliminary studies, it has been found that the inoculum size,
amounts of additional co-substrates like yeast extract, glucose, sodium pyruvate
and succinate and pH are the major factors that affected the extent and rate of TNP
degradation. Singh et al. (2011) standardized KB medium consisting of yeast
extract (0.05 %), glucose (0.045 %), K2HPO4 (0.02 %) and MgSO4.7H2O
(0.006 %) and 1.32 mM TNP (pH 6.8 ± 0.1) for degradation studies of TNP. It
was proposed that low concentration of yeast extract and glucose accelerated the
degradation of TNP, while high concentration did not (Shen et al. 2009). The
organism ignores TNP in presence of high concentration of yeast extract and
glucose, thus TNP degradation period was delayed. The concentration of the
stimulant, such as yeast extract and glucose and that of the compound to be
degraded would, therefore, be of prime importance for removal of TNP from
contaminated sites. The optimum pH and temperature for TNP degradation by
bacteria is found to be 7.0 and 30 °C, respectively.
5 RDX Toxicity
Due to its high mobility, slow volatilization from water and relative inability to
cling to soil particles, RDX contamination threatens drinking water supplies
underneath contaminated soil beds (Hawari et al. 2000; USEPA 2009). Bioaccu-
mulation of RDX has been observed in aquatic invertebrates and vertebrates
exposed to water containing RDX (Talmage et al. 1999; Lotufo et al. 2010).
Chronic or sub-chronic exposure of workers to RDX by inhalation is characterized
by generalized convulsions headaches, nausea, vomiting, and unconsciousness.
RDX has the potential to affect the central nervous system (CNS) of many different
species (Meyer et al. 2005; Johnson et al. 2007; Bannon et al. 2009; Gust et al.
2009; Quinn et al. 2009; Garcia-Reyero et al. 2011). Observed effects of RDX
exposure in the ecotoxicological model species fathead minnow include lethality,
impaired growth and reduced reproduction (Talmage et al. 1999). When orally
administered in rats and mice, highest concentrations of RDX are found in the
kidneys, followed by the liver, brain, and heart. Chronic exposure of mice to a low
dose of RDX results in carcinogenic effects by a significant increase in the inci-
dence of hepatocellular adenomas and carcinomas (Parker and Reddy 2006).
6 Microbial Degradation of RDX
The EPA has set the recommended concentration of 2 ppm for RDX in drinking
water, but up to 36 ppm is found in the contaminated water (Agency for Toxic
Substances and Disease Registry 1995). This proves that indigenous soil microbes
and fungi are incapable of complete transformation of RDX due to the accumu-
lation of toxic metabolites or low bioavailability of soil RDX (Rylott et al. 2006).
RDX is resistant to microbial degradation, due to its low volatility (dimensionless
Henry’s constant, H’ = 2 9 10-11), moderate solubility (42 mg/l), and high
mobility in aquifers (log Kow = 0.8) (Wildman and Alvarez 2001).
96 B. Singh et al.
- O -
O
O O - - 2H - -
- O 2N NO2 O 2N NO2
NO2 O 2N NO2 Tautomerase
NDFR/HT I
NDFR/HT II
F 420 F 420 + H NO2
+ - + - N
NO2 NADPH/H NO2 NADPH/H O OH 3
1 3
2
Nitrite-eliminating
enzyme
O
-
NO2
-
NO2
4
F 420
NDFR/HT I +
NADPH/H
O
NO2 + O 2N NO2
O 2N NO2 H
Hydrolase HOOC
H +
HOOC OH
6 H NO2
6
5
Fig. 3 Degradation pathway of TNP: The scheme is based on articles cited in the text. (1) TNP,
(2) H--TNP (3a) aci-nitro form 2H--TNP (3b) nitro form of 2H--TNP (4) H--DNP (5) 2,4-
DNCH (2,4-Dinitrocyclohexanone), (6) 4,6-DNH (4, 6-Dinitrohexanoate)
The biodegradation of RDX in the environment occurs both aerobically and

anaerobically. The anaerobic biodegradation of RDX has not been studied as
extensively as the aerobic technique, although it is very important to military
ranges. Anaerobic degradation rates are more rapid than aerobic rates where other
nutrients are present. RDX is a highly oxidized compound and, therefore, an initial
enzymatic denitration of RDX might destabilize the inner C–N bonds and lead to
ring cleavage. Table 2 summarizes the performance of various microbes, which
degrade RDX. Hawari et al. (2001) reported two pathways for RDX degradation,
one route involved reduction of the nitro groups in RDX to the nitroso derivatives
and another novel route involved a direct ring cleavage to produce methylenedi-
nitramine (MDNA, O2NNHCH2NHNO2) and bis(hydroxymethyl)- nitramine
[BHNA, (OHCH2)2NNO2] (Fig. 4). The metabolites, hexahydro-1-nitroso-3,5-
dinitro-1,3,5-triazine (MNX) hexahydro-1,3- dinitroso-5-nitro-1,3,5-triazine
(DNX) and hexahydro-1,3,5- trinitroso-5-nitro-1,3,5-triazine (TNX) were formed
by the stepwise reduction of NO2 in RDX (Fig. 4). This transformation then
produces formaldehyde, methanol, hydrazine, and dimethyl hydrazine. MDNA and
BHNA products were formed by enzymatic hydrolytic ring cleavage of the inner
C–N bonds of RDX. These metabolites were further degraded to produce
N-containing products (N2O, and traces of N2) and C-containing products (HCHO,
CH3OH, HCOOH, and CO2). A dead-end intermediate with the molecular formula
C2H5N3O3 was also found to accumulate which indicated that not all the com-
ponents of RDX were mineralized. The enzyme responsible for the degradation of
RDX by Rhodococcus sp. strain DN22 is a cytochrome P450 enzyme (Coleman
et al. 2002; Bhusan et al. 2003b) and the gene encode of this enzyme xplA showed
that the mechanism of action was initial denitration, followed by spontaneous ring
cleavage and mineralization (Seth-Smith et al. 2002). Numerous investigations
have documented that explosive degrading cytochrome P450 system is highly
conserved among strains of Rhodococcus sp. (Seth-Smith et al. 2008; Bernstein
et al. 2011). Roh et al. (2009) demonstrated the incorporation of 15N isotope from
labeled RDX into xplA gene amplified from total DNA extracted from bacterial
culture. Recently, conjugal transfer of RDX-degrading gene locus xplAB was
located on actinomycete bacteria from Gordonia sp. Horizontal gene transfer of
KTR9 to Gordonia polyisoprenivorans, Rhodococcus jostii RHA1 and Nocardia
sp. TW2 was studied by Jung et al. (2011). A nitrate reductase enzyme (EC
1.6.6.2) from a fungus Aspergillus niger transforms RDX under anaerobic con-
ditions using NADPH as electron donor (Hawari et al. 2000) because nitrate
reductases are ubiquitous enzymes in microorganisms. Under reduced oxygen, but
not fully anaerobic conditions, the aerobic bacteria Pseudomonas fluorescens I–C
harbouring enzyme xenobiotic reductases (XenB) degraded RDX faster than
Pseudomonas putida II-B harbouring enzyme (XenA). The results indicated that
transformation occurred when the cells were supplied with sources of both carbon
(succinate) and nitrogen (NH4+), but not when only carbon was supplied (Fuller
et al. 2009). The study suggests that these two xenobiotic reductases may be
important in the degradation of cyclic nitramines under anaerobic conditions;
however, these two organisms were not isolated from contaminated ranges.
7 Factors Effecting Microbial Degradation of RDX
According to previous studies on RDX biodegradation, there are several limiting

factors that influence the rate and extent of RDX degradation. Therefore, studies
on such factors involving the biodegradation of the RDX are necessary, if soil
bioremediation has to be carried out. Based on a series of preliminary studies, it
has been found that the inoculum size, amounts of additional co-substrates, like
fructose, glucose, glycerol and succinate and solvents are the major factors that
affected the extent and rate of RDX degradation. Addition of fructose (5.0 g/l) in
minimal salt medium supported bacterial growth regardless of the presence of a
nitrogen source (Van Aken et al. 2004). Presence of succinate (12 mM) as a
carbon source results in complete degradation of RDX after 14 h of incubation
(Fournier et al. 2002). Thompson et al. (2005) observed an increase in the rate of
RDX degradation by Williamsia sp. KTR4 and Gordonia sp. KTR9, when glucose,
glycerol, and succinate were added as carbon sources and RDX was the nitrogen
Table 2 Microorganisms capable of degrading RDX
98
Microorgaisms Degradation pathway Conditions involved (e.g. % biotransformation Degradation product Technique used References
concentration of substrate, (metabolite)
activity of enzyme, etc.)
Stenotrophomonas RDX as a sole source of The growth yield (29.2 ± 4.4 g of Methylene-N- Mass spectrometry, 1H Binks et al.
maltophilia nitrogen protein per mol of N) (hydroxymethyl)- NMR (1995)
PB1 hydroxylamine-N*-
(hydroxymethyl)
nitroamine
Williamsia sp. RDX was supplied as a 45 lM of RDX for Strains KTR4 and KTR9 degraded Metabolites nitrite, Gas Chromatography Thompson
KTR4 and sole carbon and enrichment media and 180 lM RDX within 72 h. formaldehyde, and 4- et al.
Gordonia sp. nitrogen source. 180 lM of RDX as Mineralization of [U-14C]RDX nitro-2,4-diazabutanal (2005)
KTR9 Aerobic dinitration nitrogen source only to 14CO2 was 30 % by strain
KTR4 and 27 % by KTR9 RDX
was not detectable in enrichment
cultures after 8 days. When
RDX used as both nitrogen and
carbon source degradation of
RDX is 0.60 per day for KTR4
strain and 0.65 for KTR9 strain.
When RDX used as only
nitrogen and carbon source is
glucose, glycerol, succinate,
degradation of RDX is 0.78 per
day for KTR4 strain and 1.10 for
KTR9 strain
Rhodococccus RDX as a nitrogen The gene responsible for the Complete disappearance of RDX Nitrite, Formaldehyde TLC, HPLC Seth-Smith
rhodochrous source at a degradation of RDX is a within 21 h et al.
strain 11Y concentration of constitutively expressed (2002)
250 lM cytochrome P450-like
gene, xplA
Aerobic dinitration 45 lM of RDX for
enrichment media and
180 lM of RDX as
nitrogen source only.and
formate
B. Singh et al.
(continued)
Table 2 (continued)
Aspergillus niger Two-electron reduction A nitrate reductase (EC One RDX molecule produced three Hexahydro-1-nitroso-3,5- LC/MS (ES-) Bhushan
mechanism (iii) 1.6.6.2) from Aspergillus HCHO molecules, 86 lmol of dinitro-1,3,5-triazine chromatograms et al.
where intermediate niger catalyzed the RDX produced 148 lmol of (MNX) and (2002)
is MNX biotransformation of N2O and 10 lmol of methylenedinitramine
RDX most effectively at methylenedinitramine their disappearance was
pH 7.0 and 30 °C under (equivalent to 20 lmol of N2O) accompanied by the
anaerobic conditions accumulation of nitrous
using NADPH as oxide (N2O),
electron donor formaldehyde (HCHO),
and ammonium ion
(NH4+)
Morganella Nitroso-RDX reduction O2 -depleted culture Both RDX and HMX completely Nitroso derivatives of RDX HPLC Kitts et al.
morganii B2 intermediates. conditions transformed in 2 weeks and HMX (1994)
and Produced 14CO2
Providencia from labelled RDX
rettgeri B1,
and
Citrobacter
freundii NS2
Clostridium sp. RDX (100 lM) as the Strain HAW-1 spores grew RDX transform to MNX, DNX, TNX MNX, DNX and TNX which HPLC/UV Zhao et al.
HAW-G3 sole carbon and rapidly in yeast extract with yields of 56, 7.3 and 0.2 % disappeared to form (2003)
nitrogen source and (0.1 %) medium respectively MeOH, HCHO, and N2O
hydrogen as energy containing RDX as final ring cleavage
source (104 lM) products
Acetobacterium Methylenedinitramine The homoacetogens in a Degraded 29.0 lM RDX in N2O HPLC Adrian and
malicum was observed as a mineral medium B14 days Arnett
transient containing RDX and an (2004)
intermediate, H2-CO2 (80:20)
indicating ring headspace
cleavage
(continued)
99
Table 2 (continued)
100
Rhodococcus sp. RDX used as sole RDX was converted to nitrite (30 %), Nitrite NO2, nitrous oxide GC/MS Fournier
DN22 nitrogen source. nitrous oxide (3.2 %), ammonia (N2O), formaldehyde et al.
RDX was converted (10 %), and formaldehyde (HCHO) which later (2002)
to nitrite (27 %) Converted to carbon
dioxide
Klebsiella Anaerobic dinitration. MNX was degraded to HCHO, HCHO CH3OH (12 % of LC–MS with negative Zhao et al.
pneumoniae Initial denitration CH3OH, and N2O (16.5 %) with total C), CO2 (72 % of electron spray (2002)
SCZ1 versus nitroso a removal rate [0.39 lmol/h/g total C), and N2O (60 % ionization
formation, dw (cells)] similar to that of of total N) through
denitration, RDX [0.41 lmol/h/g dw (cells)] intermediary formation
followed by ring [biomass, 0.91 g dw (cells)] of methylenedinitramine.
cleavage and The trace amounts of
decomposition in MNX also detected
water
B. Singh et al.
source than when RDX served as the carbon and nitrogen source. RDX biodeg-
radation proceeded at a rate seven times faster under water-saturated vs unsatu-
rated soil conditions (Ringelberg et al. 2003).
RDX degradation was not observed in the absence of crude extract and depends
on the presence of an RDX-specific enzyme activity. Sulfhydryl groups are
commonly found at the active site of this enzyme, as the addition of the sulfydryl-
modifying agents’ iodoacetamide (10 mM) and p chloromercuribenzene sulfonic
acid (10 nM) inhibited RDX breakdown. The inhibition of RDX breakdown by the
enzyme inhibitors iodoacetamide and p-chloromercuribenzene sulfonic acid, helps
to dispel the possibility of non-specific binding of RDX to cellular materials. RDX
degradation activity is inducible, as no degradation was observed when crude cell
extracts were prepared from cultures of S. maltophilia PB1 grown on NH4NO3 as
the sole nitrogen source (Binks et al. 1995). Kwon and Finneran (2006) observed
that electron shuttle-mediated RDX reduction is favorable and that RDX is
reduced faster by extracellular electron shuttles, including humic substance (HS)
and the HS analog anthraquinone-2,6-disulfonate (AQDS) than direct microbial
reduction. Presence of trimethylamine N-oxide (TMAO) or in the absence of
terminal electron acceptors (TEA) favoured RDX metabolism under anaerobic
conditions. Shewanella halifaxensis HAW-EB4 used periplasmic proteins and
c-type cytochromes to transform RDX to MNX, DNX, and TNX and ring cleavage
products (such as, methylenedinitramine) with more nitroso formation in cells
grown on TMAO or pre-incubated in the absence of TEA (Zhao et al. 2008).
The internal environment of all living cell is believed to be approximately
neutral. At low (4.0) or high (9.0) pH values, acids or bases can penetrate into cells
more easily, because they tend to exist in the undissociated form under these
conditions and electrostatic force cannot prevent them from entering cells
(Robertson and Alexander 1992). The optimum pH for RDX degradation by
bacteria is 7.2.
Temperature plays an important role than nutrient availability in the degrada-
tion of organic pollutants. Growth rates in general roughly double for each 10 °C
rise in temperature within range from 10 to 30 °C. Growth rates generally do not
change between 35 and 40 °C, but denaturation of proteins at higher temperatures
slows growth rates. The optimum temperature for RDX degradation by bacteria is
30 °C.
8 CL-20 Toxicity
The polycyclic nitramine CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12 hexa-

azaisowurtzitane) is a powerful explosive that may replace RDX and HMX. Toxic
effects of CL-20 were observed in the earthworm Eisenia andrei (Robidoux et al.
2004) and potworms Enchytraeus crypticus and Enchytraeus albidus (Dodard
et al. 2005; Kuperman et al. 2006).
102 B. Singh et al.
O 2N NO2 O 2N NO HCHO
N N N N
+
CH3OH
N N +
2N2O
NO2 NO2
RDX MNX
NO2
O 2N NO
O 2N NO2 N N
NH NH + N
HOH2C CH2OH
Methylenedinitramine Bis(hydroxymethyl)-nitramine N
NO
DNX
HCHO + 2N2O + H2O

ON NO
N N
CO2 + N2O + 2H2O + CH4
NO
TNX
HCHO + CH3OH
Fig. 4 Pathways of microbial degradation for RDX. The scheme is based on articles cited in the
text. Path 1 represents RDX degraded via nitroso derivatives and formation of hydrazines. Path 2
represents direct ring cleavage of RDX with formation of nitrous oxide as major end product
9 Microbial Degradation of CL-20
Biotic degradation of CL-20 occurred through the formation of its denitrohydro-

genated derivative while hydrolysis occurred through the formation of a ring
cleavage product that was tentatively identified as CH2] N–C(]N-NO2)-CH]N-CHO
or its isomer N(NO2)]CH–CH]N-CO–CH]NH (Monteil-Rivera et al. 2009). CL-20
was reported to biodegrade in soil under aerobic (Trott et al. 2003; Crocker et al.
2005; Panikov et al. 2007) and anaerobic (Strigul et al. 2006; Panikov et al. 2007)
conditions. Several strains capable of degrading CL-20 (Table 3) including Agro-
bacterium sp. strain JS71 (Trott et al. 2003), Pseudomonas sp. strain FA1 (Bhushan
et al. 2003a), and Clostridium sp. EDB2 (Bhushan et al. 2004c) have been isolated
from the soils and sediments. The white rot fungi Phanerochaete chrysosporium and
Irpex lacteus can decompose CL-20 under aerobic conditions. Three different
pathways for biotransformation of CL-20 have been suggested (Fig. 5). First
Table 3 Microorganisms capable of degrading CL-20CL-20
Microorgaisms Degradation pathway Conditions involved % biotransformation Degradation product (metabolite) Technique used References
(e.g. concentration of
substrate, activity of
enzyme, etc.)
Pseudomonas sp. FA1 CL-20 as sole nitrogen source, Biotransformation of The rates of CL-20 biotransformation by 5 N as (nitrite and nitrous oxide) HPLC Bhushan et al. (2004b)
membrane-associated CL-20 is the resting cells and the membrane- and 2 C (as HCOOH)
flavoenzyme. catalyzed an catalyzed by a enzyme preparation were
oxygen-sensitive one- membrane- 3.2 ± 0.1 nmol/h mg of cell
-1
electron transfer reaction associated biomass and 11.5 ± 0.4 nmol/
that caused initial N h mg of protein-1. In the
denitration of CL-20 membrane-enzyme-catalyzed
reactions, 2.3 nitrite ions, 1.5
molecules of nitrous oxide, and 1.7
molecules of formic acid were
produced per reacted CL-20
molecule
Agrobacterium sp. JS71 CL-20 as a nitrogen source 20 mM succinate as a After 2 days of incubation, about 80 % of HPLC-mass Trott et al. (2003)
carbon source the initial CL-20 had disappeared. spectrometry
Strain JS71 used 3 mol of nitrogen
per mol of CL-20
Phanerochaete P. chrysosporium Cl-20 biodegradation initiated after a lag [14C]-CL-20, HPLC Karakaya et al. (2009)
chrysosporium was capable of phase of about 65 h and was nearly
(white rot fungus) degrading CL- fully transformed in less than 95 h
20 in the of incubation irrespectively of initial
presence of concentration
supplementary
carbon and
nitrogen sources
Escherichia coli N-denitration Nitroreductase The rates of CL-20 biotransformation 1,4,5,8-tetranitro- LC/MS, HPLC, 14N-CL- Bhushan et al. (2004a)
catalyzed a one- under anaerobic and aerobic 1,3a,4,4a,5,7a,8,8a-octahydro- 20 and 15N]CL-20,
electron transfer conditions were 3.4 ± 0.2 and diimidazo[4,5-b:40,50- GCMS
-1
to CL-20 0.25 ± 0.01 nmol min mg of e]pyrazine [IUPAC] which
protein-1, respectively decomposed spontaneously in
water to produce glyoxal
(OHC-CHO) and formic acid
(HCOOH)
103
104 B. Singh et al.
pathway involves denitration where salicylate 1-monoxygenase (Zhao et al. 2004a),

nitroreductase (Bhushan et al. 2004c), and dehydrogenase (Bhushan et al. 2005a) are
believed to promote the transfer of a single electron to the molecule forming a free-
radical anion that releases a nitro group. The second pathway for biotransformation
of CL-20 involves a hydride ion transfer generating the denitrohydrogenated
intermediate (C6H7N11O10). This intermediate is believed to formed by dehydro-
genase enzyme isolated from Clostridium sp. EDB2 (Bhushan et al. 2005a) and a
purified diaphorase enzyme (Bhushan et al. 2005b). In the third pathway, the
mononitroso derivative of CL-20 with formula C6H6N12O11 is formed via reduction
with two redox equivalents (Fig. 5). This pathway is also catalyzed by the dehy-
drogenase enzyme of strain EDB2 (Bhushan et al. 2005a).
10 Factors Effecting Microbial Degradation of RDX
The amounts of supplied nutrient nitrogen and carbon are important factors that
control the CL-20 biodegradation. The enzyme dehydrogenase (isolated
from Clostridium sp. EDB2) responsible for CL-20 degradation was membrane-
associated and NADH-dependent and had a molecular weight of 56 kDa (Bhushan
et al. 2005a). The nitroreductase from Escherichia coli contains one molecule of
O2N N N NO2
O2N N N NO2
via two single electron

transfer two redox equivalent
1 N N
CL-20 3
dinitration O2N NO2
2
hydride
NO2 NO2 NO2 transfer
O 2N NO2
N N N N
N N O2N N N NO
+ O2N N N NO
N N N N N
N
O 2N NO2 NO2
O2N N N NO2 N N
double denitrated isomer O2N NO2
O2N N NH mononitroso derivative
N N
O2N NO2
HCOOH + OHC-CHO + N2O denitrohydrogenated product
HCOOH + OHC-CHO+ N2O+ NO2-
Fig. 5 Microbial degradation of CL-20. The scheme is based on articles cited in the text. Path 1
represents denitration of CL-20 before ring cleavage. Path 2 represents hydride transfer of CL-20
before ring cleavage. Path 3 represents reduction of CL-20 to nitroso derivatives before ring
cleavage
flavin-moiety (FMN) per enzyme monomer and catalyzes a one-electron transfer

to CL-20 to form a radical anion (Bhushan et al. 2004a). An enzyme from
Pseudomonas sp. strain called salicylate 1-monooxygenase containing a flavin
adenine dinucleotide (FAD), biotransformed CL-20 under both aerobic and
anaerobic conditions (Bhushan et al. 2004b). Under anaerobic conditions, mem-
brane enzyme(s) (NADH dependent) from Pseudomonas sp. strain FA1 showed
fivefold higher activity for CL-20 degradation than under aerobic conditions
(Bhushan et al. 2003a). Fournier et al. (2006) observed direct degradation of
CL-20 by MnP enzyme in white rot fungi under nitrogen sufficient media. The
optimum pH and temperature for CL-20 degradation by bacteria are 7.0 and 30 °C,
respectively.
11 Conclusions
The total diversity of biodegradation pathways for explosives waste remains still
unknown. The complete detoxification of high explosives has not been studied,
their assimilation as carbon or energy sources for growth by microbes still remains
an open field of study to be explored. The available information on the degradation
pathways of high explosives (cyclic nitramines) by microbes is limited by the fact
that we cannot trace the final destination of all of the carbons and nitrogens from
the molecules. Further studies with heavy or radioisotopes would be necessary to
definitively determine the final metabolites.
Much has been worked out about the microbial degradation pathways of
explosives, but several fundamental aspects regarding the evolutionary history of
their biodegradation (i.e. the sum number of changes and the amount of time and
order in which they occurred) and their integration into existing metabolic path-
ways and global regulatory control networks, like catabolite repression and
nitrogen regulation, have yet to be explored. As more energetic explosives are
discovered, it is likely that the microbes (enzymes) involved in their biodegra-
dation remains a rich field of investigation to be pursued in the future.
Knowledge of catabolic pathways of degradation, optimization of various
parameters for accelerated degradation, and design of microbe(s) through
molecular biology tools, capable of degrading explosives will lead to improve-
ments of both the qualitative and quantitative performance of bioremediation.
Research in the last 5 decades on microbes capable of degrading explosives has
led to the identification and characterization of the genes and enzymes involved in
biodegradation pathways and this information can be applied for engineering
strains with improved biodegradation capabilities. Recombinant strains are
designed using degradative plasmids for high performance and their true assess-
ment under field conditions is required to address ecological considerations for
sustainable bioremediation. Investigations in microbial ecology, chemical com-
position, and geophysical properties at contaminated environments will shed light
into adaptive pathway evolution in bacteria. This valuable information can be
106 B. Singh et al.
applied for biotreatment of explosives contamination by developing more effective

methods for stimulating or accelerating natural attenuation. For identifying niches
(where microbes bearing explosives degrading genes clusters), gene probes will be
useful in the designing of biological treatments for sites polluted with explosives
waste.
Soils contaminated with military wastes contain a plethora of different nitrate
explosives in varying concentrations. This aspect needs to be explored, if biore-
mediation is going to be proven a viable alternative for cleaning up military
ranges. Inhibition study (presence of one explosive, such as TNP, inhibits bio-
degradation of RDX etc.) is required to explore for effective bioremediation at
contamination sites. This study will also explore the toxicity of one contaminant to
another in the microbes.
Research on biostimulation has reported that removal of cyclic nitramines by
bioremediation methods can be increased by adding nutrients and/or a terminal
electron acceptor to enhance the populations already present at this site (Zhao et al.
2004; Kwon and Finneran 2006; Schaefer et al. 2007). But a great deal of research
is needed to understand designing of an effective bioremediation strategy. Biore-
mediation process (Bioattenuation, Bioaugmentation, Biostimulation) seems to be
another alternative for the in situ treatment of explosives-contaminated sites,
containing microbial strains capable of metabolizing pollutants. Phytoremediation,
in conjunction with rhizospheric microbes, may provide a cheap, fast, eco-friendly
and efficient rhizoremediation processes for the removal of explosive waste from
the upper layers of the soil (Singh et al. 2012).
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Assessment of Bioremediation Strategies
for Explosives-Contaminated Sites
O. Muter
1 Introduction
Large amounts of soil and water have been contaminated with energetic com-
pounds as a result of the manufacture, storage, testing, use and disposal of
munitions as well as the use of nitroaromatic and nitramines as chemical feedstock
for synthesis of pesticides, herbicides, dyes, and pharmaceuticals. Historically,
TNT (2 methyl-1,3,5, trinitrobenzene) has been the most widely used military
explosive (Nicklin et al. 1999; Kulkarni and Chaudhari 2007b). Since TNT is
toxic, mutagenic, and also highly energetic (Rosenblatt et al. 1991), TNT con-
tamination has a serious impact on the environment and also threatens human
health (Maeda et al. 2007).
Remediation strategies must be considered on a site-by-site basis. For example,
energy-intensive chemical treatments, such as incineration, may be too expensive
to be used for low concentrations, or may cause other environmental problems,
such as NOx emissions. Conversely, when concentrations are high, the toxicity of
nitroaromatics can limit the usefulness of bioremediation or the treatment process
may produce recalcitrant reaction by-products (Rodgers and Bunce 2001).
In recent studies, a number of abiotic approaches have shown promise for the
decontamination of polluted sites, particularly when used for the initial stage of degra-
dation followed by biodegradation (Esteve-Núñez et al. 2001; Vasilyeva et al. 2002;
Hilber et al. 2009; Boparai et al. 2010; Zhao et al. 2010). These approaches include the
transformationofHMX,RDX,andTNTbyzerovalentironbarriers,thetreatmentofTNT
redwaterbyvacuumdistillation,thesorptionofTNTbyactivatedcarbonandcharcoaland
the binding of TNT to cysteine, aniline, or crude proteinextracts.
Remediation using biological systems to decontaminate explosives-polluted
sites has attracted worldwide attention in recent years. Numerous organisms have
O. Muter (&)
Institute of Microbiology & Biotechnology, University of Latvia,
4 Kronvalda bulv LV-1010 Riga, Latvia
e-mail: olga.muter@inbox.lv

114 O. Muter
been isolated that have the ability to degrade/transform energetic compounds while
using them as a sole carbon or sole nitrogen source or through co-metabolic
processes under aerobic or anaerobic conditions (Juhasz and Naidu 2007; Kulkarni
and Chaudhari 2007b). However, TNT’s resistance to complete mineralization has
been a major obstacle to the development of an effective bioremediation method
for this explosive (Rodgers and Bunce 2001).
Because of the problems mentioned above, a combination of technological
approaches appears to be crucial for the successful biodegradation of TNT and
other explosives. This paper reviews current findings related to combined abiotic-
biotic and biotic-biotic approaches for the clean-up of sites contaminated with
explosives, mostly with TNT, RDX, HMX and CL-20.
2 Biostimulation
The most widely used bioremediation procedure is biostimulation of indigenous

and introduced microorganisms through the addition of nutrients. Carbon sources,
electron donors, salts with buffer capacity, inorganic macro- and micro-elements,
vitamins, and complex organic amendments can be used to stimulate the degrading
activity of soil microorganisms. The concentrations of both the stimulant and the
compound to be degraded are of prime importance in the design of an effective
remediation strategy (Kulkarni and Chaudhari 2007b).
2.1 Addition of Chemicals with Determined Content
Some chemicals and functions can be provided by the contaminant; for example,
TNT can serve as a sole source of carbon and energy. Similarly, TNT or sulfate
can serve as an electron acceptor and TNT or ammonium can serve as a nitrogen
source (Boopathy et al. 1993, 1997). Electron donors such as sulfides and pyruvate
increase the rate and extent of TNT reduction (Boopathy et al. 1993; Cheng et al.
1996; Rodgers and Bunce 2001).
Nitrocellulose undergoes biotransformation in the presence of nitrate- and
sulfate-reducing enrichment cultures, but the extent of denitration does not appear
to be adequate to yield a non-hazardous product (Freedman et al. 2002).
The addition of methanol and glucose during the degradation of PNP under
denitrifying conditions using anaerobic sludge blanket reactors was found to cause
a drop in the redox potential of up to -190 and -300 mV, respectively (Karim
and Gupta 2002). The addition of glucose in the range of 0.1–0.5 % generally
enhanced the degradation of PNP by Arthrobacter. However, acidification as a
result of glucose metabolism may have a negative effect on PNP depletion. In a
sudy with sequencing batch reactors, She et al. (2012) demonstrated an
enhancement in nitrophenols degradation at low glucose concentration (660 mg/l).
Assessment of Bioremediation Strategies 115
b-cyclodextrin has been shown to be a promising additive for effective PNP

bioremediation (Qiu et al. 2009).
In another study on the kinetics of CL-20 biodegradation, the highest CL-20
degradation rate was found under aerobic conditions and with the addition of co-
substrates like succinate and pyruvate which were found more efficient than
acetate, glucose, starch or yeast extract (Panikov et al. 2007).
2.2 Addition of Complex Amendments
Molasses is known to be an efficient carbon source for the co-metabolism of

explosives. Being an important by-product of sugar production, molasses contains
about 50 % sugar in the form of sucrose, glucose, and fructose, and is also rich in
mineral elements. The addition of molasses (0.3–3.3 %) to TNT-containing soils/
slurries/liquids showed different results on TNT degradation (Boopathy et al.
1998; Rodgers and Bunce 2001; Gerth et al. 2003). Molasses enhances biodeg-
radation of RDX and complete degradation occurred within a few weeks. Low
molasses dose of 1:40 (molasses to water ratio) was as effective as the higher dose
(1:20) (Lamichhane et al. 2012). Besides, corn steep liquor (1 %) has also been
used to stimulate the biodegradation of TNT in soil by Pseudomonas putida KP-
T202 (Park et al. 2003).
Crude plant extracts (e.g. spinach and parrotfeather) have been shown to transform
TNT, without the presence of the live plant (Medina et al. 2004). Cabbage leaf extract
(CLE) was shown to be an effective additive in the TNT biodegradation process
(Muter et al. 2008). Crude soybean oil and molasses stimulated the mineralization of
RDX (30–40 %) and HMX (up to 10 %), as well as the transformation of TNT to
amino-containing compounds. However, sawdust markedly decreased mineraliza-
tion regardless of the type of co-substrate used (Fuller et al. 2004).
The addition of 2 % proteinaceous material and 20 % compost to TNT-polluted
soil decreased the amount of free TNT by 87 % and the amount of water-leachable
TNT by 67 % after three weeks of anaerobic incubation (Meyns et al. 2002). To
address benzo(a)pyrene contamination, when wheat-condensed distillers soluble,
ice bran extract, and hydrolyzed poultry feathers were applied as biostimulants,
feathers had the greatest impact (Tejada et al. 2011). Likewise, three organic
wastes—banana skins, spent mushroom compost, and brewery spent grain—were
tested as amendments for the bioremediation of oil-contaminated soils, and
brewery spent grain showed the greatest effect (Abioye et al. 2010).
Complex amendments are subject to content variability. For example, a com-
parative study of the composition of CLE prepared from different cultivars and in
different harvest years demonstrated that the concentrations of the total nitrogen,
carbon, and reducing sugars in the extract were quite variable (Grube et al. 2008).
Apart from sugars, molasses may contain varying concentrations of minerals, such
as Mn, Fe, Cu, Ca, K, Mg, Se, and Zn, as well as vitamins, such as B6, pantothenic
acid, B3, B2, and folate, depending on the region of its production (Wythes et al.
1978; Olbrich 2006).
116 O. Muter
2.3 Use of Surfactants
Taking into account the hydrophobic nature of explosives, surfactants can be used
to increase their bioavailability to microorganisms. The remaining, strongly sorbed
TNT fraction is presumably sequestered and thus unavailable to the microorgan-
isms (Brannon et al. 2002; Li et al. 2004; Eriksson et al. 2004; Robertson and
Jjemba 2005; Prak 2007). Fresh mineral surfaces (and newly created mineral
microparticles) are more reactive to geochemical species than weathered soil
surfaces in river sediments and soil solutions (Stallard and Edmond 1983; Anbeek
1992; Pennington and Brannon 2002; Douglas et al. 2008).
A comparison of anionic, cationic, and non-ionic surfactants for TNT degra-
dation indicated a preference for anionic (SDS) in the concentration range of
0.1–1.0 % (Taha et al. 1997). The effect of using 1 % Tween 80 to stimulate TNT
mineralization by the white-rot fungus Phanerochaete chrysosporium (strain
BKM-F-1767) was studied by (Hodgson et al. 2000). In another study, the use of
hydroxypropyl-b-cyclodextrins as an additive resulted in the desorption of TNT
and its metabolites from topsoil and illite shale (Sheremata and Hawari 2000).
Because of their capacity to promote added microbial activity and attain further
natural attenuation in washed soils, solutions of natural humic acid were proposed
as a treatment for soil washings of highly polluted soils (Conte et al. 2005). In
some cases, surfactants can however lead to operating problems, such as precip-
itation or soil sorption of the surfactant, phase separation and foaming (Rodgers
and Bunce 2001).
3 Bioaugmentation
Bioaugmentation, the addition of microorganisms with explosives-degrading

activity to soil, can significantly promote the process of biodegradation, especially
at the initial stages of soil treatment. Many factors in contaminated soil, that
influence the viability and activity of the amended microbial biomass, include soil
type, pH, moisture, type of contamination, climatic conditions, and indigenous
microorganisms.
3.1 Microbial Enzymes Responsible for Explosives

Degradation in Microorganisms
Toxic substances, such as explosives, create extreme environments that speed up

the evolutionary process. However, bacteria with conscripted metabolic enzymes
can be used to address this problem using novel remediatiatory pathways (Symons
and Bruce 2006). TNT is a strongly electron-deficient aromatic with a positive
molecular quadruple moment and three electrophilic nitro groups. As a result, its
environmental fate hinges on specific sorptive electron donor–acceptor interac-
tions and nucleophilic, reductive (bio)transformations (Esteve-Núñez et al. 2001;
Stenuit and Agathos 2010). Removal or productive metabolism of nitro groups can
be accomplished by four different strategies: (1) Some bacteria can reduce the
aromatic ring of dinitro and trinitro compounds by the addition of a hydride ion to
form a hydride-Meisenheimer complex, which subsequently rearomatizes with the
elimination of nitrite. (2) Monooxygenase enzymes can add a single oxygen atom
and eliminate the nitro group from nitrophenols. (3) Dioxygenase enzymes can
insert two hydroxyl groups into the aromatic ring and precipitate the spontaneous
elimination of the nitro group from a variety of nitroaromatic compounds.
(4) Reduction of the nitro group to the corresponding hydroxylamine is the ini-
tial reaction in the productive metabolism of nitrobenzene, 4-nitrotoluene, and
4-nitrobenzoate (Spain 1995).
TNT denitration, using catalyst(s) of biotic origin, can be considered as a major
reaction and a driving force for beneficial biodegradation (Stenuit et al. 2009).
Studies indicate that enzymes, that exhibit denitrase activity towards TNT, belong
to the class I flavin-dependent b/a barrel oxidoreductases, also known as the Old
Yellow Enzyme family (Stenuit et al. 2005; Smets et al. 2007; Stenuit and Agathos
2010). In particular, type II hydride transferases are responsible for TNT deni-
tration (Van Dillewijn et al. 2008). Nitroreductases including aldehyde oxidase,
cytochrome b5, hydrogenases and dehydrogenases, reduce nitroaromatic com-
pounds (Esteve-Núñez et al. 2001; De Oliveira et al. 2010; Gwenin et al. 2011).
Broad specificity nitroreductases may also transform 2,4-Dinitroanisole (DNAN),
which has been tested recently as a replacement for TNT in explosive formulations
(Perreault et al. 2012).
Aerobic bacteria tend to reduce one or two of the nitro groups. The resulting
hydroxylamino or amino metabolites accumulate in the culture media without
further metabolism (Esteve-Núñez et al. 2001). The hydroxylamino metabolites
are extremely reactive and, react to form azoxy compound metabolites with
oxygen. They cause a high mutation rate and are not metabolized by any known
organism (Esteve-Núñez et al. 2001; De Lorme 2008).
3.2 Diversity of Microorganisms Capable of Explosives

Degradation
The best-studied aerobic bacteria capable of TNT biotransformation are Pseudo-

monas strains. In particular, P. aeruginosa transforms TNT with nitrite release
(Kalafut et al. 1998; Eyers et al. 2008) and P. fluorescens transforms TNT to
phloroglucinol and pyrogallol with ammonium release (Naumova et al. 1988).
Generally, Pseudomonas spp. are able to use TNT as a sole nitrogen source (Jones
et al. 1995; Rodgers and Bunce 2001). Pseudomonas sp was found capable of
118 O. Muter
metabolizing PNP as a sole source of carbon, nitrogen, and energy (Kulkarni and
Chaudhari 2007a; Zhang et al. 2008; Zheng et al. 2009). Proteomic analysis of
Pseudomonas sp. HK-6 revealed 11 protein spots induced by TNT in the soluble
protein fractions of cells (Cho et al. 2009). Recent findings indicate that P. putida
KT2440 uses two kinds of strategies to overcome TNT toxicity: (1) induction of
genes-encoding nitroreductases and detoxification-related enzymes (pnrA, xenD,
acpD), and (2) induction of multidrug efflux pump genes (mexEF/oprN) to reduce
intracellular TNT concentrations (Fernández et al. 2009). Stenotrophomonas
maltophilia has been observed to use TNT and RDX as primary nitrogen sources
(Binks et al. 1995; Oh and Kim 1998; Ho et al. 2004).
Rhodococcus strain DN22 grows on RDX as a sole nitrogen source (Priestley
et al. 2006). Rhodococcus opacus is capable of mineralizing or transforming ni-
troaromatic and nitramine compounds of importance (Weidhaas et al. 2009). RDX
biodegradation activity by Rhodococcus can be inhibited by the presence of nitrate
or/and ammonium (Bernstein et al. 2011). Catabolic pathways in rhodococci,
which are characterized for each type of aromatic (hydrocarbons, phenols, halo-
genated, nitroaromatics, and heterocyclic compounds), have been reviewed by
Martínková et al. (2009).
Arthrobacter is capable of using PNP as its sole source of carbon and energy
(Qiu et al. 2009). Raoultella terrigena strain HB removed TNT at low concen-
trations of nutrient supplements (Claus et al. 2007). In one study, some novel
strains of Methylobacterium sp. were shown to have the capacity to degrade ni-
troaromatic and nitramine compounds (Schnoor and Van Aken 2004). Another
study revealed that different species of the family Rhizobiaceae were able to
transform TNT to hydroxylaminodonitro-, aminodinitro- and diaminonitro- tolu-
enes; but mineralization was less than 2 % (Labidi et al. 2001). Enterobacter
cloacae PB2 is capable of growth using TNT as its sole nitrogen source (Nicklin
et al. 1999). Similarly, Escherichia coli cultures aerobically transformed TNT
using it as a sole nitrogen source (Yin et al. 2005).
Clostridium acetobutylicum is capable of transforming RDX with H2 as the
electron donor (Zhang and Hughes 2003). The ability to reduce TNT to TAT
appears to be a common trait of the Clostridium genera via the co-metabolism of
TNT with an energy substrate (Spain 1995; Esteve-Núñez et al. 2001; De Lorme
2008). The anaerobic bacterium Desulfovibrio was demonstrated to be capable of
mineralizing RDX while using it as a carbon and energy source for growth (Arnett
and Adrian 2009). Acetobacterium paludosum anaerobically degraded RDX faster
under autotrophic (H2-fed) conditions than under heterotrophic conditions, even
though heterotrophic growth was faster (Sherburne et al. 2005). Anaerobic bacteria
closely related to Lysobacter taiwanensis was able to grow in the presence of TNT
(Gallagher et al. 2010). Bacillus mycoides isolated from Fe-reducing bacterial
consortia was used to degrade TNT under aerobic or anaerobic conditions (Lin
et al. 2012). Denitrifying and sulfate-reducing bacteria were tested in biodegra-
dation of pentaerythritol tetranitrate (PETN), using nitrate and/or sulfate as elec-
tron acceptors (Zhuang et al. 2012).
Recent studies document that the acid-tolerant yeast Yarrowia lipolytica AN-
L15 transformed TNT through hydride ion-mediated reduction of the aromatic ring
(as the main pathway), as well as through nitro group reduction (as a minor
pathway). TNT transformation was contingent on the yeast ability to acidify the
culture medium through the production of organic acids. Rapid acidification of the
medium influences the rate and extent of TNT transformation (Ziganshin et al.
2007; Ziganshin et al. 2010). Soil isolate of the yeast Pichia sydowiorum MCM Y-
3 transformed HMX from wastewater in the fixed film bioreactor (Kanekar et al.
2009).
White rot fungus, Phanerochaete chrysosporium, has been evaluated more
extensively than any other fungal species for remediating explosives-contaminated
soil. P. chrysosporium has been shown to transform TNT using enzymes of the
lignin-degrading system including lignin peroxidase, manganese peroxidase, oxi-
dases, reductases, hydrogen peroxidase, veratryl alcohol, oxalate, and quinol
oxidases (USEPA 1993; Stahl and Aust 1995; Hawari et al. 2000; Esteve-Núñez
et al. 2001). Cladosporium resinae, Cunninghamella echinulata var elegans, Cy-
athus pallidus, and Phanerochaete chrysosporium have been grown in the pres-
ence of RDX on a vegetable juice agar (Bayman et al. 1995).
3.3 The Role of Bacterial Consortia in Biodegradation
Although many bacteria are able to metabolize organic pollutants, a single bac-
terium does not possess the enzymatic capability to degrade all or even most of the
organic compounds in a polluted soil (Fritsche and Hofrichter 2000). Different
isolation procedures provide a wide diversity of strains with target properties. The
degradation rate as well as the degradation efficiency of TNT and DNTs by the
mixed cultures is higher than that by the individual strains (Páca et al. 2008; Guo
et al. 2009). In experiments on HMX degradation under anaerobic conditions in a
mixed microbial population system, HMX was converted to methanol and chlo-
roform most quickly under mixed electron-acceptor conditions, followed in order
by sulfate reducing, fermenting, methanogenic, and nitrate reducing conditions
(Boopathy 2001).
In other experiments, the inoculation of soil samples with a mixture of bacterial
isolates had a strong effect on microbial community composition, as revealed by
16 s rDNA-DGGE analysis. Several bacterial strains present in inoculum became
dominant in TNT- and RDX- amended samples, such as Klebsiella, Raoultella,
Serratia, Stenotrophomonas, Pseudoxanthomonas, Achromobacter, and Pseudo-
monas (Limane et al. 2009; Limane et al. 2011). The amendment of soils with
TNT resulted in a shift from slower growing k-strategists towards faster growing r-
strategists. Pollution-induced community tolerance was observed as TNT con-
centrations increased (Travis et al. 2008a). Analysis of soil bacterial diversity by
DGGE showed a predominance of Pseudomonadaceae and Xanthomonadaceae in
120 O. Muter
the TNT-contaminated soils as well as the presence of Caulobacteraceae (George

et al. 2008).
Inoculation of a microorganism into the environment may have the greatest
impact on other microorganisms through competition. This is especially true when
the populations are of a similar nature to the released strain, such as when
Pseudomonas sp. is released into a population of indigenous Pseudomonas
(Naseby and Lynch 1997). Potentially hazardous events include the horizontal
dissemination of genes from the introduced bacteria to resident microorganisms by
conjugation, transduction, or transformation. The effects will vary from soil to soil.
However, there is no commonly accepted method to predict what the interactions
between an introduced organism and the indigenous population of microorganisms
will be in a particular environment (Lejbølle 2000).
4 Bioremediation Strategy and Technological Solutions
4.1 Site Assessment
Every site is different, thus site assessment is important in the development of an

effective bioremediation strategy (Parales et al. 2002). Depending on the properties
of the explosives residues in the soil, specific procedures must be followed to
ensure safety and sample integrity i.e. sampling procedure, shipping, sample
preservation, extraction, and measurement. Contamination from explosives has
certain distinctive characteristics, for example, particles larger than 3 mm diam-
eter account for 96.4 % of contamination (Fig. 1). However, many uncertainties
do remain regarding the mechanisms governing the dissolution and mobility of
particulates of explosive residues. On-site determination of nitroaromatic and
Fig. 1 Particles remaining after the sieving of soil contaminated with explosives
nitramine residues in soils are determined through a variety of approaches,

including colorimetric and immunoassay methods as well as the use of portable
gas chromatographs and ion mobility spectrometers (Jenkins et al. 1996; Hewitt
et al. 2001; Radtke et al. 2002; Jung et al. 2004; Lavoie et al. 2012).
Biosensors, although not a substitute for rigorous analytical chemistry, can
complement data collected through other methods and offer a rapid and environ-
mentally focused statement of remediation potential (Bhattacharyya et al. 2005).
Comparison of specific effect data can be difficult, because the susceptibility of test
species exposed to stressors can be highly variable (Franzle 2006). Consequently,
test organisms, that originate from the same locality, may be proven to be more
useful for the investigation of efficacy of remediation techniques (Kuncova et al.
2011).
Gas chromatography and high performance liquid chromatography are the most
commonly used laboratory methods for the determination of various nitroaromatic
and nitramine analytes (Belkin et al. 1985; Jenkins et al. 1989; Becanova et al. 2010;
Kuncova et al. 2011). Improved colorimetric methods for the rapid and unbiased
monitoring of nitrite and/or ammonium ions produced in TNT-biodegradation was
described recently (Stenuit and Agathos 2009).
4.2 Ex situ Bioremediation
4.2.1 Composting
Ex situ bioremediation methods include three categories of composting methods:

static-pile composting, mechanically agitated, in-vessel composting, and windrow
composting (USEPA 1993). In windrow composting, the waste is placed in long
piles and then mixed using conventional agricultural equipment. The contaminated
soil is screened to remove large rocks and debris, and is then mixed with organic
materials, such as manure, straw, or alfalfa which serve as a bulking agent as well
as carbon source. The windrow piles are turned regularly to control heat transfer
and aeration, and monitored for moisture content, oxygen level, pH, and tem-
perature (Esteve-Núñez et al. 2001; Rodgers and Bunce 2001).
At the Umatilla Army Depot in the United States, composting was used to
convert contaminated soil into safe humus-containing soil. The composting
feedstocks used at Umatilla consisted of 30 % contaminated soil, 21 % cattle
manure, 18 % sawdust, 10 % potato waste, and 3 % chicken manure. In other
geographical areas, substitutions may be made depending on the cost and avail-
ability of ingredients. At Umatilla, the treatment time for a 2,700 cubic-yard batch
of soil was 10–12 days (Williams et al. 1992).
Windrow composting was tested at the U.S. Joliet Army Ammunition Plant as a
cost-effective and efficient method for explosives biodegradation. A compost
blending ratio of 70 % amendments to 30 % soil was used. For the amendment
portion, a mix of approximately 18 % corn processing waste, 52 % stable bedding,
122 O. Muter
and 30 % wood chips provided the required carbon to nitogen (C:N) ratio and
moisture level. At Joliet, the process was accelerated by starting the microbial
activity prior to mixing with contaminated soil (MWH Americas, Inc 2004).
The primary criticisms of the composting technique for bioremediation are the
long incubation time, the cost of setting up and maintaining the system, and the
lack of knowledge about the bacteria and fungi involved in the process (Esteve-
Núñez et al. 2001).
4.2.2 Vermicomposting
Earthworms can improve soil quality through soil aeration and bioturbation,
increased nutritional status and fertility, and the promotion of microorganism
activity. Many studies have focused on the testing earthworms’ resistance to
explosives. These have used a wide spectrum of methodological approaches
including measuring cocoon production and juvenile hatching; tracking adult
growth, which, in general, does not correlate strongly with change in reproduction
capacity; and using non-specific biomarkers, such as DNA damage (comet assay),
neutral red retention time, a filter paper contact test, and total immune activity
(Schaefer 2004; Robidoux et al. 2001, 2004a; Fuchs et al. 2011). The products of
TNT degradation appear to bioaccumulate in earthworms with 4-ADNT having
greater toxicity than 2-ADNT; 2,4-DANT; or 2,6-DANT (Lachance et al. 2004).
Explosives such as TNT, RDX, HMX, and CL-20 do not support a common
mechanism of toxicity in the earthworm, probably due to differences in their
physical–chemical properties as well as in the metabolites formed during exposure
(Robidoux et al. 2002; Gong et al. 2010).
Through a combination of direct and indirect effects, including the promotion of
catabolically competent microorganisms, as well as biological, chemical and
physical actions, earthworm-assisted bioremediation has been proven suitable for a
wide range of organic compounds (Hickman and Reid 2008). In particular,
earthworms have been shown to be useful tools for in situ bioremediation of oil-
contaminated soils with moderate (\4,000 mg/kg) total petroleum hydrocarbon
concentrations (Schaefer and Juliane 2007). The addition of compost can promote
this effect (Ceccani et al. 2006). When three organic additives (coffee grounds,
grass and wood chips, and brewery mash) were tested, the addition of brewery
mash and the use of earthworms without additives were shown to be the most
efficient bioremediation approaches for the oil degradation (Schaefer and Juliane
2007).
The in vivo transformation of TNT to 2- and 4-ADNTs by adult white pot-
worms (Enchytraeus albidus) was accomplished, at least in part, by bacteria
associated with the host organism (Dodard et al. 2004). In another study, Lacto-
coccus lactis was isolated from the intestines of earthworms and used to anaero-
bically reduce DNTs. The in vitro production of toxic dinitroazoxytoluenes during
this process suggests that anaerobic biotransformation of DNTs could increase
environmental risk (Shin et al. 2005).
4.2.3 Slurry Bioreactor
Bioreactors are characterized by a shorter treatment time than composting, but are
more labor intensive and more expensive (Snellinx et al. 2002). Bioslurry pro-
cesses, which involve the mixture of contaminated material with water and
nutrients, enable the control of parameters, such as N, P and organic carbon source
(biostimulation); inocula (bioaugmentation); and the increased availability of
pollutants through the use of surfactants or inducing biosurfactant production
inside the slurry bioreactor (Robles-González et al. 2008). In one study, up to
99 % of TNT, which had an initial concentration 10,000 mg/kg, was removed after
182 days in a soil slurry reactor under co-metabolic condition, using molasses as a
co-substrate (Clark and Boopathy 2007).
The addition of H2 or electron donors that produce H2 (e.g. ethanol and pro-
pylene glycol) may be a useful strategy for enhancing the anaerobic biodegrada-
tion of RDX, HMX, and TNT in bioslurry (Adrian et al. 2003; Adrian and Arnett
2007). Under anaerobic conditions, the bioslurry treatment of RDX resulted in a
significant portion (35 %) of original radioactivity being incorporated into the
biomass and bound to the soil matrix (Shen et al. 1998). However, the presence of
TNT inhibited RDX mineralizing activity in the bioslurry; also, p-cresol, an
intermediate in the degradation pathway of some amino acids, was inhibited by
TNT and its metabolites in an anaerobic bioslurry to treat TNT (Shen et al. 1998,
2000).
A comparison of aerobic, anaerobic, and anaerobic/aerobic slurries indicates
that the explosives biodegradation process is dependent on slurry composition. The
highest degree of mineralization (50 %) was obtained under aerobic conditions
with a microbial consortium, phosphate, and acetic acid. Potato or corn starch were
not efficient under anaerobic conditions, but rapid mineralization ensued with
these additives under aerobic conditions (Waisner et al. 2002). The most rapid
TNT transformations and lowest redox potentials were observed in slurry reactors
under aerobic conditions (Newcombe and Crawford 2007).
A statistical study estimated that optimal slurry values for TNT biodegradation
were 6.25 g/l glucose, 4.92 g/l Tween 80, 20.23 % (w/v) slurry concentration and
5.75 % (v/v) inoculum size. An improved oxygen supply in the bioreactor sig-
nificantly reduces bioremediation time in comparison to the use of shaking flasks
(Sheibani et al. 2011a, b). The overall degradation rate in a slurry reactor is
dependent on the soil–water ratio, substrate loading rates, system operating con-
ditions, soil characteristics, substrate characteristics, system configuration, and
nature of the aqueous phase solubility (Mohan et al. 1997).
4.2.4 Packed Bed Bioreactor
Continuously working aerobic and anaerobic bed reactors have been tested for
explosives degradation, using different carriers. One study focused on TNT
anaerobic biodegradation using a reactor with a synthesized polymer carrier with a
124 O. Muter
diameter of 20 mm, ethanol as an electron donor and a hydraulic retention time of

36 h (Wang et al. 2010). In other research, porous ceramic particles with immo-
bilized Rhodococcus sp. were used for aerobic treatment of the synthetic (TNP)
wastewater. The degradation of TNP (at a maximum volumetric removal rate of
2.53 g TNP/L d) was dependent on the pH and nitrite level as well as influent
concentrations and flow rates (Shen et al. 2009). Cells of Micrococcus sp. strain
SMN-1 immobilized in various matrices, such as polyurethane foam (PUF),
sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), agar and
polyacrylamide were tested in 2-nitrotoluene degradation experiment. The PUF-
immobilized cells achieved the highest degradation as compared to other carriers.
Immobilized cells showed more tolerance to pH and temperature changes than free
cells (Mulla et al. 2012).
The use of volcanic scoria as rigid support material for attaching microbial
communities in a multi-stage packed bed biofilm reactor, has been suggested
(Mondragon-Parada et al. 2008). Non-woven, short polyester textile fibers were
used to immobilize the fungi Phanerochaete chrysosporium in a fed-batch 5 L
reactor for TNT biotransformation; the most efficient transformation occurred at a
concentration of 5 mg TNT/L d and 1,100 mg glycerol/L d feeding rate (Rho et al.
2001).
Bioreactors with immobilized fungi, meet specific problems. There are several
operational problems related to filamentous fungi, that must be overcome: the
pulsing flow, the continuous removal of excess mycelium growth, the maintenance
of a fresh, active biofilm surface with a predetermined thickness, and the ability to
maintain mild aeration while providing high levels of dissolved oxygen (Moreira
et al. 2003).
Anaerobic degradation of RDX and perchlorate in a fluidized bed reactor was
shown to be dependent on the concentrations of ethanol. Besides, there was also
competition between RDX and perchlorate in the anaerobic treatment processes
(Atikovic et al. 2008).
The immobilization and/or incapsulation of microorganisms is also of great
importance in the context of bioaugmentation. Accordingly, target cells were
encapsulated with various blends of alginate, starch, and bentonite in order to
develop controlled-release formulations. The resulting beads may be suitable for
the efficient controlled release of bacteria in real field settings (Wu et al. 2011).
4.2.5 Constructed Wetlands
Constructed wetland treatment systems are engineered systems designed and

constructed to use the natural processes involving wetland vegetation, soils, and
their associated microbial assemblages to assist in treating wastewater. The
removal mechanisms depend mainly on (1) hydraulic conductivity of the substrate,
(2) types and number of microorganisms, (3) oxygen supply for the microorgan-
isms, and (4) chemical conditions of the substrate (Haberl et al. 2003). Vegetation
plays a key synergistic role via evapotranspiration, photosynthesis, and heat

accumulation in the biomass (Low et al. 2008; Pokorný et al. 2010).
TNT and RDX have been successfully removed or retained by constructed
wetland systems (Best et al. 1997, 1999). Sludge wetlands, designed for the bio-
degradation of organic compounds, reduced the amount of sludge by 50–70 %
mainly by dewatering, with corresponding reductions in the transportation cost and
environmental impact (Gustavsson and Engwall 2012).
Technical difficulties in the use of constructed wetlands are largely related to
the design and maintenance of the hydraulic controls as well as the bioconcen-
tration of RDX or other contaminants in plant tissue and the subsequent potential
exposure (Low et al. 2008). A demonstration study concluded that gravel-based
wetlands are more applicable for explosives-contaminated groundwater than are
lagoon-based wetlands (Sikora et al. 1998).
4.3 In situ Bioremediation
4.3.1 Phytoremediation
Phytoremediation is best suited for large, contaminated surfaces or volumes with

relatively low contaminant concentrations. Phytoremediation encompasses several
different technologies: phytoextraction (bioconcentrating); phytostabilization
(binding in plant tissues), phytodegradation (enzymatic degradation of toxic
compounds by plants and plant-associated microorganisms); and phytovolatiliza-
tion. The ability of plants to tolerate, transform, and translocate explosives varies
by species (Rodgers and Bunce 2001; Bert et al. 2009; De-Bashan et al. 2011; Panz
and Miksch 2012; Perreault et al. 2012).
A conceptual model known as the Green Liver, developed for the overall
phytodegradation of xenobiotics in a plant system, postulates that a crucial
detoxification mechanism is the conjugation of glutathione and xenobiotics by
glutathione S-transferases and compartmentalization into plant organelles, such as
the vacuoles (Yoon et al. 2007). This mechanism was also shown to apply to TNT
detoxification in plants (Gandia-Herrero et al. 2008).
Vetiver grass (Vetiveria zizanioides) was tested in hydroponic systems for TNT
degradation, using urea to stimulate the process. TNT metabolites were detected in
the roots, but not in the shoots of the plants (Makris et al. 2007a, b; Das et al.
2010). An earlier study suggested that rice, which is able to grow in lagoons, could
provide an economic and ecological alternative to physico-chemical methods for
RDX removal (Vila et al. 2007). After 40 days, 89 % of the radioactivity, that was
taken up, had been translocated into the rice leaves, with 90 % in leaf extremities.
RDX uptake and accumulation in maize was found to be concentration-dependent
both in hydroponic systems and RDX-spiked soils (Chen et al. 2011).
In a simulation study on TNT removal by poplar trees (Populus fastigiata),
about 25 % of the TNT was removed from the soil over a period of 90 days. Only
126 O. Muter
about 0.1 % of the total TNT mass remained in the roots, possibly because of the
rapid biodegradation (Ouyang et al. 2007). Four-year-old trees of hybrid willow
and Norway spruce were cultivated in sand or ammunition plant soil in wick-
supplied growth vessels. Approximately 80 % of 14C (TNT) was bound up in
roots, stems, wood, and leaves or needles in a non-extractable form (Schoenmuth
and Pestemer 2004a, b).
Experiments with marine macroalgae demonstrated an ability to reduce TNT in
seawater to 2-amino-4, 6-dinitrotoluene and 4-amino-2, 6-dintrotoluene, but these
products never accounted for more than 20 % of the initial TNT (Cruz-Uribe et al.
2007).
Transgenic plants could improve phytoremediation efficiency via enhanced
growth rate and biomass, deep root systems, increased metabolism, and other
factors. One of the promising developments in transgenic technology is the
insertion of multiple genes [for phase 1 metabolism (cytochrome P450 s) and
phase 2 metabolism (GSH, GT, etc.)] for the complete degradation of the xeno-
biotics within the plant system. Major concerns over field release of such genet-
ically manipulated plants include increased invasiveness and decreased genetic
variability of native plants due to interbreeding (Abhilash et al. 2009; Vangrons-
veld et al. 2009).
Phytoremediation under field conditions can be affected by variations in tem-
perature, nutrients, precipitation and moisture, plant pathogens and herbivory,
uneven distribution of contaminants, soil type, soil pH, and soil structure. In some
cases, the contaminated soil is deeper than the rooting zone (Vangronsveld et al.
2009). Difficulties in microbial-plant selection, measuring phytoremediation rates,
predicting treatment times, and developing monitoring schemes are some of the
recognized limitations of this method, especially for multi-contaminated soils
(Lebeau et al. 2008; McGuinnes and Dowling 2009).
4.3.2 Endophyte-Assisted Phytoremediation
Plants provide the primary energy source to soil microorganisms and affect the size
and composition of microbial communities, which, in turn, have an effect on
vegetation dynamics (Esteve-Núñez et al. 2001; Rodgers and Bunce 2001; Dowling
and Doty 2009).
For example, it was found that transgenic tobacco plants overexpressing a
bacterial nitroreductase gene detoxify soil contaminated with TNT, with a signif-
icantly increased microbial community biomass and metabolic activity in the rhi-
zosphere of transgenic plants compared with wild type plants (Travis et al. 2007). In
another study, Pseudomonas sp. combined with meadow bromegrass altered the
portion of the rhizosphere community involved in nitroaromatic metabolism and
led to a reduction in soil TNT levels (Siciliano and Greer 2000). Recently, Limane
et al. (2011) reported that the addition of specific microorganisms and nutrient
amendments as well as the cultivation of rye and blue fenugreek had a positive
effect on TNT degradation in soil and on the microbial community structure.
The synergistic interaction of plants and soil microorganisms benefits micro-

organisms through the provision of nutrients by root exudates and plants through
enhanced nutrient uptake and reduced toxicity of soil contaminants (Chaudhry
et al. 2005). Good colonization and survival of the inoculums under real-life
situations are vital for this approach (Ma et al. 2011).
4.4 Combination of Different Approaches
Abiotic treatment of explosives-contaminated soil/water can be used as the first

step in remediation, followed by biological treatment. Among the advanced oxi-
dation processes (AOPs), the following techniques are actively debated: processes
based on hydrogen peroxide (H2O2 ? UV, Fenton, photo-Fenton and Fenton-like
processes), photocatalysis, processes based on ozone (O3, O3 ? UV) and elec-
trochemical processes (Ayoub et al. 2010).
In situ redox manipulation, that is, the transformation of RDX, HMX, and TNT
by iron-rich sediments following treatment with dithionite, was described by
Boparai et al. (2008) who found that reaction rates depend on the concentration of
dithionite, solid-solution ratio, pH, and the buffering matrix. Sonochemical treat-
ment in the presence of a reductant offers an effective and rapid waste remediation
option for energetic waste compounds (Qadir et al. 2003). The use of nano-TiO2,
as a photocatalytic catalyst under simulated sunlight, was shown to be effective for
the degradation of RDX in wastewater and the results were nearly identical to that
of Fenton oxidation (Liu et al. 2006). The use of combined US-Fenton process for
the treatment of wastewater collected from a regional ammunition process site was
reported by (Li et al. 2013). A combination of solar TiO2-photocatalysis (6 h) with
constructed wetlands (16 h) was able to completely treat and detoxify 4-nitro-
phenol effluents with concentrations as high as 200 ppm (Herrera-Melián et al.
2012).
A photochemical procedure combined with a biological pre-treatment of TNT
was proposed by Kröger et al. (2004) and Kröger and Fels (2007) under anaerobic
conditions. In a later study Kwon and Finneran (2008) proposed that RDX gets
degraded via a variety of intermediates but is ultimately mineralized more quickly
and completely with electron shuttling compounds, such as anthraquinone-2, 6-
disulfonate.
Zerovalent nanoiron suspension, stabilized in dilute carboxymethyl cellulose
solution, has been suggested for the chemical degradation of RDX in soil (Naja
et al. 2009). Fe(0) barriers can effectively intercept RDX plumes, and treatment
efficiency can be enhanced by biogeochemical interactions though bioaugmenta-
tion (Oh and Alvarez 2002). A strategy, that uses electron shuttles and Fe(III)- and
humic-reducing microorganisms, may work at many contaminated sites (Kwon
and Finneran 2010). However, the abiotic step could lead to a drastic reduction in
the microbial populations which may affect the success of the follow-up biological
step (Ramos et al. 2005).
128 O. Muter
Nitrobenzene reduction was performed by steel convert slag (SCS) with Fe(II)
system. The results showed that SCS with Fe(II) was an effective reductant for
nitrobenzene at pH 5.5–6.5. The amount of Ca(II) in SCS determined the
adsorption capacity for Fe(II) and further determined the reduction capacity of
SCS with Fe(II) system (Luan et al. 2012).
A reductive technology based on a mixed two-phase reactor (bimetallic particles
and aqueous stream) was developed for the treatment of aqueous effluents con-
taminated with nitramines and nitro-substituted energetic materials (Koutsospyros
et al. 2012).
Plants can be used as a phytoslurry for TNT degradation. For example, in one
study, phytoslurries of parrotfeather and spinach removed TNT faster than the
intact plants (Medina et al. 2002). A combination of food-grade surfactant and
molasses, used in a soil slurry reactor, performed better than reactors containing
slurries with either molasses or surfactant alone (Boopathy 2002). The addition of
exogenous substrates may increase phytoremediation efficiency in the early stages
when the roots do not produce exudates rapidly (Sung et al. 2004). Activated
carbon added to contaminated soil reduces the toxicity of organic pollutants and
therefore, may provide more favorable conditions for biodegradation (Paul and
Ghosh 2011).
5 Soil Quality Criteria for Assessing Bioremediation

Efficiency
Criteria for indicators of soil quality relate mainly to their (1) utility in defining
ecosystem processes; (2) ability to integrate physical, chemical, and biological
properties; and (3) sensitivity to management and climatic variations (Doran
2000). The methods used for assessing soil quality are aimed at determining soil
chemical properties; soil microbial biomass, number, and activity, metabolic fin-
gerprinting, diversity and community structure, and plant–microbe interactions
(Avidano et al. 2005; Bloem et al. 2006).
Both organic and inorganic contaminants influence potential ammonium oxi-
dation (nitrification), whereas microbial respiration is predominantly affected by
biodegradable organic contaminants (Hund-Rinke and Simon 2008). A prevalence
of r-strategist bacteria was shown for both organic- and inorganic-contaminated
soils (Avidano et al. 2005).
An assessment of soil quality, at Canadian range and training area, found that
superoxide dismutase and neutral red retention time are relevant biomarkers for
signaling exposure to soils contaminated with energetic materials and that a bio-
marker index can be used as a soil quality indicator (Berthelot et al. 2009).
The current application of molecular techniques for the characterization of
microbial communities in contaminated soil and water as well as various high-
throughput techniques and their demonstrated or possible application to assess the
biotreatment of contaminated environments are reviewed by several workers (Kirk

et al. 2004; Malik et al. 2008; Stenuit et al. 2008; Gabriel 2010).
Bacterial and fungal diversity increases soil quality by affecting soil agglom-
eration and increasing soil fertility. In soils contaminated with TNT, the disap-
pearance of most Acidobacteria was associated with a shift in Acidobacteria
community composition and a loss of diversity (George et al. 2009). A recent
study (Nõlvak et al. 2013) indicates that introduced bacterial strains, especially
Pseudomonas species, survived and multiplied throughout a 28 day experiment
that used a combination of bioaugmentation and biostimulation coupled with rye
cultivation.
6 Ecotoxicological Considerations
TNT, RDX, and HMX are toxic for most classes of organisms including bacteria,
algae, plants, earthworms, aquatic invertebrates, animals, and mammals (Torre
et al. 2008). The toxic action of TNT on cells is commonly caused by the single
electron reduction of the nitro groups, mediated by type II (oxygen-sensitive)
nitroreductase (Šarlauskas et al. 2004) leading to oxidative stress (Peres and
Agathos 2000; Ask et al. 2004; Kumagai et al. 2004; Cenas et al. 2006). In the
presence of oxygen, the nitro anion radical reacts with oxygen forming a superoxide
anion radical and the original nitro group (Peterson et al. 1979). In addition to the
oxidative stress caused by the ‘‘futile cycling’’ of one-electron reductions, partially
reduced TNT products can bind covalently to proteins and DNA (Šarlauskas et al.
2004; De Lorme 2008; Torre et al. 2008).
It is important to note that some intermediates of TNT degradation, such as
tetranitroazoxytoluene, can notably delay RDX and HMX degradation due to their
cytotoxic effect on the RDX- and HMX-degrading microbial population (Nejidat
et al. 2008; Moshe et al. 2009). Other contaminants, such as metals, may also
contribute to the global soil toxicity (Robidoux et al. 2004b; Savard et al. 2007).
The toxicity of explosives has been examined using different methodological
approaches including eco- and geno-toxicological assays and dehydrogenase
activity (Neuwoehner et al. 2007; Cyplik et al. 2011). An effective approach
includes a battery of tests using representatives of the different trophic levels,
because toxicity is species- as well as chemical-specific and it is not possible to
predict at which link(s) the biological chain will be broken (Schäfer and Achazi
1999; Frische 2002; Persoone and Chial 2003; Dubova and Zarin ù a 2004; Ek et al.
2008; Flokstra et al. 2008; Mankiewicz-Boczek et al. 2008; Persoone and Wadhia
2009). The use of several trophic levels (plants: invertebrates: mammals: birds) is
required to assess the potential for biomagnification of explosives across the food
chain (Rocheleau et al. 2008). Herbivorous animals, such as prairie voles, can
accumulate RDX and allow the movement of RDX through different trophic levels
(Fellows et al. 2006; Rocheleau et al. 2008).
130 O. Muter
Bioassays can be used to evaluate the toxic effect of explosives; the most
typical are luminescence inhibition assays with Vibrio fischeri and growth inhi-
bition assays with V. fischeri and Pseudomonas putida (Frische 2002; Oh et al.
2003; Zeng et al. 2004). Gram-positive bacteria were found to be more sensitive to
TNT than gram-negative bacteria (Fuller and Manning 1997).
Three typical indicators of TNT phytotoxicity to plant species are increased
chlorosis, leaf loss, and lack of new growth (Pavlostathis et al. 1998). Plant tol-
erance to TNT depends on the species sensitivity, growth stage of the plant, TNT
bioavailability and soil characteristics. For instance, germinating seeds, seedlings,
and mature plants of the same species tolerate different concentrations of TNT
(Ramos et al. 2005; Juhasz and Naidu 2007; De Lorme 2008).
The presence of explosives can have a stimulating effect on many plant species,
especially at lower concentrations. For example, cress and turnip were stimulated
at concentrations of 5–25 mg TNT/kg and oat and wheat showed increased growth
at concentrations of 25–50 mg TNT/kg (Gong et al. 1999). Likewise, wheat,
barley, and radish were stimulated by a concentration of 8.54 mg NA/kg (Dubova
et al. 2009). However, barley growth was inhibited by a concentration of 56 mg
TNT/kg soil in silica (Robidoux et al. 2003) while tomato and cress were inhibited
by a concentration of 8.54 mg NA/kg (Dubova et al. 2009). When concentrations
of explosives were higher (e.g. 580 mg RDX/kg and 1720 mg TNT/kg), plants,
such as corn, tomatoes, nutsedge and lettuce all died (Pennington and Brannon
2002).
At a long-term TNT-contaminated site, the varying concentrations of TNT and
its metabolites across the site were observed to impact the extent and diversity of
the vegetation dramatically, with the most heavily contaminated area being
completely devoid of vegetation (Travis et al. 2008b). Koeleria glauca, which was
the sole plant species to grow near a detonation crater at the Adazi military camp
in Latvia, was found in medium-coarse, sandy soils contaminated by explosives as
shown in Fig. 2 (Dubova et al. 2009).
Fig. 2 Vegetation near detonation crater. Koeleria glauca is the sole remaining plant species
near the detonation crater
Physiologic differences among species, particularly those related to gastroin-

testinal structure and function, can affect the absorption of explosives and hence
lead to marked differences in toxicity from exposure to the same compound
(Johnson et al. 2010). To evaluate the impact of xenobiotics, toxicogenomic
techniques should focus on selected physiological functions in several ecologi-
cally/ecotoxicologically relevant organisms that are exposed under identical
conditions (Brulle et al. 2010).
7 Development of Future Technology
Current bioremediation approaches for soils contaminated by explosives and other

organic pollutants are limited by the poor capabilities of microbial communities in
the field, lesser bioavailability of contaminants on spatial and temporal scales and
absence of bench-mark values for efficacy testing of bioremediation for their
widespread application in the field (Megharaj et al. 2011). Hence, further research
on the remediation of explosives is needed, especially in the areas of mass balance,
cost reduction, and complete mineralization (Rodgers and Bunce 2001).
To improve cost effectiveness, the phytoremediation of explosives-contaminated
soils could be combined with bio-energy production. In this context, efficient
treatment methods for the contaminated biomass could be developed that minimize
the spreading of the contaminants into the environment during post-harvest treat-
ment (Snellinx et al. 2002). Phytoremediation as a technology is still in its infancy
and has not been used commercially to an extent (Glick 2003).
Some researchers have suggested that future interest in the field of explosives
biodegradation will be focused on the optimization of the catabolic properties of
indigenous microbes rather than on the development of recombinant strains
(Rodgers and Bunce 2001). In turn, other authors consider the design of microbe(s)
through molecular biology tools as one of the most promising way to improve
bioremediation performance (Kulkarni and Chaudhari 2007b; Kim et al. 2005).
Future research on the use of biostimulation for explosives biodegradation
should analyze the main biogenic components (e.g. carbon, nitrogen, potassium,
phosphorous, reducing sugars etc.) used during the remediation processes to
carefully determine if variations in composition affect the efficacy of technique
(Muter et al. 2012).
The current understanding of remediation processes is inadequate, especially
in situations involving multiple-element and mixed-mode pollution (Mench et al.
2009). Different permulating, and contributions are being made: e.g. mixed
microbial cultures originating from petroleum and pesticide-contaminated soils,
showed an ability to transform nitroaromatic compounds (Popesku et al. 2003;
Mulla et al. 2011; Erkelens et al. 2012).
Future probing systems are expected to be integrated with geophysical and
hydrogeological methods and combined with chemical and isotopic contaminant
analysis for source localization and identification (environmental forensics)
132 O. Muter
Table 1 Technological limitations to be addressed by methods for remediating soils contami-

nated with organic compounds, particularly explosives
Biotechnological Current limitations to be Contaminant References
approach or method addressed
Bioaugmentation Poor capabilities of microbial Organic Megharaj et al.
communities in the field contaminants (2011)
Application of Column operating conditions TNT, HMX, Hilber et al. (2009),
catalysts. need to be optimized. Many RDX, organic Boparai et al.
Zerovalent catalysts show poor contaminants (2008), Liu et al.
nanoiron, an absorption and utilization (2006), Naja
activated carbon of sunlight, and require et al. (2009)
fiber cloth-loaded ultraviolet light irradiation
with nano-TiO2, during wastewater
activated degradation. Unknown
charcoal (AC) long-term stability;
unresolved problems such
as how to mix AC into soil
homogenously or how to
select AC material with
suitable quantities and
reasonable price. Various
Fe oxides and Fe (II)
species form during
reduction of sediments by
dithionite; identification of
those forms responsible for
explosives transformation
would help guide further
applications of this
technology
Alkaline conditions The concentrations of the PNP Kulkarni and
with minimal stimulant and the Chaudhari
nutritional compound to be degraded (2007a)
requirements are critical for designing an
effective remediation
strategy
Slurry phase Optimal water ratio and Pendimethalin Mohan et al. (1997)
bioreactor substrate loading rates,
system operating
conditions, nature of soil,
nature of substrate, system
configuration, operating
conditions and nature of
aqueous phase solubility
must be determined
‘‘Attached growth’’ At present, the biodegradation DNT, 2,4,6- Shen et al. (2009),
in column limits are much lower than trinitrophenol Han et al. (2011)
systems, as well the regulatory (TNP)
as suspended requirements. Inhibition is
cells in chemostat dependent on influent
concentrations and flow
rates of the influent, pH,
and nitrites
(continued)
Table 1 (continued)
Fungal growth on Operational problems limit the Moreira et al. (2003)
surface viable operational period of
the bioreactor. Excessive
growth of mycelium affects
mass transfer, metabolic
rate, and product secretion
Addition of Surfactants can cause serious TNT Prak (2007),
surfactants environmental pollution Cserháti et al.
with toxic effects to living (2002)
organisms The relationship
between their chemical
structure, physicochemical
parameters, biological
activity, and environmental
impact is not well
understood. Solubility
behavior of nitrotoluenes in
surfactant solutions is not
clear
Production of Support characteristics and Organic Abouseoud et al.
biosurfactant immobilization conditions contaminants (2008)
for microorganisms-
producers must be
determined before
conducting experiments in
continuous reactors
Constructed Technical difficulties are RDX Low et al. (2008)
wetlands largely related to
bioconcentration of
contaminants in plant tissue
and subsequent potential
exposure, hydraulic factors,
and field variables such as
seasonal and site-specific
effects
(continued)
134 O. Muter
Table 1 (continued)
Phytoremediation Multiple-element and mixed- TNT, 2,6-DNT Yoon et al. (2007),
mode pollution, plants of Ouyang et al.
economic importance, and (2007), Abhilash
interactions among et al. (2009),
contaminants, soil, plant Vangronsveld
roots, and microorganisms et al. (2009),
are insufficiently Muter et al.
understood. Induction of (2012)
genes does not guarantee
the involvement of the
genes in the detoxification
of xenobiotics by plants.
Model for field-scale
simulations should be
extended to varying soil,
water, and temperature
regimes, soil microbial
communities, and air
temperatures. Disposal of
plants with accumulated
xenobiotics
Composting It remains unclear whether the TNT Meyns et al. (2002)
complexed TNT fraction
remains biologically
unavailable or becomes
transportable
Vermicomposting The quality of raw material, TNT, RDX, Robidoux et al.
pH, temperature, moisture, HMX; DNTs (2002), Hickman
aeration, type of and Reid (2008),
vermicomposting system, Shin et al.
and earthworm species used (2005), Yadav
must be specified. TNT is and Garg (2011)
more toxic for earthworms
than RDX and HMX.
Anaerobic
biotransformation of
dinitrotoluenes could
increase environmental
risk. Research should focus
on standardized,
comparative studies and
dedicated mechanistic
studies
(continued)
Table 1 (continued)
Bioindicators Highly variable susceptibility Organic Bhattacharyya et al.
of the test species exposed pollutants (2005), Franzle
to stressors, which leads to (2006), Kuncova
difficulties in comparing et al. (2011)
specific effect data.
Catabolic sensors do not
respond in a linear manner,
hence there is no direct
correlation with the
chemical concentration
Mixture of The presence of TNT in a TNT; RDX and Atikovic et al.
contaminants mixture with RDX and HMX. RDX (2008), Moshe
HMX inhibits and et al. (2009)
biodegradation of the latter perchlorate
two explosives.
Competition between RDX
and perchlorate in
anaerobic treatment
processes
(Kästner and Cassiani 2009). However, environmental responsibility, professional

expertise, management, manpower, and related facilities are the basis for the
development and realization of efficient remediation strategies (Mahidol 2005; Tai
and He 2007). In the coming years, the management of contaminated sites will
evolve rapidly from ecological risk assessment toward restoration of ecosystem
services (Guimarães et al. 2010).
The often-cited advantages of bioremediation approaches (e.g. low cost, easy of
operation, flexibility to respond to variable environmental conditions) as discussed
by Ward (2004), should be considered in light of the limiting factors and problems
discussed (Table 1). In fact, every contaminated site has a unique combination of
climate, geography, geology/soils and contamination history. Therefore, each site
requires a site-specific remediation strategy that combines modern (and sometimes
expensive) monitoring methods, engineering skills, and a thorough understanding
of biology. In most cases, this combined technology will not be of low cost. Even
so, bioremediation has an important role to play in mainstream technologies for
explosives and other types of soil clean-up because of its ability to provide tailored
solutions for specific contamination situations.
8 Conclusion
Contamination of soil and water with energetic compounds has a serious impact on
the environment. During last few decades, various technological solutions have
been developed to help clean up explosives-contaminated sites. However, many
136 O. Muter
remediation technologies are insufficiently understood because of variable and

complex environmental conditions, improper evaluation of the level and content of
contamination, and poor capabilities of introduced microbial communities in the
field. The often-cited advantages of bioremediation approaches—low cost and ease
of operation—may no longer be applicable. Efficient bioremediation requires
professional expertise to develop adequate remediation strategies, engineering
skills, and modern (and sometimes expensive) monitoring methods. However,
relatively high cost should not exclude bioremediation from wide use because of
its ability to provide tailored solutions for specific contamination scenarios.
Acknowledgments The work was supported by the Ministry of Defence, the Republic of Latvia
(Project AĪVA 2008/220), Latvian Council of Sciences (Project 09.1177), as well as the State
Research program Nr. 2010.10-4/VPP-5 NatRes. Author is grateful to Konnie Andrews for her
suggested manuscript revisions.
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Bacterial and Fungal Degradation
of Nitroglycrine
Divya Bhatia, Anita Grewal, Meenu Rathi and Deepak Kumar Malik
1 Introduction
Nitroglycerin (NG) is also known as trinitroglycerine, glyceryl trinitrate or more

formally: 1,2,3-trinitroxypropane (Fig. 1). It is a heavy, colourless, oily, explosive
liquid. Glycerol trinitrate (GTN) is a nitrate ester formed by the action of nitro-
nium ions on the hydroxyl groups of glycerol. It is used as an energetic plasticizer
in the manufacturing of double base gun and rocket propellants. It has been widely
used for more than a century as an explosive (Urbanski 1965). Nitroglycerin is also
a major component in double-based smokeless gun-powders used by reloaders.
Combined with nitrocellulose, there are hundreds of powder combinations used by
rifle, pistol, and shotgun reloaders.
In addition to military applications, the powerful physiological and therapeutic
effects of nitroglycerin have been widely used for the treatment of blood pressure
and heart diseases. Their physiological mechanisms of action are diverse; these
drugs can benefit patients in a variety of cardiovascular diseases, including cor-
onary artery disease, congestive heart failure (CHF), and acute myocardial
infarction. In addition, organic nitrates have been found recently to be potentially
beneficial in several other diseases and conditions, such as osteoporosis, anal
fissure and cancer pain management.
On other hand, nitroglycerin is an extremely powerful explosive and highly
toxic to humans and other organisms with an aqueous solubility of 1.25 g/l. It has
profound effects on systemic as well as cardiac microcirculation. Its actions are
mediated by stimulation of soluble guanylate cyclase in vascular smooth muscle
D. Bhatia A. Grewal D. K. Malik (&)

Department of Biotechnology, University Institute of Engineering and Technology,
Kurukshetra University, Kurukshetra, Haryana, India
e-mail: deepmolbio@rediffmail.com
M. Rathi
Department of Botany, University College, Kurukshetra University,
Kurukshetra, Haryana, India

150 D. Bhatia et al.
Fig. 1 Structure of
nitroglycerol
cells. Infrequent exposure to high doses of nitroglycerin can cause severe head-
aches known as ‘NG head’ or ‘bang head’. Long-term industrial exposure to NG
has been associated with withdrawal symptoms and sudden death from cardio-
vascular accidents (Klaassen 1996). Nitroglycerin is rapidly absorbed, distributed,
metabolized and eliminated in both laboratory animals and humans. Metabolism
appears to occur in both hepatic and extra hepatic tissues via stepwise denitrifi-
cation; elimination is primarily in the urine and expired air. Absorption is some-
what less in mice than other animals (Smith 1986).
Urinary metabolites in most species consisted largely of free mononitroglycerin
(MNGs), glycerol, and other polar metabolites including glucuronides, while tri-
nitroglycerin (TNG) and free di-nitroglycerin (DNGs) were excreted only in small
amounts. Mice excreted only small amounts of free MNG and DNG- and MNG-
glucuronides, indicating their complete biotransformation in this animal species
(USEPA 1992). Nitroglycerin is absorbed through intact skin in amount sufficient
to cause vasodilation. In humans, the most prominent manifestations of NG tox-
icity are severe headaches and adverse cardiovascular effects, including organic
nitrate dependence in the case of chronic exposure (Gilman et al. 1990). In ani-
mals, the adverse effect most often observed after administration of NG at high
dosage levels is decreased weight gain (related to decreased food consumption);
effects were also seen in the liver (lesions), blood (methemoglobinemia), and testis
(lesions and aspermatogenesis) (USEPA 1992). Exposure to high concentrations of
NG can also result in testicular lesions and male infertility, and delayed devel-
opment of offspring (Smith 1986).
During its production, large quantities of washing wastewaters, saturated with
GTN, are commonly transferred to lagoons or soakaways, resulting in actual or
potential contamination of soils. Nitroglycerin may be released to the environment
from its production and use as a component of propellants and explosives and as a
pharmaceutical compound. Wastewater discharges from the manufacture of
commercial dynamite preparations, military explosives, and other production
sources may contain NG. The relatively high solubility of NG in water (1,800 mg/l
at 20 °C) suggests that environmentally significant concentrations may be dis-
solved in waste rinse water and be expected to remain in the water column
(USEPA 1992). Nitrate esters, such as GTN, are known to persist in the envi-
ronment for a long time (Williams and Bruce 2000) and their biodegradation
presents a truly xenobiotic challenge to microorganisms due to the rarity of nat-
urally occurring analogues. Chronic exposure of laboratory animals to high NG
Bacterial and Fungal Degradation of Nitroglycrine 151
concentrations resulted in adverse haematological and liver changes, and

decreased body weight gain.
Physicochemical methods of GTN destruction involve adsorption on activated
carbon, followed by reduction using inorganic chemicals (such as sodium sulfite)
or alkaline hydrolysis, which yield glycerol and nitrite or nitrate. Although several
chemical methods for the disposal of GTN have been used but they are not very
effective because of incomplete degradation, large consumption of chemicals,
evolution of toxic or offensive gases, and their relatively high cost (ADPA 1975).
A natural attenuation and in situ bioremediation have been used for remediation in
soils contaminated with certain other explosives (Spain et al. 2000). Microbially
mediated denitration and mineralization are of considerable importance for the
treatment of GTN, as low cost alternatives to thermal and chemical destruction
methods. Moreover, it can be applied to the treatment of manufacturing waste
streams, spill mitigation, and ex situ or in situ soil remediation.
Several bacterial strains, that can biodegrade GTN, have been isolated previ-
ously by several workers (Meng et al. 1995; Binks et al. 1996; White et al. 1996a;
Blehert et al. 1997). These bacteria generally utilize GTN as a sole source of
nitrogen by removing either one or two nitro groups from GTN to form isomers
of glycerol dinitrate and glycerol mononitrate. Most exhibit no biodegradation of
glycerol mononitrate and are, therefore, incapable of completely mineralizing
GTN. To date, only one axenic bacterial strain, a Rhodococcus species has been
shown to achieve complete denitration of GTN (Marshall and White 2001). Pre-
viously, complete denitration has also been demonstrated in mixed bacterial
populations (Wendt et al. 1978) and in cultures of Penicillium corylophilum
Dierckx (Zhang et al. 1997).
2 Microbial Degradation of Nitroglycerol
GTN had been regarded as either recalcitrant to microbial biodegradation or even

inherently non-biodegradable (Logan 1953). Further, during the mid-l970s, some
reports even indicated that high concentrations of the ester ([900 ppm) were
inhibitory to bacterial growth (US Army 1973, 1974). Contrary to some earlier
reports that it was recalcitrant to biodegradation, NG proved to be readily bio-
degradable in batch and continuous tests. It was speculated in earlier experiments
that NG biodegradation was not conducted at high concentration that was toxic to
the microorganisms (Wendt et al. 1978; Smith 1986; Burrows et al. 1989).
First evidence, that GTN was biodegradable, came from studies with mixed
cultures in laboratory-scale activated sewage systems. Bacteria present were
capable of achieving a significant reduction in concentration of GTN, although
doubt remained as to whether the ester was actually supporting microbial growth
(US Army 1973, 1974). First discovery and isolation of GTN degrading bacteria in
activated sewage sludge, river water, and soils led to similar work by other
workers in Europe and the United States. The bacteria, able to utilize GTN as a
sole source of nitrogen, are now well known (Meng et al. 1995; Binks et al. 1996;
White and Snape 1996; White et al. 1996b; Belhert et al. 1997).
Several studies have been conducted to investigate the ability of microorgan-
isms to degrade nitroglycerin. Various bacterial and fungal species with nitro-
glycerin degrading ability have been isolated; a few of them are listed in Table 1.
In an initial study, Wendt et al. (1978) found that both mixed and pure bacterial
cultures, obtained from domestic sewage activated sludge, were capable of
degrading NG in a stepwise fashion, via the di- and monoesters. Cultures were
unable to utilize NG as a sole carbon source, and no attempts were made to
identify the organisms involved or to characterize enzymatic pathways. A follow-
up study by Pesari and Grasso (1993), in which a mixed bacterial culture was
assayed for its ability to degrade nitroglycerin, confirmed the findings of Wendt
et al. (1978) that mixed culture bacteria unable to utilize NG as a sole carbon
source. After that sone other NG-degrading strains of Bacillus thuringiensis/
Table 1 Bacterial and fungal degradation of nitroglycrine

Bacteria/Fungus Reference
Bacteria
Agrobacterium radiobacter Samantha et al. (2004)
Arthrobacter sp. strain JBH1 Husserl et al. (2010)
Bacillus thuringiensis/cereus Meng et al. (1995)
Enterobacter agglomerans Meng et al. (1995)
Pseudomonas putida Blehert et al. (1997)
Pseudomonas fluorescence Blehert et al. (1997)
Escherichia coli 8008 Blehert et al. (1997)
Klebsiella oxytoca 8701 Blehert et al. (1997)
K. planticola Blehert et al. (1997)
E. coli 8101 Blehert et al. (1997)
K. oxytoca 8408 Blehert et al. (1997)
P. aerofaciens Blehert et al. (1997)
P. aerofaciens C16 Blehert et al. (1997)
P. fluorescens 2-79 Blehert et al. (1997)
Agrobacterium radiobacter White et al. (1996b)
Enterobacter cloacae PB2 French et al. (1996)
Bacillus subtilis Fitzpatrick et al. (2003)
Pseudomonas putida Samantha and Graham (2001)
Arthrobacter sp. Samantha and Graham (2001)
Klebsiella sp. Samantha and Graham (2001)
Rhodococcus sp. Samantha and Graham (2001)
Fungus
Geotrichum candidum Ducrocq et al. (1989)
Phanerochaete chrysosporium Ducrocq et al. (1990)
Phanerochaete chrysosporium Servent et al. (1991)
Penicillium corylophilum Dierckx. Zhang et al. (1997)
Sclerotium rolfsii Sharma et al. (1995)
Fusarium solani Sharma et al. (1995)
cereus, Enterobacter agglomerans (Meng et al. 1995), and Agrobacterium ra-

diobacter have been identified.
Studies on nitroglycerin and nitroglycol biodegradation were conducted mainly
under aerobic conditions using bacteria (Bhaumik et al. 1997; Ye et al. 2004;
Dario et al. 2010), fungi (Sundaram et al. 1997), and plants (Meagher 2000; Eapen
et al. 2007; Rylott and Bruce 2008) thus far. Under anaerobic conditions, this
process was carried out with the use of an anaerobic sludge (Christodoulatos et al.
1997).
The aerobic experiments, carried out by Wendt et al. (1978) using activated
sludge with glucose as a growth substrate, demonstrated for the first time the
feasibility of microbial degradation of GTN by mixed cultures. A mixed culture in
a two-stage bench-scale activated sewage sludge unit converted GTN to 1,3-GDN
and 1,2-GDN in roughly comparable amounts. Later, it was concluded that
breakdown of NG occurred in two stages via the isomeric di- and mononitrates,
with each successive step proceeding at a slower rate (Wendt et al. 1978; Walker
and Kaplan 1992). In the environment, NG would likely be biotransformed
through a series of successive denitration steps, and the products mineralized by
biological systems are incorporated into the microbial biomass (Walker and
Kaplan 1992).
Several groups have investigated the biological transformation of nitroglycerin
under aerobic and anaerobic conditions, with pure and mixed cultures, and mostly
in the presence of additional sources of carbon. NG undergoes a sequential
denitration pathway in which NG is transformed to 1,2 or 1,3-dinitroglycerin
(DNG), followed by 1 or 2-mononitroglycerin (MNG) and then glycerol, under
both aerobic and anaerobic conditions (Meng et al. 1995; White et al. 1996a;
Bhaumik et al. 1997; Marshall and White 2001). First, one nitro group is reduced
from the nitroglycerin molecule, converting it to one of the isomers 1,3-DNG or
1,2-DNG. A second nitro group is removed converting the molecule to 1-GMN or
2-GMN (Bhaumik et al. 1997; Accashian et al. 1998). The removal of the last nitro
group to obtain glycerol is always more difficult, but can be achieved under
aerobic conditions (Meng et al. 1995; Marshall and White 2001). Even under
reduction utilizing elemental iron, a similar chain of intermediates and final
products was observed, with nitro groups being further reduced to ammonia groups
(Oh et al. 2004).
Pesari and Grasso (1993) reported biodegradation of GTN in a sequencing
batch reactor (SBR) used to treat actual wastewater from a ball powder propellant
manufacturing facility. White et al. (1993) reported the stepwise transformations
of GTN to GDN and GMN by a strain of Pseudomonas isolated from the river
sediment. They found that successive denitration occurs forming two isomeric
glycerol dinitrates which are subsequently converted to 1-GMN and 2-GMN. The
conversion of GTN to GDN showed significant regioselectivity for the formation
of 1,3-GDN. However, the Pseudomonas species did not have the ability to
denitrate the GMN isomers, and hence decomposition ceased. Denitration of GTN,
1,2-GDN and 1,3-GDN by Agrobacterium radiobacter subgroup B was reported
by White et al. (1996b), but this strain was unable to biodegrade the mono-nitrate
isomers.
A robust biochemical treatment method is preferable provided it can ensure
complete transformation [i.e., complete denitration without accumulation of
glycerol dinitrates (GDNs) or glycerol mononitrates (GMNs)] and was economi-
cally viable. Complete denitration is preferred because GDNs and GMNs are more
soluble than GTN itself and in some instances, more toxic (Ellis et al. 1978).
Nitrate esters were undetectable in effluent samples from the continuous biore-
actor, but pure cultures isolated from the continuous bioreactor and subsequently
grown in batch bioreactors were incapable of complete GTN denitration (Wendt
et al. 1978). A little or no reduction in GTN concentration in controls without
supplemental carbon clearly suggests that biological transformation (biotransfor-
mation) of GTN was a co-metabolic process. It is still not clear whether complete
denitration of GTN was achieved because no attempts were made to quantify
GDNs or GMNs. Co-metabolism was again suggested as the mechanism of GTN
biotransformation, because GTN acclimated cultures were incapable of using GTN
as a sole carbon source in batch reactors (Pesari and Grasso 1993). Although
several of these bacterial strains were capable of removing either one or two nitro
groups from GTN to form glycerol dinitrates (GDN) and glycerol mononitrates
(GMN), none was able to biodegrade GMN to achieve complete mineralization.
However, complete biodegradation has been observed in the mixed bacterial
cultures (Accashian et al. 1998) and fungi (Zhang et al. 1997).
Meng et al. (1995) demonstrated the total conversion of GTN to glycerol using
Bacillus thuringiensis/cereus cell extracts. Their findings suggest that denitration
involves hydrolytic cleavage of the nitro group, followed by a reduction of nitrate
to nitrite by nitrate reductase. Although complete denitration was achieved, but
field application of this approach is discouraged due to serious health concerns,
since Bacillus thuringiensis is an insect pathogen and Bacillus cereus is a mam-
malian pathogen. Furthermore, continuous addition of cell extracts, which was
necessary for complete conversion, may increase treatment costs especially in
wastewater matrices where other substrates are competing for the enzymes.
The denitration of NG by pure cultures of Agrobacterium radiobacter has also
been reported (White et al. 1996b), and in vivo nuclear magnetic resonance
measurements showed that both isomers of DNG accumulated, with 1,3-DNG
preferred by a roughly 8:1 ratio (corresponding to selectivity for denitration at the
C-2 position). Subsequent conversion of the DNG isomers to 1-MNG and 2-MNG
also occurred over a long time scale. Cell extracts prepared from this organism
used a reductive pathway in the presence of NADPH to release nitrite. A purified
recombinant penta erythritol tetra nitrate (PETN) reductase (White et al. 1996b),
originally from Enterobacter cloacae PB2, has also been shown to produce nitrite
during the NADPH-dependent denitration of NG. Due to the relative insolubility
of PETN, detailed steady-state kinetic studies of the reaction with NG were
undertaken with the recombinant enzyme (White et al. 1996b), but no analysis of
the regioselectivity of reaction was reported.
Earlier, it was assumed that NG is not suitable as a source of carbon and

nitrogen, so nutrients were considered essential. But later, pure bacterial cultures
were isolated that can use GTN as sole nitrogen source (White et al. 1996a; Blehert
et al. 1997). None of these cultures, however, possessed the ability to denitrate the
GMN isomers, resulting in their accumulation. A thermodynamic evaluation of
biochemical GTN denitration was performed assuming a reductive denitration
mechanism (Smets et al. 1995). The thermodynamic feasibility of GTN mineral-
ization, as a sole carbon and energy source under both aerobic and anoxic con-
ditions, was inferred. The failure of research groups to obtain enrichment cultures,
that use GTN as a sole carbon source, is potentially caused by limitations in
experimental approach, such as attempts involving batch-type experiments
(characterized by high initial GTN concentrations) as opposed to continuous
reactor conditions in which steady, low substrate environments provide more
efficient selective pressures. Furthermore, toxicity, resulting from high initial GTN
concentrations in batch experiments, may have been an additional barrier to
overcome in the isolation of cultures that use GTN as a sole carbon and energy
source.
In anaerobic experiments, Christodoulatos et al. (1997) used bacterial consortia
from an anaerobic digester to completely remove all nitrite and nitrate compounds
of NG. Bhaumik et al. (1997) also found anaerobic digester sludge completely
capable of denitrating NG. Pseudomonas putida and P. fluoroscens could not use
NG as a sole source to denitrate the intermediate GMN (Blehert et al. 1997).
Wendt et al. (1978) saw little to no reduction of GTN concentrations in control
samples without a carbon supplement. The bacteria Arthrobacter ureafaciens,
Klebsiella oxytoca and a Rhodococcus species were able to use NG as a sole
energy source (Marshall and White 2001). Other mixed bacterial cultures from an
aerated tank sludge rapidly degraded NG in the absence of a supplemental carbon
source (Accashian et al. 1998). Pseudomonas putida and P. fluorescens, isolated
from NG contaminated soils, were able to sequentially degrade toxic levels of NG
to GDN and GMN isomers, but could not denitrate GMN isomers (Blehert et al.
1997). Marshall and White (2001) isolated the bacteria Arthrobacter ureafaciens,
Klebsiella oxytoca and a Rhodococcus species from soil samples acquired from a
wastewater disposal lagoon at a formerly used NG manufacturing plant. All of the
bacteria were able to degrade GTN, producing GDN and GMN isomers with
Rhodococcus achieving complete removal of all nitrate esters.
Fungi use the GTN as a source of nitrogen. A variety of fungi have been
demonstrated with the capacity to degrade GTN. Biological treatments have
included biostimulation of existing indigenous microflora and bioaugmentation
where an explosive degrading microbial inoculum is added to the contaminated
environment (Kaplan 1990). Meng et al. (1995) reported that GTN can be com-
pletely denitrated during a long-term incubation with cell extracts of either
Bacillus thuringiensis and Bacillus cereus or Enterobacter agglomerans. Although
the method of Meng et al. (1995) appears to degrade GTN effectively, it must be
pointed out that B. cereus and E. agglomerans are mammalian pathogens whereas
B. thuringiensis is an insect pathogen. Further, the workers pointed out clearly that
this method is suitable only for the small-scale degradation of GTN.
Parrish (1977) isolated 190 fungi which have the explosive degrading capa-
bility. He concluded that the degrading capability of the fungi was affected by the
concentration of the nitroglycerine. Concentration of the nitro-glycerine above
20 ppm inhibits the growth of the fungi; that’s why Parrish discounted their use in
bioremediation. However, some studies suggest that under the right conditions,
fungi are capable of achieving mineralization explosive at rates far higher than
bacteria (Hawari et al. 2000). However, more recent studies have shown that the
ability to degrade explosives to some degree is distributed across many genera
within the Zygomycota, Ascomycota and Basidiomycota (Weber et al. 2002). A
few fungi, reported as having some degradative effects on nitrate esters, are dis-
tributed across the mitosporic fungi and wood rotting Basidiomycete genera. In all
cases of fungal degradation of nitrate esters, additional carbon sources have to be
supplied, but still degradation is only partial. Even when a cellulolytic species is
combined in co-culture with de-nitrating species (Sclerotium rolfsii and Fusarium
solani), decomposition is still incomplete (Sharma et al. 1995). Geotrichium
candidum was shown to have denitration capability, generating glycerol dinitrate
and glycerol mono nitrate with glycerol-2 mononitrate as the pre-dominant
products (Ducrocq et al. 1990).
The complete mineralization of GTN was reported by mixed cultures in
anaerobic microcosms, when supplied as the sole carbon and energy source
(Christodoulatos et al. 1997). Toxicity, in that case, may have been minimized
because of low initial GTN concentrations and relatively high biomass concen-
trations, thus limiting the effective GTN concentration per unit biomass. An earlier
study was carried out on a mixed microbial culture capable of complete denitration
and growth on GTN as sole carbon, energy, and nitrogen source under aerobic
conditions (Accashian et al. 1998). Although kinetics of aerobic denitration
exceeded that of anaerobic denitration, accumulation of GMN suggested toxicity
at initial GTN concentrations exceeding 0.3 mM. NG was metabolized to DNG
and MNG intermediates by various bacterial cultures (Gorontzy et al. 1994), and in
some cases, all glycerol nitrates could be removed from the culture medium.
Accashian et al. (1998) used sequential batch and packed bed reactors to study
NG degradation under aerobic conditions. Bhaumik et al. (1997) examined the
bioconversion of NG using mixed bacterial cultures and the fungus, Phanero-
chaete chrysosporium. The mixed cultures and P. chrysosporium completely
denitrified NG, forming GDN and GMN isomers which remained in the solution.
Accashian et al. (2000) reported that mixed microbial cultures required an inoc-
ulum from a wastewater treatment plant to initiate complete NG mineralization.
Complete denitration of NG by mixed microbial cultures showed a ten-fold faster
removal rate under aerobic than under anaerobic conditions from aeration tank
sludge (Accashian et al. 1998).
Phanerochaete chrysosporium was capable of denitrifying GTN under aerobic
conditions without an added carbon source, but conversion improved substantially
when a source was added (Bhaumik et al. 1997). Christodoulatos et al. (1997)
reported that mixed bacterial cultures in anaerobic with NG as the sole carbon
source, completely mineralized NG in 114 days compared to 26 days with an
addition of 2,000 mg/l of glucose. Penicillium corylophilum Dierckx is the only
single fungus culture reported to achieve complete denitration of all NG esters to
glycerol (Blehert et al. 1997; Marshall and White 2001).
Nitroglycerin degradation has been successful using natural and inoculated
organisms under both aerobic and anaerobic conditions, with and without the aid
of supplemental carbon sources. Although some microbes used in the studies were
isolated from contaminated army ammunition plant soil (Blehert et al. 1997) and
wastewater lagoon soil (Marshall and White 2001), no field soils were used as test
media. The test media used included laboratory culture media (Blehert et al. 1997;
Accashian et al. 1998, 2000; Marshall and White 2001), digester sludge (Bhaumik
et al. 1997; Christodoulatos et al. 1997) and wastewater from NG manufacturing
plants (Smith et al. 1983). However, investigations of NG degradation in field soils
have not been reported.
A soil isolated microbial consortium capable of biodegrading various organic
compounds and to reduce nitrate to atmospheric nitrogen under anaerobic condi-
tions was used. Complete removal of nitrates with simultaneous elimination of
nitroglycerin and ethylene glycol dinitrate (nitroglycol) was achieved (Cyplik et al.
2012). Saad et al. (2010a) studied the degradation of tri-nitroglycerin (TNG) with
zero-valent iron nanoparticles (ZVINs) in water either present alone or stabilized
on nanostructured silica SBA-15 (Santa Barbara Amorphous No. 15). X-ray dif-
fraction (XRD) showed that iron in both ZVINs and ZVINs/SBA-15 was present
mostly in the a-Fe0 crystalline form considered responsible for TNG degradation.
Transmission Electron Microscopy (TEM) showed that iron nanoparticles were
well dispersed on the surface of the nanosilica support. Both ZVINs and ZVINs/
SBA-15 degraded TNG (100 %) in water to eventually produce glycerol and
ammonium. Saad et al. (2010b) utilized nano-structured silica material [Mobil
Composite Material no. 48 (MCM-48)] prepared by mixing tetra ethyl ortho sil-
icate (TEOS) and cetyl trimethyl ammonium bromide (CTAB) to remove TNG
from water. In conclusion, the nano-structured silica based sorbent, with high
sorption capacity and remarkable reusability, should constitute the basis for the
development of an effective technology for the removal of TNG from the con-
taminated water.
3 Degradation of Nitroglycerin by Plants
The ability of plants to metabolize the xenobiotic nitrate ester, glycerol trinitrate
(GTN, nitroglycerin), was examined using cultured plant cells and plant cell
extracts (Goel et al. 1997). Intact cells rapidly degrade GTN with the initial
formation of glycerol dinitrate (GDN) and later formation of glycerol mononitrate
(GMN). A material balance analysis of these intermediates indicates little, if any,
formation of reduced, conjugated or cell-bound carbonaceous metabolites. Cell

extracts were shown to be capable of degrading GTN with the simultaneous for-
mation of GDN in stoichiometric amounts. The intermediates observed, and the
timing of their appearance, are consistent with a sequential denitration pathway
that has been reported for the microbial degradation of nitrate esters. The degra-
dative activities of plant cells are only ten-fold less than those reported for bac-
terial GTN degradation.
The uptake and transformation of nitroglycerine (NG) and ethylene glycol di-
nitrate (EGDN) from wastewater by plants using in vitro regenerants of Juncus
inflexus and Phragmites australis were investigated (Podlipna et al. 2010). The
plants were exposed to the NG (600 mg mg/l), the parent compound disappeared
during 20 days and degradation products as dinitroglycerine (DNG) and monon-
itroglycerine (MNG) were identified in the medium. During 20 days, the initial
concentration of 100 mg/l EGDN disappeared in the case of J. inflexus or
decreased to 5 % in the case of P. australis. Ethylene glycol mononitrate as the
degradation product was also identified.
It is highlighted that the possible employment of plants and fungi is limited for
the removal of nitroglycerin and nitroglycol from the soil. Moreover, these
methods have not found application in waste treatment techniques because of a
high nitrate level. Since nitrates act as methane fermentation inhibitors, their
presence limited the application of anaerobic sludge.
4 Conclusion
Cleaning up of nitroglycerin in the subsurface environment is a real world prob-

lem. A better understanding of the mechanism of biodegradation has a high eco-
logical significance that depends on the indigenous microorganisms to transform
or mineralize the organic contaminants. Based on the preformed studies, it can be
stated that selection of proper micro-organisms consortium, resistant to toxic
xenobiotics, is crucial for the removal of nitrates with a simultaneous nitroglycerin
and nitroglycol elimination from environment. Gradual usage of organic com-
pounds contained in wastes as a carbon and energy source for denitrifying
microorganisms allowed for the reduction of toxicity of the wastes. The use of
genetically modified (GM) bacteria represents a research frontier with broad
implications. The potential benefits of using genetically modified bacteria are
significant. But the need for GM bacteria may be questionable for many cases,
considering that indigenous species often perform adequately but we do not tap the
full potential of wild species due to our limited understanding of various phyto-
remediation mechanisms, including the regulation of enzyme systems that degrade
pollutants.
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Bioremediation of Perchlorate
Contaminated Environment
Atreyi Ghosh, Kannan Pakshirajan and Pranab Kumar Ghosh
1 Introduction
Perchlorate is a highly oxidized chlorine oxy-anion manufactured for use as the

oxidizer in solid propellants for rockets, missiles, explosives and pyrotechnics
(Urbansky 2000; Gullick et al. 2001; Logan et al. 2001). Approximately 90 % of
all perchlorate salts are manufactured as ammonium perchlorate for use in rocket
and missile propellants. The periodic replacement and use of solid propellant has
resulted in the discharge of more than 15.9 million kg of perchlorate salts into the
environment since the 1950s. Perchlorate salts are highly soluble in water. Since
sodium perchlorate has a solubility of about 2 kg/l, a large amount is readily
transported to surface and ground waters. The United States Environmental Pro-
tection Agency (USEPA) has identified perchlorate users and manufacturers in 44
states, and perchlorate releases in at least 20 states (USEPA 2005). Such per-
chlorate releases are estimated to have affected the drinking water supply to
15 million people.
Perchlorate can be detected by many methods including ion-selective elec-
trodes, ion chromatography, capillary electrophoresis, HPLC and spectropho-
tometry (Urbansky 2000) and among these methods, ion chromatography is the
most commonly used detection method for perchlorate. Besides, the anthropogenic
source of perchlorate is known to be Chilean caliche which is used in fertilizers.
A. Ghosh
Centre for the Environment, Indian Institute of Technology Guwahati,
Guwahati, Assam 781039, India
K. Pakshirajan (&)
Department of Biotechnology, Indian Institute of Technology Guwahati,
e-mail: pakshi@iitg.ernet.in
P. K. Ghosh
Department of Civil Engineering, Indian Institute of Technology Guwahati,

164 A. Ghosh et al.
The EPA developed a method for measuring perchlorate concentrations in fertil-

izers. It was concluded that most fertilizers did not contain perchlorate. Therefore,
it could not be an extensive source for perchlorate contamination in the environ-
ment (Urbansky et al. 2000).
Although there is currently no federal drinking water standard for perchlorate,
perchlorate has been included in the federal Contaminant Candidate List (USEPA
1998). High concentrations of perchlorate are known to affect the function of the
thyroid gland in humans by inhibiting the uptake of iodide (Wolff 1998). Recent
studies have also indicated that low concentrations of perchlorate significantly
inhibit iodide uptake in humans and animals (Losi et al. 2002; USEPA 2005).
Thus, perchlorate contamination of the environment poses a threat to not only the
indigenous wildlife as well as human health, but also to the normal growth and
development of amphibian populations. The Office of Environmental Health
Hazard Assessment in California EPA has proposed a public health goal of 6 mg/l
for perchlorate in drinking water. In a recent perchlorate risk assessment draft
report, the USEPA (2005) proposed a draft reference dose of 0.03 mg/kg of body
weight per day, which could produce a drinking water equivalent level of 1 mg/l to
protect human health. Based on this information, Department of Health Service in
California decreased the action level for perchlorate in drinking water from 18 to
4 mg/l (DHS 2002). In New Mexico, the action level was set at 1 mg/l. In the last
10 years, several reviews have been published on various perchlorate issues that
include: bacterial degradation (Herman and Frankenberger 1998; Kim and Logan
2001) chemistry and analytical chemistry (Urbansky 2000) toxicological studies
and drinking water standards (Urbansky 2000; USEPA 2005); and contamination
sources and occurrence data (Wolff 1998; Urbansky 2000; Gullick et al. 2001;
Logan et al. 2001). Many important advances made in the treatment of perchlo-
rate-contaminated water after existing treatment technologies were reviewed by
Herman and Frankenberger (1998). Besides, many other reports have also high-
lighted the biological treatments of perchlorate which are capable of removing
perchlorate down to levels to be suitable for drinking water i.e., 4 mg/l (Attaway
and Smith 1993; Herman and Frankenberger 1999; Logan et al. 2001; van Ginkel
et al. 2008, 2010). In the current review, we have mainly focussed on recent
developments that have improved our understanding of the bacteria responsible for
perchlorate reduction, the pathways used and the treatment systems developed
using perchlorate respiring bacteria (PRB). In addition, we provide a brief back-
ground on the chemistry, occurrence, health issues, and drinking water issues
related to perchlorate contamination.
2 Perchlorate and its Various Sources
Perchlorate ion consists of a tetrahedral array of oxygen atoms around a central

chlorine atom. It is a strong oxidizing agent owing to the +7 oxidation state of the
chlorine. In this respect, perchlorate is slightly weaker than dichromate or
Bioremediation of Perchlorate Contaminated Environment 165
permanganate. However, perchlorate reduction is extremely slow and can usually

be observed only under strong acidic condition. In fact, the redox behavior of
perchlorate is so rarely observed in chemical systems that sodium perchlorate is
used to adjust the ionic strength of solutions prior to electrochemical or other
laboratory studies (Urbansky 1998). Complexes of perchlorate are usually rare. As
an oxidant, perchlorate is kinetically non-labile, which means the reduction of the
central chlorine atom from an oxidation state of +7 (perchlorate) to -1 (chloride
ion) occurs extremely slowly. Perchlorate sorption is not expected to attenuate
because it absorbs weakly to most soil minerals. Thus, natural chemical reduction
in the environment is not expected to be very significant. These two factors
account for perchlorate being both very mobile in aqueous systems and persistent
for many decades under typical ground and surface water conditions. The acti-
vation energy to perchlorate reduction is so high that it cannot be expected to act as
an oxidant under human physiological conditions (i.e., diluted solution, ambient
temperatures and neutral pH). This is supported by absorption, distribution,
metabolism, and elimination studies that show perchlorate is excreted virtually
unchanged in the urine after absorption. Thus perchlorate appears to be an another
addition to a growing list of halogenated chemicals that persist in the environment,
but the chemical characteristics of this inorganic anion make it quite unusual.
Typical chlorinated aliphatic compounds, e.g., trichloroethylene (TCE), are rela-
tively insoluble carbonaceous compounds, strictly used for industrial purposes. In
addition, these compounds are volatile in nature and capable of being absorbed and
can be reduced by metals, such as zerovalent iron. In contrast, perchlorate is a
highly soluble inorganic anion (2.09 kg/l for NaClO4) that adsorbs poorly to
mineral surfaces and activated carbon and is not retarded during groundwater
transport. Perchlorate salts have the origin from both natural and anthropogenic
sources. The natural sources are mostly confined in arid and semi-arid environ-
mental conditions. Natural perchlorate was first identified in Chilean nitrates over
100 years ago. Perchlorate has also been detected in both drinking water and saliva
samples collected in India. Concentrations of perchlorate measured in drinking
water in India are one to two orders of magnitude lower than the concentrations
reported for many industrialized countries, like USA, Japan and Korea. However,
concentrations of perchlorate in water samples did not exceed the USEPA’s
interim health advisory level of 15 lg/l for perchlorate in drinking water. Based on
the mean concentration of 0.1 lg/l in drinking water from India, exposure of
perchlorate for a 70 kg adult drinking 2 l/d of water would be 0.003 lg/kg bw/d,
which is \1 % of the reference dose established by the EPA. However, concen-
trations in saliva exceeded the concentrations in the water samples with several
saliva samples containing concentrations above 1 lg/l, suggesting the presence of
other sources of perchlorate exposure for the Indian population. Studies have
reported foodstuffs to be a source of perchlorate in the United States (Sanchez
et al. 2005) and further investigation is needed to examine the sources of exposure
of the Indian population to perchlorate.
166 A. Ghosh et al.
3 Perchlorate: A Potent Environmental Pollutant
Perchlorate is manufactured in large quantities as ammonium perchlorate, pri-

marily for use as an oxidizer in solid rocket propellants. Its contamination is
mostly associated with military activities or defense contractors (Gullick et al.
2001). It is also used in vehicles, electroplating operations, perchloric acid pro-
duction, electro-polishing, production of matches, flash powder for photography,
bleaching agent, leather tanning, oxygen generators, ejection seats, paints and
enamels, etching of copper and brass, road flares and fireworks. Wastes from the
manufacture and improper disposal of perchlorate-containing chemicals are
increasingly being discovered in soil and water. Ammonium perchlorate
(NH4ClO4) has been used as an energetic booster in the rocket fuels and it appears
that most perchlorate contamination is the result of discharge from rocket fuel
manufacturing plants or from the demilitarization of weaponry (missiles). Potas-
sium perchlorate (KClO4) can be used as a solid oxidant for rocket propulsion and
it was the original source for perchlorate contamination. However, most of the
contamination appears to have come from the legal discharge decades ago of then
unregulated waste effluents containing high levels of ammonium perchlorate.
Although ammonium perchlorate was released initially, the salt is highly soluble
and dissociates completely to ammonium and perchlorate ions upon dissolving in
water. Perchlorate has been found in ground waters in the United States at typical
concentrations of 50–200 mg/l, primarily as a result of production and use in solid
rocket propellant (Urbansky 2000). Most of the affected regions have perchlorate
concentrations below 0.5 g/l; however, concentrations as high as 3.7 g/l have also
been detected (Urbansky 2000). Perchlorate in sewage sludge, rice, bottled
drinking water and milk was detected in China while investigating the perchlorate
pollution status (Shi et al. 2007). Perchlorate is known to interfere with the uptake
of iodine in the thyroid at the (Na+)-iodide (I-) symporter, or NIS of thyroid gland,
thereby causing a reduction in the hormones thyroxine (T4) and tri-iodothyronine
(T3) (USEPA 2005). Hyperthyroidism due to iodine deficiency during pregnancy
is a cause of cretinism, a permanent cognitive impairment of the developing fetus.
Perchlorate is thought to be responsible for abnormal fetal and child growth and
development (Urbansky 1998, 2000). In some cases, thyroid gland tumors can be
caused due to the disruptions in thyroid hormone levels. Despite the fact that the
appreciation of widespread perchlorate contamination emerged only a few years
ago, considerable progress has been made in hazard identification and quantitative
dose–response characterization for both the human health and ecotoxicological
risks of potential perchlorate exposures. The thyroid has been confirmed as the
target tissue in humans, laboratory animals and wildlife. The key event of the
mode of action for perchlorate is iodide uptake inhibition at the NIS with the
potential for both subsequent neurodevelopmental and neoplastic sequelae. A
harmonized human health reference dose has been proposed to be protective for
both sequelae based on a mode of action model. Additional research is needed to
determine the contribution of sources of perchlorate exposure other than drinking
water. This requires more progress in the area of analytical methods to extend
current approaches to other media. The office of Environmental Health Hazard
Assessment in California EPA has proposed a Public Health Goal of 6 lg/l for
perchlorate in drinking water. In the National Academy of Science’s (NAS’s)
January 2005 report, maximum permissible dose for perchlorate was proposed to
be 0.7 lg/kg/d (Gu et al. 2007).
4 Perchlorate Treatment Technologies
Perchlorate treatment technologies can be divided into two primary categories,

destruction and removal. Removal technologies encompass broadly the physico-
chemical and biological treatment. The physico-chemical methods include elec-
trochemical reduction, ion-exchange, membrane filtration, electrodialysis, catalytic
reduction and biological treatment including phytoremediation and microbiological
treatment processes. A recent report by USEPA indicates that ion exchange and
bioremediation are among the most commonly used technologies to remove or
degrade perchlorate from the contaminated media.
4.1 Membrane-Based Techniques
Membrane-based techniques can be also effective for perchlorate removal but they
suffer from several drawbacks. While reverse osmosis (RO) would effect sufficient
remediation, it can be impractical for a municipal treatment system because of the
fouling of membranes and the associated cost. Moreover, RO-treated water has to
be remineralized with sodium chloride, sodium bicarbonate and other innocuous
salts to prevent degradation of the distribution system and to make the water
palatable, since deionized water generally is considered to have an unpleasant
taste. Therefore, as long as sufficient salts are taken in from food and other sources,
consumption of deionized water is not likely to pose a threat to the normal elec-
trolyte balance. As with RO, electrodialysis also might be used in this fashion.
These two techniques are probably best suited for point-of-use or small systems.
4.2 Anion Exchange
Although perchlorate ion is strongly retained by quaternary ammonium resins, the

low initial concentration of perchlorate limits application of this method in several
cases. For example, it might be necessary to reduce perchlorate concentration from
1 mg/ml to 20 ng/ml. However, bicarbonate, carbonate, chloride, and a host of
other anions are all likely to be present at much higher concentrations. Assuming
168 A. Ghosh et al.
that a chloride-form resin is used, the presence of phosphates, carbonates, and

sulfate remains an issue. Although it may be possible to produce a resin salt that
matches the proportions of the major anions in the influent water, it would be
extremely inconvenient. In addition, the low concentration of perchlorate in the
raw water substantially reduces the driving force for its removal. In other words,
adequate removal of perchlorate may require essentially demineralizing and
remineralizing the water depending on its anion content. It is possible to modify
resins so as to improve their selectivity for particular anions. Selectivity of the
resin Dowex 1X-8 for perchlorate is 100 times better for chloride and 10 times for
nitrate. In addition to selectivity in a thermodynamic context, there is a matter of
serious concern for rapid equilibration and anion exchange. If the rate of exchange
is too slow, a resin will not be usable no matter how high its selectivity. The U.S.
Department of Energy developed a mixed triethylammonium-trihexylammonium
resin that is capable of removing pertechnetate down to the parts-per-trillion (ppt)
level (Brown et al. 2003). Tethered triphenylarsonium or phosphonium moieties or
a tethered (through a phenyl group) nitron might work in an anion-exchange resin
to selectively preconcentrate perchlorate as a step in the remediation. The disad-
vantage of the tethered triphenylarsonium group is that normal degradation of the
resin would lead to the release of arsenic into the treated water. Although the
health effects of nitron are unknown, it will undergo biodegradation; furthermore,
it would be destroyed readily by UV irradiation (185 nm), whereas arsenic would
remain as an inorganic oxyanion even if the organic portion of the species are
destroyed.
4.3 Precipitation
The low solubility of the HNitClO4 (complex of nitron and perchlorate) ion pair
reveals a strong association between the protonated nitron cation and the per-
chlorate anion. All insoluble ion pairs and complexes exist at same level in
solution. It may be possible to exploit this pairing for purposes of remediation. If
the addition of nitron to perchlorate-containing waters results in the formation of
the soluble ion pair, it may be possible to subsequently induce an intramolecular
reaction in which both the perchlorate and the nitron are destroyed. Photoactiva-
tion of the perchlorate by UV or laser irradiation may promote an intramolecular
redox reaction (probably by oxygen atom transfer). The proximity of the HNit+-
ClO4- ion pair within a solvent (water) cage means that it is not necessary to form
an encounter complex. In addition, the local concentration of the two species is
very high within the solvent cage. This helps in reducing the effects of the per-
chlorate kinetic barrier (discussed below). Indeed, irradiation with UV light also
promotes destruction of the nitron by hydroxyl radical formation. Ideally, the
largest possible wavelength (lowest frequency and energy) light would be used to
reduce side reactions that would destroy the nitron. Unfortunately, nitron has
potential to remediate only those sites with very high perchlorate concentrations.
However, the cost of nitron is a limiting factor. At present, nitron is about 52 times
more expensive than an equal mass of reagent-grade sodium chloride. At some
of the sites, where the perchorate concentration is 0.037 M, nitron could readily
be used as a precipitant since the nitron-hydrogen perchlorate salt has a solubility
of only 0.19 mM. Although the action level of 18 ng/ml corresponds to 0.18 M, a
level of 0.19 mM is certainly preferable to 37 mM. Of course, one drawback is
that a source of acid (usually 5 % acetic acid) must be present. On the other
hand, vinegar is probably preferable to 0.037 M ClO4-. Moreover, such post-
remediation acetic acid would be biodegradable. In addition to cost, all physical
separation processes have one major problem i.e., waste disposal. Presumably, the
regenerant from ion exchange and the concentrate from RO or electrodialysis
would contain perchlorate at concentrations too high to be released into a sewage
system. This waste presents a problem in terms of cost and post-treatment
needs. Although these techniques take the perchlorate out, they concentrate it
somewhere else.
4.4 Chemical and Electrochemical Reduction
Chemical reduction ion, specifically in the redox sense of adding electrons, is

simply too slow in case of perchlorate and therefore does not appear to play any
role to the drinking water treatment unless safe new catalysts become available.
Commonly used reductants, such as iron metal, thiosulfate, sulfite, iodide and
ferrous ions, do not react at any observable rate and the more reactive species are
too toxic. In addition, any reductant has oxidized by-products and the toxicity of
the by-products must be considered before its application. Thus, there is a hope for
electrochemical reduction with certain advantage. A definite advantage of the
technique is the large amount of control over kinetics that results from control of
the operating potential. Electrode reduction kinetics can be viewed as being lim-
ited by three factors: (1) diffusion of the ions to the electrode surface (2) associ-
ation with the electrode surface, and (3) activation past the overpotential required
to reach the transition state. Among these factors, overpotential is a major limiting
factor which can still be overcome. Although some may be affected by electro
reduction, this probably does not present a significant obstacle. To date, this option
has not been explored for low-concentration treatment at anything approaching
pilot scale. Although electrochemical technologies are well established for other
industries (e.g., electroplating of metals, electrolysis of brine), these techniques
have not yet found a place in the drinking water treatment. By reduction method,
the most successful strategies for remediating perchlorate contamination utilize
metal cation-catalyzed reduction either chemical or electrochemical. Several metal
chelates have this potential, especially if embedded in an electrode for use in
electrochemical reduction.
170 A. Ghosh et al.
4.5 Biological Treatment Method
All physico-chemical techniques to treat perchlorate contamination have high

capital and maintenance cost and generates of a large quantity of brines and spent
resin having high perchlorate concentration. In addition, membrane fouling by
alkaline earth and transitional metal compounds can present a problem depending
on their concentration in the water. Under this scenario, bioremediation serves to
offer the best solution as revealed by batch studies on the effects of several
environmental factors and co-pollutants on perchlorate reduction by a bacterial
mixed consortium (Ghosh et al. 2011). Bioreduction of perchlorate in the engi-
neered systems offers the greatest potential for inexpensive and complete per-
chlorate removal. Several reactor technologies have shown a potential to treat
perchlorate. There are total 5 full scale and 15 pilot scale in situ bioreactors
implemented at different states in the US where perchlorate contamination in
ground water has been detected above the permissible limit. Although a bioreactor
system is technically feasible for perchlorate removal, it can be proven to be
ineffective or costly for treatment at its low concentration (e.g., at 100 ppb)
because a highly reducing environment is required for perchlorate removal using
microorganisms. Additionally, certain microbiota can irreversibility foul or dam-
age the membrane material, necessitating its complete replacement.
4.5.1 Perchlorate Reduction Pathway
Perchlorate respiring bacteria (PRB) have been found in many different environ-
ments making it possible to bioremediate perchlorate-contaminated environments
(Attaway and Smith 1993; Herman and Frankenberger 1999; Hatzinger et al.
2000). As shown in Fig. 1 perchlorate is used as a terminal electron acceptor by
pure and mixed cultures of microorganisms (Logan et al. 2001; Herman and
Frankenberger 1998). The reduction of perchlorate or chlorate to chloride by
bacteria was also subsequently confirmed by many other researchers (Korenkov
et al. 1976; Rikken et al. 1996). However, none of the intermediates accumulates
in solution (Attaway and Smith 1993; Herman and Frankenberger 1998; Logan
et al. 2001). Several bacterial strains belonging to the genera Vibrio, Wolilella,
Dechloromonas, Dechlorosoma, Dechlororspirilium and Citrobacter have been
identified and tested for perchlorate reduction. In all these bacteria, chlorite dis-
proportionation to chloride and oxygen is a non-energy yielding step catalyzed by
chlorite dismutase enzyme (Rikken et al. 1996).
4.5.2 Bioreactor Systems for Perchlorate Removal
The conventional bioreactors for ex situ treatment of perchlorate employ either

fixed or fluidized film bioreactors in plug flow or recirculation mode with acetate
Fig. 1 Schematic showing

the pathway involved in the
perchlorate reduction by
perchlorate reducing bacteria
(PRB)
or H2 as the electron donor. In the fixed film packed-bed bioreactor, sand, plastic,
glass bead, activated carbon or elemental sulfur is used as support media (Wallace
et al. 1998; Min et al. 2004; Sahu et al. 2009; Chung et al. 2010). However, in
fluidized-bed bioreactors either sand or GAC is used for microbial colonization
and high recycle rates used to keep the support medium suspended and mixed
(Xiao et al. 2010). Many of these bioreactors are efficient in reducing perchlorate
to acceptable levels along with removal of several co-contaminants. Positive
enrichment of highly specific perchlorate reducing bacteria has been demonstrated
under different operational conditions (Nerenberg et al. 2008; Ahn et al. 2009;
Xiao et al. 2010). Recently, McCarty and Meyer (2005) developed a numerical
model based on the relative penetration of competing electron acceptors (O2,
nitrate and perchlorate) in the biofilm of a fluidized-bed reactor for the treatment of
ground water. The ex situ treatment process is particularly suitable for the treat-
ment of highly concentrated waste streams originating from the perchlorate
manufacturing units or the munitions handling facilities. However, direct appli-
cation of this process for the treatment of drinking water is questionable at the
moment. The high operational cost and excess biomass build-up and clogging
resulting from the use of organic electron donor limits a large scale application of
this technology. Possibility of secondary contamination of treated water with
microbial cells and their metabolic by-products is also a major concern. Carryover
of organic residues can increase the demand for chlorine for disinfection, leading
to formation of unacceptable disinfection by-products in the treated water.
Moreover, presence of residual ethanol and methanol can be unacceptable in
drinking water supplies. To circumvent some of these problems, in recent years, a
whole range of newer generation bioreactors have been developed. These include
H2-based hollow-fiber membrane biofilm reactor (MBfR) (Nerenberg et al. 2008;
van Ginkel et al. 2008, 2010; Sahu et al. 2009; Chung et al. 2010), ion exchange
172 A. Ghosh et al.
Fig. 2 Shcematic typical

flow through reactor treating
pechlorate contaminated
ground water (Schaefer et al.
2007)
membrane bioreactor (IEMB) (Matos et al. 2006) and bioelectrical reactor (BER)
(Thrash et al. 2007) or a microbial fuel cell (MFC) (Butler et al. 2010). The MBfR
systems offer benefits in terms of less biomass production and lower solubility of
H2 in water (1.62 mg/l at 25 °C and 1 atm H2), there by eliminating the need for
post-treatment removal. Similarly, the insoluble elemental S°-based chemolitho-
trophic perchlorate reduction can be suitable for ensuring a long-term sustained
supply of electron donor in low maintenance bioreactors (Sahu et al. 2009). The
IEMB process simultaneously combines advantages of Donnan dialysis and bio-
logical perchlorate reduction and selectively removes ionic pollutants. Figure 2
shows a schematic typical flow through reactor system used for treating per-
chlorate contaminated water (Schaefer et al. 2007).
Many heterotrophic biological treatment systems have been tested to degrade
perchlorate in suspended, fixed-bed and fluidized-bed reactors (Attaway and Smith
1993; Wallace et al. 1998; Green and Pitre 1999; Hatzinger et al. 2000; Logan
et al. 2001). Organic electron donors that have been used in the bioreactors include
simple compounds, such as acetate and ethanol as well as more complex organic
substrates, as found in the compost piles. Perchlorate degradation has also been
achieved in bioreactors using only inorganics. These reactors are sustained by
hydrogen gas delivered by pressurization, gas transfer across liquid films or syn-
thetic membranes (Miller and Logan 2000; Nerenberg et al. 2008). These
hydrogen-based technologies are promising technologies for water treatment
because less biomass is produced by autotrophic processes than heterotrophic
processes. Large-scale tests are needed to evaluate process efficiency and the
economics of different hydrogen-based systems. Although at least one biological
Table 1 Laboratory-scale bioreactor systems studied for perchlorate removal from contaminated waters
Bioreactor system Microorganism used Water/wastewater source Bioreactor operating conditions Treatment References
efficiency
Hydraulic Influent Effluent ClO-4 Electron
(%)
retention time ClO-4 (mg/ (mg/l) Donor(s)
(HRT) l)
Fluidized bed reactor with Mixed culture Drinking water well 3.1 h 6.7 \0.400 Acetate, 99.94 Greene and Pitre
sand and activated Methanol, (1999)
carbon media Ethanol
Up flow reactor packed with Mixed culture Synthetic water 51 min 100–1,000 \0.005 Lactate 99.99 Logan et al. (2001)
sand
Autotrophic packed bed Pure culture Synthetic water 1.1–1.3 min 0.740 0.460 Hydrogen 37.83 Miller and Logan
biofilm reactor (2000)
Suspended growth reactor Wollinella succinogenes Synthetic water 3h 0.13,0.738 0.01, 0.031 BYF-100 98.64, Attaway and
HAP-1 in mixed culture 96.00 Smith (1993)
Fixed bed (sand GAC) Mixed culture Synthetic water 10 h 0.13, 0.01, 0.031 Acetate 98.64, Herman and
0.738 96.00 Frankenberger
(1999)
Fixed bed (GAC) Tapwater Synthetic water 1h 0.051 \0.004 A mixture of 76.15 Brown et al.
acetate, (2003)
lactate and
pyruvate
Bioremediation of Perchlorate Contaminated Environment
Fixed bed (cylindrical pall Primary sludge and and Synthetic water 0.11 \0.004 Acetate 99.60 Burns et al. (2001)
rings) effluent from
wastewater treatment
plant
Fixed bed (celite pellets) Perclace Synthetic water 0.8 \0.004 Acetate 99.50 Losi et al. (2002)
Ion exchange membrane Polluted water Enriched mixed culture 0.25, 1.4, 2.0, 1 \0.004 Ethenol 99.98 Matos et al. (2006)
from municipal sludge 4.0, 8.3 h
Fixed bed (glass beads) Mixed culture Synthetic wastewater 0.073 \0.004 Hydrogen 94.52 Logan and Lapoint
(2002)
(continued)
173
Table 1 (continued)
174
Bioreactor system Microorganism used Water/wastewater source Bioreactor operating conditions Treatment References
efficiency
Hydraulic Influent Effluent ClO-4 Electron (%)
retention time ClO-4 (mg/ (mg/l) Donor(s)
(HRT) l)
Fluidized bed (sand or Biological solids from an Synthetic wastewater 25 Ethanol, 99.98 Hatzinger et al.
GAC) anaerobic digester Methanol or (2000)
mixture of
the two
alcohols
Hollow-fiber membrane Ralstonia eutropha Synthetic wastewater 1h 0.1 \0.0003 Hydrogen 96.00 Nerenberg et al.
bioreactor. (2008)
Fixed bed (sand) Dechlorosoma KJ. Synthetic wastewater 2.1 min 20 \0.004 Acetate 99.98 Kim and Logan
(2001)
Fixed bed (GAC) Mixed culture Drinking water 18 min 20 \0.004 Acetate 99.98 Kim and Logan
(2001)
Fluidized bed ractor with Mixed culture Synthetic wastewater 154 min 300 \0.3 Acetate 99.90 Patel et al. 2008
GAC as packing
material
Hollow fibre membrane Contaminated drinking Contaminated drinking 90 min 1 0.01 Hydrogen 99.92 Nerenberg and
biofilm reactor (MBfRs) water water Rittmann
(2004)
Hollow fibre membrane Synthetic wastewater Mixed consortium 4, 8, 20, 24, 5, 30, 40, \0.004 Acetate 99.92, van Ginkel et al.
biofilm reactor (MBfRs) 29 h 100 99.98, (2010)
99.99,
99.99
Ion-exchange brine using Synthetic wastewater Backwash sludge from 1, 0.4 h 20, 100, 2, 45,6 Yeast extract, 90,55, van Ginkel et al.
the membrane biofilm perchlorate degrading 8.2 citric acid 26.82 (2008)
reactor (MBfR) packed bed reactor
A. Ghosh et al.
treatment process has been approved for use in the state of California for drinking
water treatment (DHS 2002), little has been done to study the removal of PRB
from the treated water. Membrane bioreactors can be used to keep the bacteria
separated from the contaminated water (Batista and Liu 2001), but these systems
are at a less advanced stage of development than other biological perchlorate
treatment systems. It has been suggested that reactors based on enzymes to reduce
perchlorate could avoid the potential health problems associated with biological
treatment. Both perchlorate reductase, which can reduce both perchlorate and
chlorate, and chlorite dismutase (Stenklo et al. 2001; van Ginkel et al. 2008) have
been purified. However, no such enzyme-based systems have been reported in the
literature for treatment of perchlorate contaminated water. The presence of alter-
nate electron acceptors in perchlorate contaminated water will be an issue for all
types of biological reactors. Oxygen is an important intermediate in the perchlorate
degradation pathway (Rikken et al. 1996). It is well known that for PRB, oxygen is
a preferential electron acceptor to perchlorate, but a high concentration of dis-
solved oxygen inhibits perchlorate reduction (Song and Logan 2004). It is not clear
yet what concentration of dissolved oxygen will completely inhibit perchlorate
reduction, how long bacteria can withstand exposure to high concentrations of
oxygen before losing the ability to reduce perchlorate, or how long it would take
oxygen-exposed bacteria to regain the ability to reduce perchlorate. However, the
presence of oxygen, nitrate, or sulfate in bioreactor feed streams does not appear to
be a problem for the steady operation of such systems. In a pilot-scale test for ex
situ groundwater treatment, it was found that oxygen, nitrate and perchlorate were
all completely reduced, but sulfate was not measurably degraded (Logan et al.
2001). Thus, it is likely that the main impact of oxygen and nitrate on the treatment
system will be to increase the requirement of substrate (such as acetate or
hydrogen) that is oxidized by the bacteria. Table 1 summarizes the different lab-
oratory scale reactors which have been studied to treat perchlorate contaminated
waters.
5 Conclusions
Unregulated anthropogenic activities in the past have resulted in ubiquitous

presence of perchlorate in the environment. Percholorate is highly toxic and affects
functioning of human thyroid gland even at microgram quantity. Currently, ion
exchange and biological reduction are the two candidate processes for the removal
of perchlorate from drinking water and wastewaters. Based on the current state of
process development, it can be concluded that perchlorate decontamination can be
realized only through integration of one or more of physico-chemical and bio-
logical processes. Integration of ion exchange with bioregeneration of spent resins
and brine can help reduce salt consumption, waste volume and overall operational
costs. This can be realized through identification of novel autotrophic and het-
erotrophic halophilic perchlorate reducing bacteria. Similarly, biological reduction
176 A. Ghosh et al.
processes can be further optimized in terms of reactor detention time, loading rate,
selection of appropriate electron donor and identification of minimum electron
donor concentrations. The successful application of H2-based processes will
require development of safe H2 storage technologies. On the other side of the
spectrum, the in situ processes can be made highly effective by judicious use of
available monitoring tools. In addition, technologies, such as electrical stimulation
which supplies energy without chemical interventions, is an area that warrants
further research and development.
One of the most important issues for designing a perchlorate treatment system
will be the regulatory requirement for perchlorate removal. It now appears likely
that the removal of perchlorate to very low levels will be very necessary. The
USEPA is expected in the near future to finalize a draft of its final assessment of its
toxicological effects of perchlorate, which could lead to recommendation for
perchlorate removal to, 1 mg/l for drinking water (Renner 2002). Both biological
and chemical treatment systems can be used to treat perchlorate contaminated
water to low levels. The most appropriate system will be site and case-specific,
with economic, social and political factors playing a role in the selection of each
treatment system.
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Bioremediation of Nitroaromatics
(NACs)-Based Explosives: Integrating
‘-Omics’ and Unmined Microbiome
Richness
Debasree Kundu, Chinmay Hazra and Ambalal Chaudhari
1 Introduction
The global population crossed beyond 7.2 billion by November 2012 and is
expected to reach 9.1 billion by 2050 (Stenuit et al. 2008). Thus, the quest for
survival will consistently tamper natural as well as unnatural resources. Envi-
ronmental contamination with nitroaromatic compounds (NACs)-based high
explosives (HEs), ordinance related compounds (ORCs), munitions and explosives
of concern (MECs) and unexploded ordnances (UXO) are the results of (1) large-
scale industrial manufacture, loading, assembling, packing (LAP), storage, testing
and deployment of devices containing explosives and the burial of obsolete
munitions, (2) past and current defense related activities, (3) rapid industrialization
coupled with increased urbanization and altered agricultural practices for a more
comfortable lifestyle and (4) recent pursuit for demilitarization of nuclear and
conventional munitions.
Basically, Chemical Warfare Agents (CWAs) are categorized into two groups:
organic and inorganic explosives. The former include monocyclic nitramines like
hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 1,3,5,7-Tetranitro-1,3,5,7-tetr-
azacyclooctane (HMX), 2,4,6-trinitrotoluene (TNT), pentaerythritol tetranitrate
(PETN), triacetone triperoxide (TATP) and 2,4,6,N-Tetranitro-N-methylaniline
(Tetryl), while the inorganic entails glycerol trinitrate (nitroglycerin), sulfur
mustard [bis(2-chlorethyl) sulfide, HD], soman [(3,30 -dimethylbutan-2-yl)-meth-
ylphosphonofluoridate, GD], O-ethyl S-[2-(diisopropylamino)ethyl] methylphos-
phonothiolate (VX) and ethylene glycol dinitrate. While polycyclic nitramine 2,4,
6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (HNIW) or China Lake
20 (CL-20) are being identified as potential alternatives for RDX and HMX, 2,4-
Dinitroanisole (DNAN) is recognized as promising substitute for TNT by the
military based industries (Perreault et al. 2012).
D. Kundu C. Hazra A. Chaudhari (&)

School of Life Sciences, North Maharashtra University, P.O. BOX 80 Jalgaon 425001, India
e-mail: ambchasls@yahoo.com

180 D. Kundu et al.
The huge production of nitroaromatic explosives and related war activities

continuously poured the energetic materials in excess amount to the environment
causing extensive contamination of soil and groundwater (Bernstein and Ronen
2011; Singh and Singh 2011; Rylott et al. 2011a, b; Singh et al. 2012). The world
wars are recognized for continuous incorporation of ammunition lying in
(1) unlined wastewater ponds and creeks; (2) soil layer (2 m below the surface);
(3) 45 m thick vadose zone; and (4) groundwater reservoirs (around 1.35 km long
contamination plume with peak concentrations of up to 2,100 lg/l) (Sagi-Ben
Moshe et al. 2009). The estimated clean up of unexploded ordinance, discarded
military munitions and active constituents spread on 10 million hectares area
would cost between $16 and $165 billion (Lal and Srivastava 2010). In addition to
active ranges, about 6 million hectares of unexploded ordinance contaminated
land is identified by the US Defense Science Board (Rylott and Bruce 2008; Rylott
et al. 2011a). Europe and USA have about 6 9 105 contaminated sites (USEPA
2004; EEA 2007; Bombach et al. 2010), while India has about 20,000 abandoned
sites contaminated with 60 different nitroaromatics. Now an environmental
watchdog survey ranked Russia, China and India among the ‘top ten’ most pol-
luted countries in the world (Prasad et al. 2010). These high energy explosive
materials are (1) toxic, (2) bind tightly to the organic matter in soil, (3) recalcitrant,
and (4) identified as potent carcinogens, mutagens and teratogens (Fahrenfeld et al.
2012). Hence, these explosive materials are recognized as priority pollutants of
hazardous category (National Priorities List; USEPA 2011).
Although physico-chemical treatments have been developed for removal of
high energy pollutants, but they are expensive, non-specific, and also have the
potential to incorporate secondary contaminations (Kulkarni and Chaudhari 2006,
2007; Singh et al. 2008; Kundu et al. 2011). As a result, there has been an
increasing interest in eco-friendly and bio-based treatments commonly known as
bioremediation (natural attenuation, biostimulation and bioaugmentation), utiliz-
ing pan-microbiome, a diverse and dynamic collection of microbes that reside in
and around us. However, the implementation of such bioremediation techniques
in situ is not always successful, due to (1) difficulty to control and scale up key
biodegradative processes from the laboratory to full-scale and (2) poor success rate
of bioaugmentation trials in terms of predictability, dynamics of catabolic
microbial populations and process monitoring. Thus, despite of concerted efforts,
the isolation of potential microorganisms to utilize high energy materials (RDX,
HMX, TNT and CL-20) for growth has remained elusive (Rylott et al. 2011a, b).
Due to their astonishing catabolic diversity, versatility and plasticity for
adaptation, microbes are the best candidates among all living organisms for bio-
remediation. However, effective biological decontamination needs (1) a better
know-how of the physico-chemical characteristics of the contaminated environ-
ment, (2) detailed profile of the microbial communities involved in key physio-
logical processes, and (3) characterization of microbial communities in terms of
structure, phenotypic potential, function(s) and interactions with the environment
(Eyers et al. 2004; Rittmann et al. 2006; Stenuit et al. 2008). Although various
applications of culture-independent molecular tools are explored to study the
Bioremediation of Nitroaromatics (NACs)-Based Explosives 181
diversity and dynamics of microbial communities in the last two decades, they
have been proved to be invaluable for the qualitative (e.g., fingerprinting tech-
niques), quantitative [e.g., dot blot, fluorescence in situ hybridization (FISH), real-
time PCR etc.,] description of environmental microbial communities and analysis
of new catabolic operons of xenobiotics in environmental bacteria (Cao et al.
2009; Kapley and Purohit 2009). However, the applications of these modern tools
appeared to be time-consuming and inconclusive for characterization of an eco-
system. In contrast, high-throughput approaches offer miniaturization, automation
and simultaneous ‘real-time’ analysis of numerous samples at a reasonable price.
In addition, whole genome sequencing of bacteria involved in the elimination of
recalcitrants and entire community reduced genome-sequencing costs. Both
genomic approaches with post-genomic tools will provide a comprehensive
understanding of the composition and functioning of microbial communities
(Stenuit et al. 2008).
This review substantiates the potentialities of new molecular approaches in
exploring the genetic diversity of microbes to degrade recalcitrant high energy
materials by high-throughput molecular techniques and critically to asses the
bioremediation of sites/effluents contaminated with hazardous and/or recalcitrants.
2 High-throughput Approaches for Molecular Surveys

of Microbial Communities and Monitoring
of Bioremediation Efficacy with Respect
to Nitroaromatics
Maintaining, evaluating and predicting the optimal biodegradation performance of

a biotreatment process necessitates lineage between the physico-chemical envi-
ronmental conditions with the stability and resilience of the degrading microbial
communities through available tools. A number of culture-independent molecular
techniques, currently used to study complex microbial communities, are compat-
ible with a high-throughput setup and meet the above-suggested criterion. An
overview of contemporaneous integrative molecular and ‘x-omics’ technologies
employed to survey intrinsic microbial communities in bioremediation of con-
taminated sites is illustrated in Fig. 1.
2.1 Fingerprinting Techniques
Based on the separation of amplicons after PCR amplification of phylogenetic (for

instance, 16S rRNA) or functional genes using universal or specific primers,
genetic fingerprinting techniques usually provide a specific pattern of a microbial
community (Nocker et al. 2007; Stenuit et al. 2008). Some high-throughput ver-
sions used in several bioremediation cases include (a) terminal restriction fragment
length polymorphism (T-RFLP) in 2,4-DNT and TNT-contaminated soil
182 D. Kundu et al.
Fig. 1 Schematic illustration of bioremediation strategies at sites contaminated with hazardous

NACs-based waste with special emphasis on the potential of high-throughput technologies
(Fahrenfeld et al. 2012), (b) length heterogeneity analysis by PCR (LH-PCR) in

stimulated bioremediation in nitrotoluene contaminated groundwater, (c) single
strand conformation polymorphism (SSCP) in nitrobenzene degradation,
(d) fluorescent single strand conformation polymorphism (F-SSCP), (e) denaturing
gradient gel electrophoresis (DGGE)) at sites contaminated with RDX and TNT
(Fahrenfeld et al. 2012) and abundances of dsrB (dissimilatory sulfite reductase
b-subunit)-genes, (f) denaturing high performance liquid chromatography
(D-HPLC) in fermentor sludge, compost and soil samples (Wagner et al. 2009),
(g) ribosomal intergenic spacer analysis (RISA), (h) serial analysis of ribosomal
sequence tags/ribosomal DNA (SARST or SARD, respectively) (Yu et al. 2006;
Ashby et al. 2007) and (i) single-point genome signature tags (SP-GSTs) (van der
Lelie et al. 2006). Applications of these tools for in situ analysis of xenobiotic
degrading bacterial communities and quantification of their catabolic genes have
been reviewed (Stenuit et al. 2008; Desai et al. 2010).
2.2 Real-Time PCR
Real-time PCR, a high-throughput design with high analytical sensitivity for the
detection and quantification of specific genes in complex DNA mixtures
(environmental samples), is highly sensitive (limit of quantification is typically

1–2 genome copies) and does not require any time-consuming post-PCR steps for
the quantification of PCR products as the amount of amplicons is monitored in
real-time.
Owing to the ease and relatively low cost compared to microarrays, qPCR
remains the most favourable technique for fast analysis of microbial numbers from
in situ samples. Recent examples include microfluidic cards that contain 384
miniaturized qPCR assays, microfluidic-dynamic-array systems allowing 2,304
qPCR gene expression measurements on a single chip, OpenarrayTM accommo-
dating 3,072 33-nl qPCR reactions, and the SmartChipTM real-time PCR system
equipped with high-density chips containing 5,000–30,000 nanowells. Although
qPCR is a highly sensitive technique, it is prone to errors and validity of resulting
data sets should be considered with regard to specificity of primers, efficiency in
DNA extraction methods and errors arising from PCR methodology and instru-
mentation (Maphosa et al. 2010).
Successful examples of detecting target catabolic genes/genotype using PCR
includes: ntdAcAd genes encoding the Rieske-type dioxygenase, 2-nitrotoluenene
2,3-dioxygenase (2NTDO) a and b subunits for 2-nitrotoluene (2NT) in Acido-
vorax sp. strain JS42 (Ju and Parales 2011), NfsA and NfsB in Escherichia coli,
SnrA and Cnr in Salmonella enterica serovar Typhimurium, NfsI in Enterobacter
cloacae, RdxA in Helicobacter pylori, flavin reductase P in Vibrio harveyi, Frase I
in Vibrio fisherii, nitrobenzene nitroreductase in Pseudomonas pseudoalcaligenes
(Somerville et al. 1995), PnrA and PnrB in Pseudomonas putida and NitA and
NitB in Clostridium acetobutylicum (Smets et al. 2007).
Moreover, a quantitative fingerprinting method combining real-time PCR and
T-RFLP was developed for simultaneous determination of microbial abundance
and diversity within a complex wastewater community (Yu et al. 2005). In
addition, the integrated approach was successfully coupled with the stable isotope
probing technique (SIP) to develop a quantitative assay for concomitant identifi-
cation and quantification of active microorganisms involved in naphthalene deg-
radation in soil microcosms (Yu and Chu 2005), TNT contaminated sediment and
monitoring of the biodegradation rates of RDX in contaminated soils and aquifers.
Analysis of isotope fractionation in NACs, however, has been focused predomi-
nantly on 15N fractionation during abiotic reduction under anoxic conditions. The
magnitude of isotope fractionation associated with NAC biodegradation and its
variability for structurally different compounds due to specific enzyme-substrate
interactions, however, has not yet been investigated (Friemann et al. 2005). Taking
this cue, Hofstetter et al. (2008) combined the evaluation of d13C and d15N
changes in nitrobenzene, based on the isotope enrichment behavior for assessing
the extent of nitrobenzene biodegradation via competing pathways in contami-
nated environments. Further application of this technique, combined with advan-
ces in metagenomics and transcriptomics technologies, should facilitate the
discovery of new explosive degrading activities (Rylott et al. 2011a).
184 D. Kundu et al.
2.3 Microarrays for Genome and Transcriptome Analysis
In the field of environmental genomics, five major classes of microarrays have

been developed: (1) phylogenetic oligonucleotide arrays (POAs), containing oli-
gonucleotide probes to target taxonomic genes (e.g., 16S rRNA gene), (2) func-
tional gene arrays (FGAs) target genes encoding key enzymes involved in specific
processes, (3) community genome arrays (CGAs), constructed from whole geno-
mic DNA of multiple cultured strains or species, (4) whole genome (open reading
frame) array/WGA and (5) metagenomic array (MGA) (Stenuit et al. 2008; Desai
et al. 2010).
The application of POA targeting bacterial groups potentially involved in the
bioremediation processes can detect (1) all lineages of sulfate-reducing prokary-
otes, (2) Geobacter chapellei in the soil RNA pool, (3) direct profiling of envi-
ronmental communities by hybridizing soil RNA extracts to POA and (4) shifts in
a soil microbial community associated with TNT-contamination using a rRNA-
targeted POA. Loy et al. (2005) used a 16S rRNA gene-targeted oligonucleotide
microarray (RHC-PhyloChip), consisting of 79 probes for diversity analysis of
b-proteobacterial order Rhodocyclales in activated sludge samples from an
industrial wastewater treatment plant.
FGA-based detection of catabolic genes, on the other hand, is yet to be
employed to detect catabolic genes in NACs-based explosives contaminated sites.
In this direction, a comprehensive FGA containing [24,000 probes for all func-
tionally known geochemical, ecological and environmental processes, including
metal reduction and resistance and organic contaminant degradation called
‘Geochip’ microarray paved the way for rapid monitoring of whole community
(He et al. 2007). Regardless of the challenges associated with microarray tech-
nology (i.e., suitable probe design, specificity, sensitivity, hybridization behaviour
and quantification of target populations), it is anticipated that FGAs, in combi-
nation with other techniques, such as high-throughput non-gel-based proteomics
and metatranscriptome sequencing, will considerably enhance our understanding
of microbial degradation of nitroaromatics (Maphosa et al. 2010).
In addition to the detection of specific microorganisms or genes in contami-
nated environments, the responsiveness of specific organisms to contaminants can
be exploited to detect the presence of pollutants in hostile environments alongwith
their toxicity (Stenuit et al. 2008). For this purpose, in a DNA-microarray based
screening of the expression levels of all yeast genes to the fungicide thiuram
(Kitagawa et al. 2002), a yeast microarray was used to evaluate the potential
toxicity of unknown chemicals present in open burn and detonation sites (Kim
et al. 2004). Also, a whole-genome microarray of the soil nematode Caenorhab-
ditis elegans was developed to study the effect of xenobiotics on gene expression
profiles (Reichert and Menzel 2005). Evaluation of microarray to study the
physiology of pure environmental cultures or physiological studies of explosives
contaminated samples is still scanty.
2.4 Metagenomics: Access, Analyse and Exploit
The concept of environmental genomics is based on simultaneous analysis of

genes within environmental microbes (genomics) or analysis of collective
microbial genomes in an environmental sample (metagenomics) (Desai et al.
2010). Metagenomics includes (1) shotgun sequencing of microbial DNA isolated
directly from a given environment, (2) high-throughput screening of expression
libraries, constructed from cloned community DNA in order to identify specific
functions, such as antibiotic resistance (functional metagenomics), (3) profiling of
RNAs and proteins produced by a microbiome (meta-transcriptomics and
meta-proteomics) and (4) identification of a community’s metabolic network
(meta-metabolomics). Availability of whole genome sequences from several
environmental microorganisms relevant to bioremediation has been used to
determine the gene pool of enzymes involved in degradation of anthropogenic
pollutants (Galvao et al. 2005; Desai et al. 2010). Out of 3,788 prokaryotic gen-
omes (bacterial: 3,434 and archae: 172) sequenced as of November 2012, 70
genomes are pertaining to biodegradation of NACs-based explosives (http://www.
genomesonline.org).
Engineering of regulator variants either to increase their specificity or to
broaden their effect or profile constitutes an elegant strategy to discover new
catabolic activities in metagenomic libraries. For example, the inducer-binding site
of the transcriptional regulator DntR, which activates the oxidative denitration of
DNTs, can be modified in order to increase its sensitivity and specificity. The
potential production of DNtR variants, responsive to 2,4-DNT, could be further
exploited in high-throughput screening of metagenomic libraries in search of new
pathways of TNT mineralization (Smirnova et al. 2004; Galvao and de Lorenzo
2006; Stenuit et al. 2008). Further, substrate-induced gene expression screening,
metagenomic library screenings, and sequenced bacterial genomes to discover new
degradation pathways are yet to be applied in bioremediation of NACs-based
explosives. Besides, scaling up of sequencing projects from individual genomes to
community genomes is expected to provide novel biocatalytic activities and
massive valuable information to understand microbial communities with catabolic
activities towards NACs.
2.5 Metabolic Engineering and Protein/Enzyme Engineering
Metabolic engineering combines logical analysis of metabolic and other pathways

with molecular biological techniques to improve cellular properties by designing
and implementing rational genetic modifications (Rayu et al. 2012). Current efforts
devoted in metabolic pathway engineering of NACs are: (1) accelerating the
existing pathways or design a ‘new’ effective pathway/hybrid pathway with
superior catalytic abilities for recalcitrant; (2) development of engineered
186 D. Kundu et al.
microorganisms which possess improved catalytic activity combined with (a)

advanced capacity to survive under extreme environmental conditions and/or (b)
ability to produce suitable biosurfactants, a trait particularly useful in cases where
the limited bioavailability of NACs constitute an obstacle for effective biodegra-
dation (Rayu et al. 2012); and (3) deletion strategies for eliminating competitive
reaction pathways.
The fast emergence of new enzymes for the degradation of synthetic aromatic
compounds is difficult to explain only by horizontal transfer of genes and elevated
mutation frequency under stressful conditions. Under the formulated concept of
evolution based on the promiscuity of proteins, protein evolution towards a new
function involves transitions from a specialized enzyme into a generalized inter-
mediate and thereafter to a new, specialized enzyme. This is further supported by
the results of several directed evolution studies on catabolic enzymes. Enzyme
promiscuity indicates that (1) the assembly of a catabolic operon and the acquisition
of efficient transcriptional control over the system are independent events and (2)
evolution of new effector-responsive regulators is inter-related. Based on this, it was
demonstrated that the toluene-activated transcriptional activator XylR, encoded by
TOL plasmid pWW0, is able to acquire de novo responsiveness both to 2,4-DNT
and its mono-substituted precursors and to the unrelated isomer of 3-NT (Galvão
et al. 2007). Interestingly, these mutations did not alter amino acids in the effector-
binding pocket of the regulator, but amino acid substitutions were located at the
protein surface. These changes were believed to affect conformational shifts that
follow effector binding and modulate signal transmission between XylR domains
(Galvão et al. 2007), thereby indicating an existing but unproductive binding site in
XylR for DNT and many others in the toluene catabolic pathway (Kivisaar 2009). In
contrast, 2-NT degradation genes in Acidovorax sp. JS42 are controlled by the
LysR-type specific regulatory protein NtdR, which is 98 % identical to NagR, the
activator of the naphthalene degradation operon in Ralstonia sp. strain U2 (Jones
et al. 2003; Lessner et al. 2003). Based on how nitrotoluene-responsive regulator
NtdR can be generated from a NagR-like ancestor by just a few mutations, Ju et al.
(2009) showed stepwise broadening of the effector range without loss of the original
activity by reconstructing pathway of the evolution of NtdR from NagR.
On this premise, recombinant strains were constructed for the mineralization of
2,4-DNT by combining pathway from various bacteria via conjugation into a
single recombinant host. Such assemblage includes: (1) introduction of the genes
encoding the 2,4-DNT degradation pathway from Burkholderia sp. strain DNT
into Pseudomonas fluorescens ATCC 17400 for its complete degradation (Monti
et al. 2005), (2) cloning and expressing the genes encoding a novel partial
reductive pathway for nitro-toluene from Comamonas sp. strain CNB-1 in E. coli,
(3) introduction of the TOL plasmid pWWO (P. putida mt-2) into P. putida F38/D
creating a hybrid strain P. putida TB101, and (4) construction of hybrid P. putida
TB105, constructed by cloning the genes encoding the TOD pathway on broad
host range multicopy vector RSF1010 and introducing into the TOL strain
P. putida mt-2. Interestingly, genes for TNT denitration have not yet been probed,
but, metagenomic libraries may unravel the same in the near future (Eyers et al.
2004). Function-driven screening approach was adopted to identify ntnMA, ntnD,

ntnC, pnbA and pnbB involved in 4-NT biodegradation, ntdAa, ntdAb, ntdAc and
ntdAd from 2-NT biodegradation pathway, dntA, dntB, dntD, dntG and dntE from
2,4-DNT biodegradation and xenB for 2,4,6-TNT biodegradation. The ‘substrate-
induced gene expression screening’/‘guilt by association’ based on genetic
reporters rather than on direct enzymatic screening or on DNA sequence simi-
larity, yielded 33 positive clones induced by benzoate and two positive clones
induced by naphthalene from a total of 152,000 clones (Uchiyama et al. 2005).
A newer strategy devised transcriptional traps for tracing 2,4-dinitrotoluene
(2,4-DNT) using a bacterial transcriptional factor other than DntR (Garmendia
et al. 2008) by (1) production of variants of the XylR protein evolved in vivo for
responsiveness to 2,4-DNT and (2) re-introduction of such variants into P. putida
bearing a transcriptional fusion between the Pu promoter and a reporter gene. Such
innovative traps integrated with stable orthogonal sensor circuits in P. putida
(de las Heras et al. 2008) was successfully used for the detection of 2,4-DNT
residues in a soil microcosm. Using long-term laboratory evolution experiments,
Ju and Parales (2011) obtained JS42 mutants that grew on 4-nitrotoluene via a new
degradation pathway. The underlying basis for this new activity stemmed from the
accumulation of specific mutations in the gene encoding the dioxygenase that
catalyses the initial oxidation of nitroarene substrates, but at positions distal to the
active site and previously unknown to affect activity in this or related enzymes.
Overall, various strategies illustrate that DNA fragments from an entire microbial
community may provide novel genes capable of degrading pollutants (Stenuit et al.
2008).
2.6 Proteomics and Metaproteomics
Characterization of aromatic degradation pathway has been conventionally

reported based on key metabolite identification, key enzymes purification and
biochemical characterization, mutagenesis, gene cloning and sequencing. With the
whole genome sequence and some of the catabolic plasmids available, high-
throughput proteome analysis has emerged in elucidating the pathways involved in
the biodegradation of aromatic compounds (Chauhan and Jain 2010).
Only a few environmental metaproteomic studies have been achieved so far
(Stenuit et al. 2008; Desai et al. 2010) focusing on three targets: (1) environmental
stress response, (2) catabolic protein(s) identification and (3) community structure
analysis. Proteomic approaches, although still in their infancy in explosives bio-
degradation studies, are increasingly being applied with the aim of elucidating the
metabolism of organisms that are unable to grow in sufficient amounts to perform
standard biochemical analyses, such as enzyme purification and activity mea-
surements. Proteomics has begun to play a crucial role in identifying nitroreduc-
tase and oxygenase that are involved in aerobic/anoxic degradation of different
substrates. Non gel-based proteomics could be used as quantitative biomarkers
188 D. Kundu et al.
with high specificity and sensitivity to differentiate closely related strains in cul-
tures and environmental samples. Proteomics will further aid in functional char-
acterization of the large diversity of Rieske-type dioxygenases and ring
hydroxylating dioxygenases. Proteomics is emerging as a powerful tool in biore-
mediation allowing studies of protein–protein interactions and cell surface pro-
teomics toward the discovery of new biomarkers (genes and proteins) and
providing insight into metabolic pathways of denitration, that are, at present, not
fully understood. Also, proteomic analysis of stress responses in various micro-
organisms exposed to explosives may be suggested as a promising way to discover
biomarker proteins of exposure (Nesatyy and Suter 2007) that could act as
‘detectors’ of pollution.
2.7 Metabolomics and Fluxomics
The screening of a global metabolome within a single analytical platform are

now-a-days realized by molecular-level metabolite resolution techniques: isotope
distribution analysis of metabolites and molecular connectivity analysis using
ultra-high mass accuracy techniques for elucidating biodegradation pathways of
xenobiotics (Breitling et al. 2006; Villas-Bôas and Bruheim 2007; Stenuit et al.
2008).
Now, transcriptome and metabolome analyses have been successfully used to
study genome-wide expression profiles and metabolic profiles of bacteria relevant
to aromatic compounds metabolism, such as exposure to aromatic pollutants and
metabolism of aromatic amino acids (Wood 2008).
Metabolic pathway engineering via rational design of nitrobenzene 1,2-dioxy-
genase by substituting amino acid at the position 293 (F293Q) expanded substrate
specificity, resulting in 2.5-fold faster oxidization rate against 2,6-dinitrotoluene
and also, site-directed mutagenesis and the replacement at the position 258
(N258 V) of 2-nitrotoluene dioxygenase significantly changed the enantiospeci-
ficity (Singh et al. 2008). On the basis of the importance of the A206 position of a
naphthalene dioxygenase on regioselectivity, the analogous position at I204 of 2,4-
dinitrotoluene dioxygenase (DNTDO) broadens the specificity for substrates
(Leungsakul et al. 2005). Similarly, improvements for 2,3-DNT, 2,5-DNT, 2,6-
DNT, 2NT, and 4NT were obtained by saturation mutagenesis. Leungsakul et al.
(2006) applied error-prone PCR to obtain mutant with changes in two residues
(M22L/L380I) of methy nitrocatechol (MNC) monooxygenase in DNT catabolic
pathway. This led to transformation of 4- nitrophenol and 3-methyl-4-nitrophenol
with 11-fold efficiency than wild type enzyme.
Relatively new approach of genome shuffling achieved five-fold rate of TNT
transformation in Stenotrophomonas maltophilia OK-5 via nitro reduction (Lee
et al. 2006). In spite of an elusive ring cleavage and mineralization pathway for
TNT, ambiguity over the use of degradation against transformation terminologies
still persists (Rylott et al. 2011a).
A fully fused self-sufficient RDX degrading cytochrome P450 encoded by a

single gene was found to be attractive for expression in plant systems for envi-
ronmental clean up. In order to improve the rates of RDX degradation in plants,
the genes, xplA and xplB have been engineered into Arabidopsis, as a model plant
system and yielded very rapid RDX removal (Singh et al. 2008; Rylott et al.
2011b). In near future, these systems are expected to be transferred to microbial
machinery for enhanced explosive removal. Another promising approach is to
exploit the specialized membrane structure ‘superchannels’ from Sphingomonas
sp. A1 for enhanced pollutant uptake. Introduction of these superchannels in
dioxin-degrading S. wittichii RW1 and the polypropylene glycol degrading
S. subarctica IFO 16058T, resulted in substantial enhancement in bioremediation
capacity. This could be a general approach and be applied to other engineered
nitroaromatics degrading microorganisms. However, energy dependent transport
through these superchannels might limit their applicability (Singh et al. 2008).
2.8 From Systems to Synthetic Biology: An Approach

for Programmed Bioremediation
After an eventual standstill due to recurrent unpredictable failure and multiscale

complexity, onset of systems and synthetic biology has, however, relaunched the
objective of creating the laboratory designer microorganisms (de Lorenzo 2008)
(Fig. 2). Under the framework of bioremediation, user-friendly bioinformatics
Fig. 2 Top-down and bottom-up approach in systems biology: from molecules to ecosystems
190 D. Kundu et al.
tools such as, Optkock framework, Optstrain, DESHARKY (a Monte Carlo

algorithm), Qiime (qiime.sourceforge.net) and PhyloTrac (www.phylotrac.org/)
are available to engineer central physiological functions in accordance to pre-
dictions from in silico metabolic modeling (Fulekar and Sharma 2008; Desai et al.
2010).
Current efforts on synthetic biology include the programming of complex
interplays between two bacterial strains in artificial consortia, multispecies con-
sortia and plant leaf-endophyte engineering (de Lorenzo 2008). At present, about
184 identified sites in the USA, 51 in Europe and a few more in Japan and China
are actively exploring newer synthetic biology-based bioremediation of DNT and
TNT (http://www.synbioproject.org/).
A comprehensive database of all available genomics, proteomics, and meta-
bolomics information from bioremediation research may provide a shared platform
for exchanging database information, analysis methods and pipeline through
concerted efforts of all elements. Ultimately, such tools may foster accurate
interpretation of the ‘x-omics’ data, leading to generation of judicious predictive
models and strategies for successful implementation of bioremediation applica-
tions in the near future (Chakraborty et al. 2012).
2.9 Whole Cell-Based Biosensors for Environmental

Biomonitoring
A biosensor is a self-contained integrated device designed for a concentration-

dependent response in the presence of a chemical analyte (Purohit 2003; Lei et al.
2006; Singh 2007). A microbial biosensor is an analytical device which integrates
microorganism(s) with a physical transducer to generate a measurable signal
proportional to the concentration of analytes. It is mainly based on amperometry,
potentiometry, conductometry, voltammetry, microbial fuel cell, fluorescence,
bioluminescence, and colorimetry. Whole-cell bioreporters, whether existing
within the walls of the laboratory or applied as bonafide environmental sensors,
have earmarked as practical tools for the detection and monitoring of contaminants
of ecological concern. The emerging biosensor market is expected to grow at over
9 % and market in Asia–Pacific is estimated to reach $794 million by 2012
(Mongra and Kaur 2012).
Several whole cell-based biosensors have been proposed to track toxic explo-
sives since 1990. Bioreporters offer several advantages over routine analytical
tools in terms of bioavailability, specificity and flexibility. Besides monitoring
general toxicity, biosensors can detect the presence of pollutants undetected by
conventional analytical methods. Alternatively, an array of bioreporters integrated
with pattern learning algorithms or decision tree models can identify a chemical
either by the unique pattern or ‘fingerprint’ of bioreporter signals or providing
rapid ‘snapshots’ of contaminant presence/absence. Now, high-throughput
biomonitoring of multiple contaminants is made possible using different types of

bioreporters and multi-well platform techniques (e.g., 384-well plates) (Stenuit
et al. 2008). Further, bioreporters are integrated in customized chip-based devices
which are equipped with photodetectors to detect low-level bioluminescence. In
contrast, absolute chemical measurements are not achieved with biosensors as
required by the current legislations. Hence, the best approach to characterize a
contaminated site or biotreatment process is likely to combine an on-line, real-time
monitoring biosensor system with periodic chemical measurements (Ron 2007;
Stenuit et al. 2008). A non-exhaustive list of biosensors developed for the
detection of NACs-based explosives has been summarized in Table 1.
Recently, newer biosensors based on artificial recognition elements, such as
fluorescent conjugated polymers, small molecule fluorophores, metal–organic
frameworks and covalent organic frameworks, hold a great promise for the
development of miniaturized, mass-produced biosensor chips and devices in the
environmental applications (Zhang et al. 2012). Also, the possibility of introducing
innovative outputs and microengineering to biosensor design in addition to
rewiring bacterial signaling systems to function in other organisms may also widen
the use of bacterial reporter assays in on-line and in situ environmental monitoring
and remediation (Checa et al. 2012).
3 Bioinformatics in Bioremediation of NACs
Several in silico softwares, pipelines, web resources and algorithms have been
developed to interpret or correlate molecular and x-omics data. Nonetheless,
bioinformatics resources, exclusively committed to bioremediation of NACs-based
explosives, are still scarce. The University of Minnesota Biocatalysis/Biodegra-
dation Database (UMBBD) has enlisted 200 pathways, 1,350 reactions, 1,195
compounds, [1,000 enzymes, 491 micro-organism entries and 259 biotransfor-
mation rules encompassing microbial bioremediation (http://umbbd.msi.umn.edu/)
(Gao et al. 2011). MetaRouter is yet another system for maintaining heterogeneous
information related to bioremediation and biodegradation in a framework that
allows updating query modification (Desai et al. 2010). The system can be
accessed and administrated through a web interface (Pazos et al. 2005). Other
software platforms are: Kyoto Encyclopedia of Genes and Genomes (KEGG) at
http://www.genome.ad.jp/kegg/kegg.html (Moriya et al. 2010); Boehringer
Mannhein Biochemical Pathways (BMBP) on the ExPASy server, Switzerland
(http://www.expasy.org/cgi-bin/search-biochem-index); International Society for
the Study of Xenobiotics (http://www.issx.org); PathDB: Metabolic Pathways
Database at NCGR (http://www.ncgr.org/pathdb/) etc.
Table 1 Biosensors for the detection of NACs-based explosives
192
Input/target Sensor module Transducer module Output Design strategy Reference

TNT Fluorescent porous polymer Fluorescence quenching Fluorescence Fluorescence-based optical-fiber Yang and
films sensor Swager
(1998)
TNT and Antibody-coated fiber optic Analyte/antibody interaction Plasmon resonance/ Competitive immunoassay; Bakaltcheva
RDX probes fluorescence/ fluorescent antigen competes et al.
bioluminescence with free antigen of unknown (1999)
concentration
TNT Porous silica microspheres Fluorescence quenching Fluorescence Fluorescence-based optical-fiber Keith and
with physisorbed nile red sensor David
dye (2000)
TNT and Antibodies Activated porous membranes Fluorescence induction Membrane-based displacement Rabbany
RDX immunoassay et al.
(2000)
DNT DntR DntR/PDNT::gfp GFP Rational re-design of the inducer- Ng and
binding site of DntR Forsman
(2000)
TNT Maltose binding protein Electrode modified with an Amperometric and cyclic Enzymatic immobilization Naal et al.
(MBP) nitroreductase electropolymerized film of voltammetry (2002)
(NR) fusion (MBP-NR) N-(3-pyrrol-1-ylpropyl)-
4,40 -bipyridine (PPB)
TNT Fluorescently labeled TNT Anti-TNT antibody Fluorescence Competitive displacement of Goldman
analogue labeled TNT by TNT et al.
(2003)
TNT Enzyme labeled antibodies Anti-TNT antibody (Ab), Electrochemiluminescence Competition between the labeled Wilson et al.
bound to paramagnetic fluorescein labeled TNT and and unlabeled TNT for Ab (2003)
beads unlabeled TNT binding
TNT Chlorophyll a Dictyosphaerium chlorelloides Fluorescence The inhibition of chlorophyll Altamirano
DcG1wt a fluorescence of PSII by et al.
TNT (2004)
D. Kundu et al.
(continued)
Table 1 (continued)
Input/target Sensor module Transducer module Output Design strategy Reference
DNT Olfactory receptor (WIF-1a) Engineered Saccharomyces GFP Saccharomyces cerevisiae strain Radhika
cerevisiae strain with gfp with primary components of et al.
the mammalian olfactory (2007)
signaling pathway
DNT/ DntR DntR/PDNT::phzMS Pyocyanin Novel output-redox active Gu et al.
salycilate compound (2010)
TNT Phenothiazine oligomers Fluorescence quenching Fluorescence Fluorescence-based optical-fiber Zhang et al.
sensor (2010)
TNT and Chlorophyll a Spinach leaves PSII particles Photo-electrochemical Gold screen-printed electrodes Bhalla et al.
RDX measurements (Au-SPE) in a droplet (2011)
biosensor
TNT Riboflavin Self-assembled monolayer AC voltammetry Enhancement of redox currents Sumner and
(SAM) modified gold by electrochemistry Chu
electrode (2011)
TNT Conjugated polymer poly (2- Fluorescence quenching Fluorescence Coiled shaped plastic optical Chu and
methoxy-(20 - fiber was employed as sensor Yang
ethylhexloxy)-p- head to detect TNT (2012)
phenylene-vinylene)
Bioremediation of Nitroaromatics (NACs)-Based Explosives
(MEH-PPV)
DNT and Benzothiophene based Fluorescence quenching Fluorescence Electrospun nanofibrous Long et al.
TNT conjugated polymer P explosive sensor with (2012)
fluorescent probe in
polystyrene supporting matrix
193
194 D. Kundu et al.
4 Biosafety Issues and Regulatory Challenges
Critical research questions, pertaining to the development and implementation of

genetically engineered (GE) bacteria for enhanced bioremediation, have been
identified and poised for possible future research (Singh et al. 2011). Since the
introduction of biomolecular engineering into bioremediation, intensive debates on
ethics and risks of the release of engineered strains into the environment were
realized as major issue due to (1) possibilities of the persistence of naturally
undesired genes in the environment and (2) their undesirable transfer to indigenous
species (Cao et al. 2009). These potential risks can be minimized by bio-con-
finement system, viz., induction of suicide genes and nucleases-containing killing
system. Surprisingly, the R & D efforts about bio-confinement systems for engi-
neered bacteria were stopped around a decade ago for unknown reasons (Com-
mittee on the Biological Confinement of Genetically Engineered Organisms 2004;
Cao et al. 2009). Besides potential risks, government permission and stringent
regulatory norms pose a major impediment to actualizing field release studies and
prevented the integration of state-of-art engineered microbes into field. Never-
theless, a comprehensive and cautious risk assessment is necessary before
releasing the engineered strains into the environment to avoid unknown
consequences.
5 Conclusions
For efficient and consistent bioremediation strategies, an in depth understanding of

the parameters governing the community structures and metabolic performance of
intrinsic microbial communities are required. The potentials of specialized
‘x-omics’ approach provide an overview to: (1) explore catabolic activities,
(2) gauge (qualitative and/or quantitative) microbial community composition,
(3) identify the biodegradation capabilities and the function of a specific microbial
community, (4) relate microbial community analysis to the metabolic function of
specific groups of bacteria, and (5) monitor in ‘real-time’ the performance of a
bioremediation process.
Presently, most of high-throughput approaches are still at the nascent stage and
the ‘data storm’ generated by the same warrant efficient data management software
toolbox to avoid the data deluge dilemma. Hence, rapid advances in computation
and modeling are urgently needed in order to harness the potential of high-
throughput technologies.
Acknowledgments Debasree Kundu and Chinmay Hazra are grateful to University Grants
Commission (U.G.C.), New Delhi, and Department of Science and Technology (D.S.T.), New
Delhi for providing RFSMS and INSPIRE fellowship, respectively.
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Bioremediation of 2,4,6-Trinitrotoluene
Explosive Residues
Sikandar I. Mulla, Manjunatha P. Talwar

and Harichandra Z. Ninnekar
1 Introduction
A large scale manufacturing and use of a variety of synthetic chemicals in the form
of pesticides, herbicides, plasticizers, explosives, dyes, drugs, and industrial
products in our daily life, continuously pollute soil, water, and air which have
direct or indirect adverse impact on our and animal health. Many of these
chemicals are reported to be toxic, mutagenic or carcinogenic to humans and
animals. Nitroaromatic compounds are a major group of pollutants of the envi-
ronment because of their widespread use, toxicity, recalcitrance and bioaccumu-
lation. Nitroaromatics, such as nitrotoluenes, nitrobenzene, nitrophenols,
nitrobenzoates, and nitroanilines, are extensively used in industry for the synthesis
of explosives, pesticides, dyes, plastics and pharmaceuticals (Zylstra et al. 2000;
Nishino and Spain 2004; Ye et al. 2004; De Lorme and Craig 2009; Mulla et al.
2011a). These compounds are also produced by incomplete combustion of the
fossil fuels (Kulkarni and Chaudhari 2007). There have been several reports of
widespread contamination of soil and groundwater by explosives, such as 2,4,6-
trinitrotoluene (TNT) which is synthesized using both mono- and dinitrotoluenes
(Fig. 1) (Neuwoehner et al. 2007; De Lorme and Craig 2009; Mulla et al. 2011b,
2013). TNT has been identified in at least 20 of the 1,397 hazardous waste sites by
the U. S. Environmental Protection Agency (USEPA 2011). Bioremediation of
explosive chemicals may provide an effective method for their detoxification
(Mulla et al. 2012).
S. I. Mulla M. P. Talwar H. Z. Ninnekar (&)

Department of Biochemistry, Karnatak University, Dharwad, Karnataka 580003, India
e-mail: hzninnekar@yahoo.com

202 S. I. Mulla et al.
CH3 CH3 CH3

O2N NO2 O2N NH2 H2N NO2
NO2 NO2 NO2

2,4,6-Trinitrotoluene(TNT) 2-Amino-4,6-dinitrotoluene 6-Amino-2,4-dinitrotoluene
CH3 CH3
CH3
H2N NO2 H2N NH2
H2N NH2
NH2 NH2
NO2
2,6-Diamino-4-nitrotoluene 4,6-Diamino-2-nitrotoluene 2,4,6-Triaminotoluene(TAT)
CH3 CH3
CH3 CH3 CH3
NO2
O2N NO2 NO2
NO2
NO2 NO2
2,6-Dinitrotoluene 2,4-Dinitrotoluene 2-Nitrotoluene 3-Nitrotoluene 4-Nitrotoluene
O2N NO2
NO2
2,4,6-Trinitrobenzene(TNB)
Fig. 1 Structure of TNT and its intermediates
1.1 Physico-Chemical Properties of 2,4,6-Trinitrotoluene
2,4,6-Trinitrotoluene (TNT) and its isomers were first prepared in the year 1863 by
Joseph Wilbrand and was originally used as a yellow dye (Wilbrand 1863). Later,
it was purely synthesized in 1880 by Hepp and its chemical structure established in
1883 by Claus and Becker. TNT was first synthesized on an industrial scale in
1891 in German country. Subsequently, aluminium was mixed with TNT to
manufacture a high energy explosive which was adopted by all military powers
(Kirk and Othmer 1951). At present, these explosives are known as primary,
secondary or tertiary based on their susceptibility to initiation. TNT is included in
secondary explosive with other explosive chemicals. During World War I, the
production of TNT was limited by the amount of toluene available as a by-product
of the coke industry (Kirk and Othmer 1951). However, its potential use as an
explosive was not utilized for several years, mainly because of difficulty with
Bioremediation of 2,4,6-Trinitrotoluene Explosive Residues 203
detonation. After 1940, when toluene became readily available as a by-product of

the petroleum industry, TNT was manufactured extensively during World War II
(Kirk and Othmer 1951, 1993). TNT was also used as raw material with other
chemicals to synthesize various high energy producing explosives as mentioned in
Table 1 (Meyer et al. 2007; Pichtel 2012).
TNT is synthesized by nitration of toluene with the mixer of nitric acid and
sulphuric acid (Davis 1943). There are two processes used for the manufacture of
TNT: (1) continuous process and (2) three-stage batch process. To produce pure
TNT, the undesired isomers and residual dinitrated species must be removed from
the reaction mixture. The purity of the products was identified by its solidification
point (80.2–80.8 °C) (Urbanski 1984). This chemical is relatively insensitive to
shock and hence cannot be exploded without a detonator (Yinon and Zitrin 1993).
It is a non-hygroscopic chemical compound. On exposure to air at relative
humidity of 90 % and temperature 30 °C, only 0.03 % of water was absorbed
(Yinon 1990). It is thermally stable having low melting point and is amenable for
melt casting. Therefore, it is the most favoured chemical explosive in the military
and industry, because of its insensitivity to shock and resistance which reduces the
risk of accidental detonation (Pichtel 2012).
The presence of three nitro groups on the aromatic ring of TNT at position 2,4
and 6 makes it a highly electron deficient molecule and can be reduced both
biotically and abiotically (Hwang et al. 1998; Esteve-Nunez et al. 2001; Heiss and
Knackmuss 2002). The partial positive charge on nitrogen atom and the high
electronegativity of atoms assist TNT reduction (Esteve-Nunez et al. 2000, 2001).
In aerobic conditions, nitro groups of TNT are first reduced to form hydroxylamino
groups and finally converted amino groups containing aromatic compound. In
contrast, in anaerobic conditions, TNT is reduced sequentially to 2,4,6-Triamino-
toluene (TAT) (Wikstrom et al. 2000; Esteve-Nunez et al. 2001; Rieger et al. 2002).
The resistance of TNT to complete mineralization is because of reduction of the
nitro groups to produce by-products that are not chemically favourable for reduc-
tion. The by-products of TNT, such as dinitroaminotoluenes, diaminonitrotoluenes
Table 1 Use of TNT in the Explosive products Composition

synthesis of military high
energy producing explosives Amatex TNT, ammonium nitrate, RDX
(Leggett et al. 1977; Pichtel Ammonal TNT, ammonium nitrate, aluminium
2012) Anatols TNT, ammonium nitrate
Baratol TNT, barium nitrate
Composition B RDX (60 %), TNT (39 %), wax (1 %)
Cyclotol RDX, TNT
HTA-3 HMX, TNT, aluminium
Minol TNT, ammonium nitrate, aluminium
Octol HMX (70–75 %), TNT (25–30 %)
Pentolite Ammonium picrate, TNT
Tetrytol Tetryl, TNT
Torpex RDX, TNT, aluminium
Tritonal TNT (80 %), aluminium (20 %)
and TAT, can bind to the soil and become unavailable to microbes or self-poly-
merize into chains that are also not usually available for biological degradation
(Fig. 1) (Esteve-Nunez et al. 2001; Heiss and Knackmuss 2002). In fact, such
binding and self polymerization occur more rapidly and more frequently in oxic
conditions than anoxic conditions. Because of these reasons, the biological deg-
radation of TNT most likely occurs under anaerobic condition. In aerobic systems,
the degradation of TNT is minimal, as the soil and sediments adsorb and/or
covalently bind to many of the metabolites present in the soil (Wikstrom et al.
2000).
1.2 2,4,6-Trinitrotoluene as a Major Environmental

Pollutant
TNT is the one of major pollutants of the environment, because of its widespread
use as a military explosive (Fant et al. 2001; Travis et al. 2007; Kalderis et al.
2011). TNT was found in the atmosphere due to detonation and burning techniques
used in the demolition of armaments (US Army 1986). In addition to this, dust
particles of TNT and vapours found in the atmospheric air specifically at the places
of their manufacture and explosion (Fig. 1) (Hathaway 1985; Kannan and Kapoor
2006). TNT is also released into soil from spills, disposal of solid waste, open
incineration and detonation of ordnances and demolition of armaments (Kraus
et al. 1985; US Army 1986; USEPA 1989). Demolition of armaments can result in
the contamination of surface soils by the activities, such as open burning and
detonation or land filling of solid wastes generated during burning and non-
destructive reprocessing of armaments containing 2,4,6-trinitrotoluene (US Army
1986). TNT concentration was found to be 60–700 g/kg in both soils and sedi-
ments at military installations in the United States and Europe (Green et al. 1999;
Conder et al. 2004; Stenuit and Agathos 2010).
The presence of high concentration of TNT in the environment is of global
concern. TNT and its intermediates are found to be harmful to human beings,
animals, plants, and microorganisms (Meng et al. 2012). Infact, even low con-
centration of TNT is sufficient to inhibit the growth of the organisms present in the
ecosystem (Davies 2005). This can affect the primary production of phytoplankton
in the marine environment. TNT is released in large quantities in the aqueous
effluents of explosives, synthesizing amenities and grenades and also through field
use/disposal. Its relative solubility in water makes it to interact and contaminate
both water and soil (Frische 2003). The soil, groundwater and aquatic ecosystems
contaminated with TNT were classified as yellow water, red water, and pink water
based on their coloration (Barreto-Rodrigues et al. 2009). TNT contaminated red
water contains mainly dinitrotoluene sulfonates [DNTS, including 2,4-dinitrotol-
uene-3-sulfonate (2,4-DNT-3-S) and 2,4-dinitrotoluene-5-sulfonate (2,4-DNT-5-S)]
as well as other low-concentration of nitro group containing aromatic compounds
and inorganic salts (Meng et al. 2012). Because of its high toxicity, red water is not
permitted to be discharged into the environment without proper treatment (Meng
et al. 2012). Usually, TNT contamination occurs at the site of industrial plants,
because of its high production, military installations, munitions storage areas and
other areas where unexploded ordnances (UXOs) are present (Boopathy et al.
1997; Esteve-Nunez et al. 2001; Fant et al. 2001; Heiss and Knackmuss 2002;
Zaripov et al. 2002). Hence, decontamination of TNT in the system using different
remediation techniques depends on TNT concentrations and also the areas of the
contamination.
1.3 Toxic Effects of 2,4,6-Trinitrotoluene
TNT and its metabolites are found to be highly toxic to various organisms
including mammals, fish, insects, and bacteria (Lachance et al. 2004). The human
health risk factors of TNT exposure primarily affect the workers at the munitions
factories and disposal sites and are classified as Group C carcinogens (USEPA
1993). TNT toxicity is analyzed based on its lethal dose, route of exposure, length
of exposure, damage to the target organ and the health problems that may arise at
the later stage. TNT and its metabolites may enter the human system orally and
through inhalation, and skin absorption (Ryon 1987). There is an exposure risk to
military soldiers who handle bombs, grenades and RDX that contain TNT.
Quantifiable amount of TNT and its intermediates also exist in the rest rooms of a
munitions disposal factory and hand grenade intermediate storage area (Letzel
et al. 2003). After its exposure, TNT persists in the body for a longer time. TNT
was found in gastrointestinal tract, skin, liver, kidneys and lungs (El-Hawari et al.
1981). Its metabolites have been identified in the urine of munition factory workers
even after more than half month and away from the work place, indicating TNT
and/or its metabolites are slowly excreted from the body (Woollen et al. 1986).
Their toxic effects on humans result in disorders, such as anaemia, haemolysis,
impairment of the nervous system, cataracts, and liver toxicity.
In human beings, the toxic effects of TNT on the central nervous system are
observed in the form of seizures, hepatotoxicity, and immune system dysfunction
(Johnson et al. 2000; Letzel et al. 2003; Lachance et al. 2004). These effects differ
from those present in invertebrates where reproduction is affected at lower lethal
doses in chronic dosing experiments (Lachance et al. 1999, 2004; Robidoux et al.
1999; Steevens et al. 2002; Conder et al. 2004). The toxic action of TNT on cells is
commonly caused by the single electron reduction of the nitro groups, leading to
oxidative stress (Peres and Agathos 2000; Purohit and Basu 2000; Kumagai et al.
2004). TNT reduction reaction is carried out by type II nitroreductases which are
found both in bacterial and human cells (Ask et al. 2004; Sarlauskas et al. 2004).
Generally, the reaction proceeds in the presence of oxygen, the nitro anion radical
reacts with oxygen to form a superoxide anion radical and the original nitro group
(Peterson et al. 1979). Such type of reaction was termed as a futile cycle because
the reducing equivalents are oxidized without net reduction in the nitro groups
(Schenzle et al. 1999). The partially reduced TNT products can also bind cova-
lently to proteins and DNA (Sarlauskas et al. 2004). Bakhtiar et al. (1997) have
observed that TNT can form heme adducts in vitro when it was reduced to a
nitrosodinitrotoluene by sodium hydrosulphite. However, no heme adducts were
observed in the presence of unreduced TNT. TNT is believed to exhibit its toxicity
via the oxidation of haemoglobin iron (Peres and Agathos 2000). Its toxic effect
has been also studied in several animal models, including rats, mice, guinea pigs,
rabbits, dogs, and fish. The effects of TNT on these animals showed anaemia,
enlarged spleens and livers, decreased body weight, elevated blood cholesterol and
reduced serum glutamic-oxaloacetic transaminase. However, testicular atrophy
was observed only in rats. While a majority of the toxicity signs were reversible
after a month of recovery, but testicular atrophy in rats was not (Dilley et al. 1982).
The higher concentration of TNT is found lethal to the invertebrates. Fishes are
very sensitive to TNT and have a low LC50. The toxic effects of TNT on
microorganisms (bacteria and phytoplankton) are detrimental to primary produc-
tivity and alter the community structure in soils and aquatic ecosystems (Green
et al. 1999; Sagi-Ben Moshe et al. 2009). Therefore, it is necessary to understand
the damage that occurs on TNT contamination of eco-geological system (Green
et al. 1999; Johnson et al. 2000). If the ecosystem is damaged at lower tropic
levels, it makes an impact also on the higher tropic levels which destroy the
phytoplankton primary production in the soil (Esteve-Nunez et al. 2001; Sagi-Ben
Moshe et al. 2009). Infact, it is difficult to analyze overall effect of TNT con-
tamination on specific soils as their physical characteristics are not uniform in the
contaminated zones. For the determination of microbial toxicology, soil profiles
are used to assess the diversity present in the contaminated soils with respect to
non-contaminated soils (Siciliano et al. 2000; Sagi-Ben Moshe et al. 2009).
Denaturing gradient gel electrophoresis (DGGE) and/or terminal restriction frag-
ment length polymorphism (T-RFLP) are also used for such assessments. A
reduction in the diversity of bacteria or phytoplankton has been observed due to
the contamination of sites with TNT and its intermediates (Siciliano et al. 2000;
Sagi-Ben Moshe et al. 2009). The mutagenic properties of TNT and its interme-
diates have been observed in the order 2,4-diaminonitrotoluene and 4-aminodi-
nitrotoluene \ 2,6-diaminonitrotoluene \ 2-aminodinitrotoluene = TNT \ trini-
trobenzene (Lachance et al. 1999).
2 Biodegradation of TNT
The biodegradation of TNT occurs by various pathways in different microorgan-

isms. It involves transformation of the nitro groups of TNT without cleavage of the
aromatic ring (Hawari et al. 2000; Bernstein and Ronen 2012). The nitro group
present on ring has strong electron-withdrawing properties that promote high
electron deficiency with electrophilic characteristics on the p-electron system
which makes the aromatic ring as more stable (Rieger and Knackmuss 1995). The
four functional groups (three nitro groups plus one methyl group) provide high
stability to the aromatic ring and make it more difficult for enzymatic action
(Stenuit et al. 2005). Generally, the degradation of TNT occurs in the biological
system by reductive mechanism. The biodegradation of TNT occurs differently
under aerobic and anaerobic conditions.
2.1 Anaerobic Biodegradation
The ability of bacteria to transform TNT under anaerobic condition has been
investigated by several research groups (Table 2). The biodegradation of TNT
under anaerobic condition occurs by reduction of the nitro group to form the
corresponding mononitroso, monohydroxylamino and monoamino derivatives
(Fig. 2). Such reactions involve two-electron transfers from nitro substitutes to
TNT. These monoamino derivatives were further transformed into diamino and
triamino derivatives through reductive mechanism (Fig. 2). The reactions are
catalyzed by a wide range of enzymes, like nitroreductase, aldehyde oxidase,
dihydrolipic amide dehydrogenase, cytochrome b5 reductase, diaphorases,
hydrogenases, xanthine oxidase, and carbon monoxide dehydrogenase (Esteve-
Nunez et al. 2001). In addition, the reduction occurs sequentially in two steps
involving single electron transfer with the formation of nitroanion, followed by
nitroso-metabolite catalyzed by nitrotroreductse (oxygen sensitive). These amino
derivatives were further transformed to triaminotoluene (TAT) under strictly
anaerobic conditions at less than -200 mV redox potential (Hawari et al. 2000).
Boopathy and Kulpa (1992) have studied the degradation of TNT by a sulphate
reducing bacterium, Desulfovibrio sp. which utilized TNT as source of nitrogen for
growth and also as terminal electron acceptor (Fig. 2). This organism converted
TNT to diaminonitrotoluene by reduction process. They predicted that diamino-
nitrotoluene may be further converted to triaminotoluene though they could not
identify this compound in the culture medium. However, they could identify tol-
uene in the culture medium under nitrogen limiting conditions. Under nitrogen-
rich conditions (i.e., in presence of ammonium), TNT was converted to diami-
nonitrotoluene, but toluene could not be produced. There are reports of alternative
transformation of TNT to partly reduced dihydroxylamine derivatives with for-
mation of phenolic amine products (Hughes et al. 1998). Esteve-Nunez and Ramos
(1998) have studied the metabolism of TNT by Pseudomonas sp. JLR11 which
utilizes TNT as a sole source of nitrogen and suggested that the removal of methyl
group occurs in the initial step of degradation of TNT under anaerobic condi-
tions. 1,3,5-Trinitrobenzene and 3,5-dinitroaniline were identified as metabolic
products in degradation process.
Table 2 Anaerobic degradation of TNT by various microorganisms

Organism References
Cellulomonas sp. ES6 Borch et al. (2005)
Clostridium acetobutylicum Hughes et al. (1998)
C. acetobutylicum Khan et al. (1997), Hughes et al. (1998)
C. bifermentans Lewis et al. (1996), Regan and Crawford (1994)
C. bifermentans ATCC 638 Ederer et al. (1997)
C. bifermentans KMR-1 Ederer et al. (1997)
Clostridium bifermentans LIP-1 Esteve-Nunez et al. (2001)
C. nitrophenolicum Sagi-Ben Moshe et al. (2009)
Clostridium paterianum Esteve-Nunez et al. (2001)
C. pasteurianum DSM 525 Preuss et al. (1993)
C. sordellii Ederer et al. (1997)
C. sporogenes Ederer et al. (1997)
C. thermoaceticum Huang et al. (2000)
Desulfovibrio sp., (B Strain) Boopathy et al. (1993)
D. gigas Boopathy and Manning (1996)
D. desulfuricans Boopathy and Manning (1996)
D. vulgaris Boopathy and Kulpa (1994)
Desulfovibrio sp. Drzyzga et al. (1999)
Desulfobacterium indolicum Boopathy et al. (1997)
Escherichia coli Ederer et al. (1997)
Klebsiella sp. C1 Kim et al. (2002)
Lactobacillus acidophilus Ederer et al. (1997)
L. casei Ederer et al. (1997)
L. lactis Ederer et al. (1997)
Lysobacter taiwanensis Gallagher et al. (2010)
Methylobacterium sp. BJ001 Van Aken et al. (2004)
Methanococcus strain B, Boopathy (1994)
M. deltae Boopathy (1994)
M. thermolithotrophicus Boopathy (1994)
Pseudomonas sp. Strain JLR 11 Esteve-Nunez et al. (2000)
Raoultella terrigena Claus et al. (2007)
Serratia marcescens Montpas et al. (1997)
Sulphate-reducing bacterium Preuss et al. (1993)
Veillonella alkalscens Esteve-Nunez et al. (2001)
2.2 Aerobic Biodegradation
There are several reports of aerobic degradation of TNT by microorganisms

(Table 3). Under aerobic condition, transformation of TNT involves formation of
the mono- and diamino derivatives, which were not further metabolized (Fig. 3).
However, their partially transformed nitroso and monohydroxylamino metabolites
can react themselves in the presence of oxygen to form azoxytetranitrotoluene
(Fig. 3) (McCormick et al. 1976; Haidour and Ramos 1996). Such transformations
remove TNT, but at the same time, the recalcitrant metabolites accumulate at high
CH3 CH3
O2N NHOH
CH3
NHOH NH2
NO2 Toluene
2-Hydroxylamino-4,6-DNT
NH2
2,4-DA-6-hydroxylaminotoluene
CH3 CH3
O2N NO2 O2N NH2 CH3
H2N NH2
CH3
NO2 NO2
O2N NH2
TNT NH2
2-Amino-4,6-DNT
TAT
NH2
CH3
2,4-DA-6-NT
O2N NO2
CH3
O2N NO2
NH2
CH3
CH3
NHOH 4-Amino-2,6-DNT O2N NO2
HO OH
4-Hydroxylamino-4,6-DNT
NHCOCH3
OH
4-Acetamido-2,6-
dinitrotoluene Methylphloroglucinol
CH3
O2N NHOH
CH3
O2N NH2
NHOH
2,4-Dihydroxylamino- HO
6-nitrotoluene CH3
NHOH
2-A-5-hydroxy-4-hydroxylamino-6-NT
OH
4-Hydroxytoluene
Fig. 2 Proposed pathways for anaerobic degradation of TNT by microorganisms (Esteve-Nunez

et al. 2001; Kalderis et al. 2011; Bernstein and Ronen 2012)
concentration. The amino intermediates may follow an alternative route through

deamination with the formation of benzoic acid or N-acetylamino derivatives
(Fig. 3) (Gilcrease and Murphy 1995; Vanderberg et al. 1995). However, these
Table 3 Aerobic degradation of TNT by various microorganisms

Organism References
Achromobacter Muter et al. (2012)
Acinetobacter johnsoni Fuller and Manning (1997)
Acinetobacter junii A8 Soojhawon et al. (2005)
Acinetobacter sp. VT11 Solyanikova et al. (2012)
Agrobacterium sp. 2PC Fuller and Manning (1997)
Alcaligenes eutrophus Fuller and Manning (1997)
Anabaena sp. Pavlostathis and Jackson (1999)
Arthrobacter globiformis Fuller and Manning (1997)
Arthrobacter sp. RP17 Fuller and Manning (1997)
Bacillus cereus Fuller and Manning (1997)
B. subtilis Fuller and Manning (1997)
Bacillus sp. Kalafut et al. (1998)
Clavibacterium agropyri (R.L2) Gh and Moussa (2011)
Corynebacterium glutamicum Fuller and Manning (1997)
Corynebacterium sp. Nap2 Fuller and Manning (1997)
Cytophaga pectinovora Fuller and Manning (1997)
Enterobacter cloacae PB2 French et al. (1998)
Escherichia coli Fuller and Manning (1997), Stenuit et al. (2005)
Flavobacterium odoratum Fuller and Manning (1997)
Klebsiella Muter et al. (2012)
Klebsiella sp. 1PC Fuller and Manning (1997)
Klebsiella sp. Strain C1 Chang et al. (2002)
Klebsiella pnueomoniae Litake et al. (2005)
Kocuria palustri RS32 Solyanikova et al. (2012)
Micrococcus luteus Fuller and Manning (1997)
Mycobacterium sp. HL4-NT-1 Vorbeck et al. (1994)
M. vaccae strain JOB5 Vanderberg et al. (1995)
Myxococcus xanthus Fuller and Manning (1997)
Nocardiodes CB22-2 Behrend and Heesche-Wagner (1999)
Pantoea sp. Strain Thu-A Zou et al. (2012)
Pantoea sp. Strain Thu-B Zou et al. (2012)
Pantoea sp. Strain Thu-C Zou et al. (2012)
Pantoea sp. Strain Thu-Z Zou et al. (2012)
Pseudomonas Duque et al. (1993)
Pseudomonas Muter et al. (2012)
Pseudomonas aeruginosa Kalafut et al. (1998)
Pseudomonas aeruginosa Oh et al. (2001)
Pseudomonas aeruginosa Fuller and Manning (1997)
P. aeruginosa MA01 Alvarez et al. (1995)
P. cepacia Fuller and Manning (1997)
P. fluorescens Fuller and Manning (1997)
P. fluorescens Pak et al. (2000)
P. pseudoalcaligenes JS52 Fiorella and Spain (1997)
P. putida Fuller and Manning (1997), Park et al. (2003)
P. putida HK-6 Cho et al. (2008)
(continued)
Table 3 (continued)
Organism References
P. putida strain KP-T201 Park et al. (2002)
Pseudomonas sp.clone A Haidour and Ramos (1996)
Pseudomonas sp. DFC49 Fuller and Manning (1997)
Pseudomonas sp. JLR11 Esteve-Nunez and Ramos (1998)
Pseudomonas sp. Tol1A Fuller and Manning (1997)
Pseudomonas sp. JS150 Fuller and Manning (1997)
Pseudomonas DFC49 Fuller and Manning (1997)
Pseudomonas sp. strain TM15 Kubota et al. (2008)
Pseudomonas sp. Esteve-Nunez et al. (2001)
Pseudoxanthomonas Muter et al. (2012)
Raoultella Muter et al. (2012)
Rahnella aquitilis BFB Fuller and Manning (1997)
R. erthropolis Vorbeck et al. (1998)
R. erthropolis Fuller and Manning (1997)
R. globerulus Fuller and Manning (1997)
R. rhodocrouss Fuller and Manning (1997)
Rhizobium sp. T10 Labidi et al. (2001)
Rhizobium sp. B5 Labidi et al. (2001)
Rhizobium sp. M8 Labidi et al. (2001)
Rhodococcus sp. TF2 Fuller and Manning (1997)
Rhodococcus erythropolis Esteve-Núñez et al. (2001)
Rhdococcus opacus 1G Solyanikova et al. (2012)
Rhdococcus sp. VT-7 Solyanikova et al. (2012)
Salmonella typhimurium Litake et al. (2005)
Serratia Muter et al. (2012)
Serratia marcescens Montpas et al. (1997)
SP1b (coryneform) Fuller and Manning (1997)
Sphingomonas capsulata Fuller and Manning (1997)
Sphingomonas sanguinis (R.L2) Gh and Moussa (2011)
Staphylococcus sp. Kalafut et al. (1998)
Stenotrophomonas Muter et al. (2012)
Streptomyces albus Fuller and Manning (1997)
S. chromofuscus A11 Pasti-Grigsby et al. (1996)
S. griseus Fuller and Manning (1997)
amino intermediates, that accumulates in the environment, were not considered as

dead-end products. Tront and Hughes (2005) have investigated a novel pathway
for TNT metabolism where TNT served as the sole carbon, nitrogen, and energy
source under an aerobic condition. Their results showed the ability of microor-
ganisms to oxidize TNT directly through removal of a nitro group and oxygenation
of the aromatic ring. A metabolic intermediate, 3-methyl-4,6-dinitrocatechol, was
identified through stable isotope mass spectrometry and tandem mass spectrometry
(Fig. 3). Radio labeled tracers were used to demonstrate that TNT-derived carbon
O2N NO2 CH3

NH2
CH3
H3C
O2N NHOH
N O
NO2
N 2-Amino-4-
H 3C NO2 nitrotoluene CH3
2-Hydroxylamino- H 2N NH2
4,6-dinitrotoluene
O2N NO2
CH3
4,4',6,6'-Tetranitro- NO2
O2N NH2
2,2'-azoxytoluene
2,6-Diamino-4-
O2N NO2 nitrotoluene
NO2
H3C 2-Amino-4,6-
CH3 CH3 dinitrotoluene
N O
O2N NO2 O2N NH2
N
CH3
NH2 O2N NH2

NO2
O2N NO2 TNT 2,4-Diamino-6-dinitrotoluene
CH3
NHCOCH3
2,4',6,6'-Tetranitro-
CH3 4-Acetamido-2-
4,2'-azoxytoluene
amino-6-nitrotoluene
O2N NO2
CH3
O2N NO
NH2
4-Amino-2,6-dinitrotoluene
CH3
CH3
O2N NO2 CH3 NH2
O2N OH 2-Nitroso-4-amino-
O2N NO2
6-nitrotoluene
OH
N O NO2
NHOH
N 3-Methyl-4,6-dinitrocatechol
4-Hydroxylamino- CH3 CH3
2,6-dinitrotoluene
O2N NHOH O2N NHOH
O2N NO2
CH3
NHOH NH2
2,2',6,6'-Tetranitro- 2,4-Dihydroxylamino- 2-Hydroxylamino-4-
4,4'-azoxytoluene 6-nitrotoluene amino-6-nitrotoluene
Fig. 3 Proposed pathways for aerobic degradation of TNT by microorganisms (Esteve-Nunez

et al. 2001; Tront and Hughes 2005; Kalderis et al. 2011; Bernstein and Ronen 2012)
was incorporated into biomass and that metabolic products included CO2 and NO2,
thus demonstrating an alternative aerobic metabolic pathway leading to mineral-
ization of TNT.
2.3 Biodegradation of TNT by Fungi
There are also reports of mineralization of TNT by white rot fungus, Phanero-
chaete chrysosporium and other fungi under ligninolytic conditions (Fernando
et al. 1990; Bumpus and Tatarko 1994; Hawari et al. 1999; Hodgson et al. 2000;
Esteve-Nunez et al. 2001; Kim and Song 2001). The lignin degrading fungus,
Phanerochaete chrysosporium produces various enzymes, such as lignin peroxi-
dase, manganese peroxidase, oxidases, reductases, hydrogen peroxidase, veratryl
alcohol, oxalate, and quinol oxidases (Fernando et al. 1990; Bumpus and Tatarko
1994; Stahl and Aust 1995; Hawari et al. 1999). Most of fungi prefer transfor-
mation of TNT through reductive mechanism rather than oxidative mechanism.
Stahl and Aust (1993a) demonstrated that P. chrysosporium mycelium can
transform TNT to a mixture of 2-ADNT, 4-ADNT, 4-hydroxylamino-2,6-dini-
trotoluene, and azoxytetranitrotoluenes (Fig. 4). TNT is reduced by extracellular
enzymes synthesized by P. chrysosporium that requires live and intact mycelia
(Stahl and Aust 1993a, b, 1995). If any system destroys the integrity of the
extracellular membrane, it leads to inactivation of nitroreductase. The compounds,
which are known to be inhibit extracellular systems, also suppress the activity of
nitroreductase. Rieble and co-workers demonstrated that NAD(P)H acts as co-
substrate in absence of oxygen for extracellular-bound TNT nitroreductase (Rieble
et al. 1994). Alternatively, there is a report on NAD(P)H-dependent intracellular
TNT reductase (Michels and Gottschalk 1995). However, the exact mechanism, by
which fungus mineralizes TNT, is not known though its preliminary work has been
demonstrated (Michels and Gottschalk 1995; Hodgson et al. 2000). P. chrysos-
porium transforms 4-ADNT to 4-formamide-2,6-DNT and then to 2-amino-4-
formamide-6-nitrotoluene, which disappears rapidly under lignolytic systems, but
not under nonligninolytic conditions. In nonligninolytic systems, ADNT inter-
mediates are gradually reduced to DANTs and hence, an increased concentration
of azoxytetranitrotoluenes is observed (Fig. 4). TNT-derived intermediate hy-
droxylaminodinitrotoluene inhibits the activity of veratryl alcohol oxidase of lig-
nin peroxidise (Bumpus and Tatarko 1994; Michels and Gottschalk 1994). Due to
this activity, lignin peroxidase is protected from H2O2 inactivation, and also it
explains why the presence of the hydroxylaminodinitrotoluenes makes ADNT
more readily mineralizable than TNT (Esteve-Nunez et al. 2001).
Lee et al. (2009) used white-rot fungus, Irpex lacteus to transform TNT to
4-amino-2,6-dinitrotoluene (4-ADNT) and 2-amino-4,6-dinitrotoluene (2-ADNT))
which undergo further degradation. In addition to other enzyme systems, man-
ganese peroxidase plays a significant role in the TNT transformation. The man-
ganese peroxidase enzyme, prepared from white-rot fungi, Nematoloma frowardii
CH3
O2N NH2
CH3
CH3
O2N NH2
O2N NH2
NH
NH2 CHO
NHCOCH3 2-A-4-Formamido-6-NT
2-A-4-acetamido- 2,4-DA-6-NT
6-nitrotoluene
CH3 CH3
O2N NO2 O2N NO2
CH3 CH3
+
O2N NO2 O2N NO2
HONCOCH3 NHOCOCH3
4-N-AcHDNT 4-N-AcoxyDNT
NO2 NH
TNT CHO
CH3 4-Formamido-2,6-DNT
CH3
O2N NO2
O2N NO2
CH3
NHOH O2N NO2

N
O
4-Hydroxyl-2,6-DNT
4-Nitroso-2,6-DNT
NH2
O2 4-A-2,6-DNT
_ CH 3
O
O2N NH2
Ar-N=N-Ar
Azoxytetranitrotoluene
HO
NH 2 CH 3
O2N NHOH
5-Hydroxy-2,4-DA-6-NT
O2N NO2
NHCOCH3
H3C N= N CH3
CH3 CH3
2-Hydroxylamino-4-
O2N Azotetranitrotoluene NO2 O2N NH2 O2N NH 2
acetamido-6-NT
+
OH H O
NO2 NO2
Ar-HN=NH-Ar 4-Amino-5-hydroxy- 2-Amino-5-hydroxy-

2,6-dinitrotoluene 4,6-dinitrotoluene
Hydrazotetranitrotoluene
Fig. 4 Proposed pathways for the degradation of TNT by fungi (Spain 1995; Esteve-Nunez et al.
2001; Kalderis et al. 2011)
and Phlebia radiat, was used to mineralize TNT and its reduction products
(Scheibner et al. 1997; Scheibner and Hofrichter 1998; Van Aken et al. 1999). The
fungal strains, belonging to wood- and litter-decaying basidiomycetes, were also
used for their capability to metabolize and mineralize TNT (Scheibner et al. 1997;
Van Aken et al. 1999). Under lignolytic conditions, significant mineralization was
caused by Clitocybula dusenii TMB12 and Stropharia rugosoanulata DSM11372
which suggests a vital role of ligninolytic systems in the mineralization process by
these fungi. Ziganshin et al. (2010) have isolated a yeast strain Geotrichum can-
didum AN-Z4 from an anthropogenically polluted site that was capable of trans-
formation of TNT.
2.4 Biodegradation of TNT by Plants
Generally, plants can mineralize TNT through three main phases; phase I
(transformation), phase II (conjugation), and phase III (compartmentation) (Fig. 5)
(Sanderman 1994; Ohkawa et al. 1999). In Phase I, plants detoxify TNT by
reductive mechanism with the formation of hydroxylaminodinitrotoluene (HAD-
NT) via nitroso intermediates and finally to aminodinitrotoluene products. Though
the final products are stable, but the HADNT intermediates are unstable at
25–35 °C. A wide range of nitroreductases present in the plants may detoxify
TNT. Myriophyllum aquaticum is an aquatic plant that metabolizes TNT through
oxidation of methyl group or hydroxylation of aromatic ring (Bhadra et al. 1999).
When Myriophyllum aquaticum was incubated with radiolabelled ADNT or
HADNT, the products of oxidative transformation of these substrates were not
detected. These results suggested that direct transformation of TNT occurred by
the oxidative mechanism (Bhadra et al. 1999). Phase 2, involves the conjugation of
transformed intermediates with polar substrate molecules like sugars, glutathione
and amino acids (Coleman et al. 1997). Initially, these hydrolysable TNT conju-
gates were identified in Phaseolus vulgaris. 2- and 4-ADNT conjugated to one or
more six carbon units were identified in Madagscar Periwinklle extracts (Fig. 5)
(Harvey et al. 1990; Bhadra et al. 1999). The glycosyltransferase enzymes may be
involved in the conjugation. Further studies revealed the formation of mono- and
diglycoside conjugates of the unstable 2- and 4-HADNT intermediates (Fig. 5)
(Wayment et al. 1999; Vila et al. 2005; Subramanian et al. 2006). In Arabidopsis
thaliana (Arabidopsis), glycosyl transferases (UGTs) catalyzed the conjugation of
TNT-transformation products 2- and 4-hydroxylaminodinitrotoulene and these
enzymes play an important role in TNT detoxification (Gandia-Herrero et al.
2008). Phase 3, involves compartmentation of the conjugated metabolites which
may be deposited in vacuoles or undergo hydrolysis and release of the compound
into the apoplast region. The final phase of metabolism has been characterized into
two independent phases, first one involves only transport and storage in the vac-
uole, and in second phase, the final reaction occurs, such as binding to cell wall or
excretion (Schroder 2007).
CH3 UPTAKE CH3 UPTAKE CH3

O2N NO2 O2N NO2 O2N NO
NO NO2 NO2
4-Nitroso-2,6- 2-Nitroso-4,6-
dinitrotoluene TNT
dinitrotoluene
CH3 PHASE I-TRANSFORMATION CH3

O2N NO2 O2N NHOH
NHOH NO2
4-Hydroxylamino- 2-Hydroxylamino-
2,6-dintrotoluene 4,6-dintrotoluene
4-Hydroxylamino- 4-Hydroxylamino-
2,6-dintrotoluene 2,6-dintrotoluene 2-Hydroxylamino- 2-Hydroxylamino-
O-glycoside C-glycoside 4,6-dintrotoluene 4,6-dintrotoluene
C-glycoside O-glycoside
CH3 CH3
O2N NO2 O2N NH2
NH2 NO2
4-Amino-2,6- 2-Amino-4,6-
dinitrotoluene dinitrotoluene
PHASE II-CONJUGATION
4-Amino-2,6-dinitro- 2-Amino-4,6-dinitro-
toluene glycoside toluene glycoside
PHASE III-COMPARTMENTATION
Fig. 5 Proposed pathways for the degradation of TNT by plants (Spain et al. 2000; Gandia-
Herrero et al. 2008; Rylott and Bruce 2009)
3 Enzymes Involved in the Degradation of TNT
At present, there is no evidence of complete degradation of TNT, but it can be

transformed into various metabolites by the different microorganisms and plants.
The biological systems contain various enzymes, such as pentaerythritol tetrani-
trate (PETN) reductase, morphinone reductase, old yellow enzyme (OYE),
xenobiotic reductase XenA and XenB, flavonitro reductases and N-ethylmaleimide
(NEM) reductase NemA. These enzymes may catalyze the reduction of nitro
substitutes in TNT with the formation of hydroxylamine and amine derivatives. A
group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD)-
dependent nitroreductase enzymes are able to metabolize TNT using the reducing
power of nicotinamide adenine dinucleotide [NAD(P)H]. In Enterobacter cloacae
PB2, the enzyme catalyzing reduction of TNT is known as pentaerythritol tetra-
nitrate reductase (PETNr), a monomeric 40 kDa flavoenzyme (Binks et al. 1996;
French et al. 1996). PETNr enzyme is capable of catalyzing reduction of both nitro
groups and aromatic ring of TNT (Fig. 6) (Symons and Bruce 2006). This enzyme
was isolated based on its ability to confer resistance to PETN, a nitrite ester
explosive on the bacterium (Binks et al. 1996) and it was found to be a member of
the Old Yellow Enzyme family of flavoenzymes (Williams et al. 2004). The
enzyme is capable of reductive liberation of nitrite from TNT. Caballero et al.
(2005), determined that PnrA was a NADPH dependent nitroreductase, homolo-
gous to nfsA, the E. Coli nitroreductase, capable of transforming TNT to yield
4-hydroxylamino-2,6-dinitrotoluene via a ping-pong bi–bi mechanism (Fig. 6).
+ +
_ CH3 NAD(P)H + H NAD(P) CH3
CH3 +
2e + 2H H2O O2N NHOH
O2N NO
O2N NO2 _ +
2e + 2H
+ + Nitroso-dinitrotoluene NO2
NO2 NAD(P)H + H NAD(P) NO2 reductase
TNT 2-Hydroxylamino-4,6-
TNT 2-Nitroso-4,6- dinitrotoluene
reductase dinitrotoluene
_ + +
2e + 2H NAD(P)H + H
2-Hydroxylamino-4,6-
dinitrotoluene reductase
+
H2O NAD(P)
CH3
O2N NH2
NO2
2-Amino-4,6-dinitrotoluene
Fig. 6 Nitroreductase-catalyzed initial reactions in the biodegradation of TNT (Spain et al.

2000; Symons and Bruce 2006)
Reductive transformation of TNT via nitroso intermediate to form 2- or 4-HADNT

was found to be catalyzed by FMN-containing nitroreductase. The nitroreductase
enzyme encoded by nfsI gene similar to onr was also cloned from E. cloacae
(Bryant and DeLuca 1991). Nitroreductases can transform TNT much faster than
PETNr and these enzymes, when expressed in transgenic plants, showed higher
activity with TNT than PENTr (French et al. 1998, 1999; Hannink et al. 2001;
Abhilash et al. 2009; Rylott and Bruce 2009).
Hydride transferase enzyme plays a significant role in such type of reductive
transformation of TNT. The partial reduction of di- or trinitro substitutes of the
aromatic ring through addition of hydride ions, catalyzed by XenB, NemA and
PETN reductase tends to initiate formation of a hydride-Meisenheimer complex
(Spain 1995). This complex was observed in TNT because the oxygenolytic attack
is difficult due to its electron deficiency. The protonation of hydride-Meisenheimer
complex leads to enzyme-catalyzed rearomatization of the molecule and release of
nitrite, which can be utilized by the bacteria (Fig. 7) (Rieger and Knackmuss
1995). A reductive biotransformation of TNT is catalyzed by some members of
Old Yellow Enzyme family including PETNr (French et al. 1998; Pak et al. 2000;
Williams et al. 2004). The white-rot fungus Irpex lacteus on incubation with TNT
showed the formation of 2,4-dinitrotoluene as a metabolic product of TNT’s
hydride-Meisenheimer complexes (Kim and Song 2000). The formation of
hydride- and dihydride-Meisenheimer TNT complex with the release of nitrite was
catalyzed by TNT hydride transferase (Fig. 7) (Pak et al. 2000; Stenuit et al. 2006;
Van Dillewijn et al. 2008; Wittich et al. 2008). The nitrite released in the reduction
was originated from the dihydride-Meisenheimer complex rather than from the
hydroxylamine (Wittich et al. 2009). Stenuit et al. (2009) have extracted extra-
cellular enzyme from Pseudomonas aeruginosa, which on incubation with TNT
and NAD(P)H, showed the release of nitrite. Recently, Ziganshin et al. (2010)
have demonstrated that a yeast strain Geotrichum candidum transformed TNT
through the formation of unstable intermediate hydride-Meisenheimer complexes
with their subsequent destruction and accumulation of nitrite and nitrate (Fig. 7).
4 Bioremediation of TNT
Bioremediation is the process whereby waste products are biologically degraded

under specific conditions to a non-toxic form or to levels below concentration
limits established by regulatory authorities (Mueller et al. 1996). Bioremediation
involves use of biological systems to catalyze degradation or transformation of
toxic chemicals to less harmful forms. Bioremediation is an eco-friendly and cost-
effective pollution control technology. Unlike conventional technologies, biore-
mediation can be carried out on-site. Bioremediation of TNT contaminated soils
includes the use of slurry (Boopathy et al. 1998a, b), composting (Rezaei et al.
2010), land farming (Clark and Boopathy 2007), bioaugmentation and biostimu-
lation methods and also use of transgenic or genetically engineered plants
CH3 CH3
NO2
O2N NO2 O2N
H H H
TNT Hydride H H
H
NO2 transferase NO2
CH3 TNT Hydride Hydride-Meisenheimer Dihydride-Meisenheimer

transferase complex _ complex _
O2N NO2 (2H-TNT)
(H-TNT)
NO2
TNT
TNT Hydride
transferase
CH3
H CH3
O2N NO2 O2N NO2
H H
+
H H
H H
NO2 NO2
_ _
3H-TNT 1H-TNT
TNT-trihydride complex TNT-monohydride complex
TNT Hydride
transferase
O2N NO2
H H
H H
+
N
_
O OH
_ +
Isomers of 3,5--2H-TNT.H
Fig. 7 Hydride transferase-catalyzed initial reactions in the biodegradation of TNT (Spain et al.
2000; Symons and Bruce 2006; Ziganshin et al. 2010)
(Van Dillewijn et al. 2008; Wang et al. 2009). Composting has been used for the
field-scale bioremediation of TNT contaminated sites. The initial anaerobic
transformation of TNT, followed by humification under aerobic conditions, was
found to be more effective (Kalderis et al. 2011).
Biological treatments of TNT consist of composting and bioslurry mechanisms
(Kalderis et al. 2011). Both composting and bioslurry systems proceed through co-
metabolism based on the reduction of the nitro groups of TNT by microbes. In this
process, hydroxylamine and amine groups, present on the nitroaromatic ring, react
with quinones and carbonyl substitutes of the humic fraction of the soil leading to
formation of immobilized TNT derivatives that are not biologically available and
thus exhibit decreased toxicity. In composting process, the soil is mixed with
alternative degradable substrate for the growth of organisms in the soil to trans-
form TNT. The windrow composting is a better process which involves both
anaerobic and aerobic phases. The first phase proceeds anaerobically with the
reduction of TNT and condensation of the amine derivatives to the humic material.
In the second phase, TNT products, formed under anaerobic conditions, were
metabolized by the soil microbes to harmless products. The bioslurry system uses
a mixture of contaminated substance with water and nutrients. As compared to
composting system, the bioslurry treatment is much faster and high rate of TNT
reduction can be achieved. The sequential anaerobic bioremediation, which also
uses both anaerobic and aerobic process, consists of a consortium of facultative
anaerobic organisms. Hess and Schrader (2002) have studied a combined process
having a fast abiotic pre-treatment with more concentrated hydroxyl radicals,
followed by a bioslurry treatment to degrade the products from the first step. In this
process, TNT can be mineralized up to 97 %. Pseudomonas putida was found to
be superior bacterial strain for TNT removal. Bioremediation of clay soil con-
taminated with TNT was tested in the slurry phase, resulting in more than 89 %
removal of TNT during a period of 15 days (Sheibani et al. 2011). Erkelens et al.
(2012) have studied to assess the potential of a previously bioremediated hydro-
carbon contaminated soil (PBR) to increase TNT degradation rates. This was
performed by adding TNT chips to PBR and uncontaminated soils (PNC) in
laboratory based studies (up to 16 weeks). Residual TNT chip analysis showed
higher TNT degradation in PBR soils (70 %) than in PNC soils (30 %).
4.1 Microbial Remediation of TNT
Microorganisms have evolved diverse pathways for degradation of nitroaromatics.

TNT can be mineralized or transformed by the microbes under anaerobic condi-
tions either by removal of the NO2 substitutes to be used as a nitrogen source/
terminal electron acceptor or by respiration of the NO2 substitutes (Boopathy et al.
1997; Hawari et al. 1999; Esteve-Nunez et al. 2001; Heiss and Knackmuss 2002;
Rieger et al. 2002; Gonzalez-Perez et al. 2007). In anaerobic system, Type I
nitroreductase can be used for effective removal of nitro groups present on the
TNT due to its insensitivity to oxygen. In this process, the nitroreductase enzyme
(Type I) catalyzes the reduction of nitro groups to amino groups (Shah and Spain
1996). Microbes have been cultured which are able to transform TNT to
monoaminonitrotoluenes, diaminonitrotoluenes, and possibly to triaminotoluene
(Esteve-Nunez et al. 2001). The two bacterial genera capable of mineralizing TNT
are Clostridia and Desulfovibrio (Rieger et al. 2002). In Clostridia, TNT can be
metabolized to TAT alongwith other unknown products (Esteve-Nunez et al.
2001). E. coli species are also capable of enzymatically reducing TNT to TAT and
use amino groups as a nitrogen source for growth when glucose is present as a co-
substrate (Esteve-Nunez et al. 2001; Gonzalez-Perez et al. 2007). A reduction of
TNT results in the release of nitrogen from the nitroaromatic ring as nitrite or
ammonia (Esteve-Nunez et al. 2001). Nitroreductases usually catalyze this
reduction, however, there are also other enzymes, such as aldehyde oxidase, di-
hydrophilic amide dehydrogenase, cytochrome b5 reductase, diaphorases,
hydrogenases, xanthine oxidase, and carbon monoxide dehydrogenase (Honeycutt
et al. 1996; Esteve-Nunez et al. 2001; Gonzalez-Perez et al. 2007). These enzymes
are present in several of bacterial strains and eukaryotes that carry out the initial
steps of metabolism of TNT both in laboratory and environmental conditions.
Several Desulfovibrio strains have been isolated which are capable to utilize TNT
as a sole source of nitrogen. Desulfovibrio sp. strain B utilized TNT as a sole
source of nitrogen and formed toluene as observed in the culture medium (Bo-
opathy and Kulpa 1992). A wild type strain E. coli AB1157 was found capable of
utilizing TNT as a sole nitrogen with glucose as a carbon source in the medium
(Gonzalez-Perez et al. 2007). Pseudomonas sp. strain JLR11 was capable of uti-
lizing TNT as a sole source of nitrogen by releasing nitrite and reducing to
ammonium with its incorporation into carbon skeletons when glucose was present
as a co-substrate (Esteve-Nunez et al. 2000). TNT transformation can occur when
nitrate, sulphate or carbon dioxide were present as an electron acceptor. On the
removal of amino substitutes, TAT can be converted to toluene, p-cresol, and
methylphloroglucinol (Esteve-Nunez et al. 2001). Herrmann et al. (2007) have
demonstrated that Pseudomonas aeruginosa SH-2, Pseudomonas putida MC-I, and
Pseudomonas sp. X. Burkholderia cepacia SH-1 are capable of transforming about
80 % of TNT into metabolic products and addition of glucose or succinate
improved both the growth of cells and TNT uptake. Recently, Cho et al. (2012)
have observed that reductive degradation of TNT can be enhanced by bio-reduced
iron bearing soil minerals (IBSMs) using Shewanella putrefaciens CN32. Further,
Muter et al. (2012) mentioned that an increase in nutrient amendment concen-
tration led to an increase in the TNT degradation. After inoculation of bacterial
consortium AM 06 and incubating for 14 days, an initial TNT concentration of
100 mg/l in liquid samples was reduced from 72.7 mg/l (no added nutrients) to
58.2 mg/l (10 % nutrient solution added), 40.0 mg/l (50 % nutrient solution
added), and 8.0 mg/l (100 % nutrient solution added). The presence of TNT at
concentrations less than 100 mg/l did not influence sugar consumption in the
liquid samples. In soil samples with high initial concentrations of TNT (500 mg/
kg), the contribution of bioaugmentation for the degradation of TNT has been
demonstrated for the soil samples amended with 50 and 100 % nutrient solutions
(Muter et al. 2012).
Biostimulation is a major soil remediation technology. There are also some
reports available on the degradation of TNT by immobilized microbes. Ullah et al.
(2010) have studied biodegradation of TNT by immobilized Bacillus sp. YRE1 at
different temperatures. It was found that both charcoal and polystyrene immobi-
lized bacteria degraded TNT more efficiently at 37 °C. However, a maximum
reduction of 73.35 % was observed in case of charcoal immobilized Bacillus sp.
YRE1 at 37 °C whereas, polystyrene immobilized bacteria showed only 70.58 %

reduction. Bacillus sp. YRE1 immobilized on charcoal showed maximum degra-
dation at pH 7 with 93.81 % reduction in TNT. Similarly, pH 5 was found to be
optimum for 94 % degradation of TNT by polystyrene immobilized bacteria.
Charcoal immobilized cells showed an enhanced transformation with 96 %
reduction in the presence of Tween 20 whereas, polystyrene immobilized cultures
showed 87.77 % reduction in TNT. Wang et al. (2010) studied the combined
process of immobilized microorganism-biological filter which can be used to
degrade TNT in an aqueous solution. The results showed that the process could
effectively degrade TNT-contaminated effluents.
4.2 Phytoremediation of TNT
Phytoremediation is the process whereby toxic chemicals like TNT can be

degraded to less non-toxic forms by plants. Therefore, it is considered as an eco-
friendly and low-cost alternative to the other remediation techniques (Rylott and
Bruce 2009). During phytoremediation process, plants either degrade TNT or
immobilize it through incorporation into vacuoles and cell walls (Rittmann et al.
1994; Mueller et al. 1995). Before applying this technique, one should know the
interaction of plants with TNT and their tolerance to TNT. It also depends on the
toxic effects of TNT on the plant species. Many of plant species show tolerance to
TNT in the range of 0.05–0.1 g/kg in the soil. The type of soil also has significant
effect on binding: high humic containing soil binds more TNT. Its removal from
biologically available pool and lowering of its toxicity mainly depend on the plant
species, growth stage of the plant, biological availability of TNT and soil quality
(Hannink et al. 2002; Thorn and Kennedy 2002; Rocheleau et al. 2006). For
example, germinating seeds and mature plants of the same species tolerate various
concentrations of TNT. Although the high concentration of TNT is bioavailable in
aqueous conditions, but plant tends to tolerate lower concentrations of TNT in
water than in soils where bioavailability is more restricted. Basically, the phyto-
remediation of explosives is performed in wetlands. A test was conducted for the
feasibility of treating contaminated groundwater with wetlands that includes two
types of wetlands for comparison, lagoon system with submergent plants and a
gravel-bed wetland with emergent plants (Richman 1996). In both the cases, TNT
removal was observed below detection limit, but its mineralization was found low.
In addition, Best and co-workers have demonstrated the aquatic and wetland plant
treatments photolysis of TNT (Best et al. 1999). Gong and co-worker have studied
the toxic effect of TNT on different plant species and their results indicated low
levels of TNT (5–25 mg/kg) for Lepidium sativum (cress) and Brassica rapa
(turnip) and high levels (25–50 mg/kg) for Acena sativa (oat) and Triticum aes-
tivum (wheat) for causing toxicity (Gong et al. 1999). However, Acena sativa was
capable of tolerating up to 1,600 mg TNT/kg and thus demonstrated a potential
ability to detoxify soil. Therefore, compared to other plant species, Acena sativa
appears to be a promising bioremediation tool in low-level TNT contaminated

soils (Gong et al. 1999).
Plants detoxify TNT through their biochemical transformation, followed by
conjugation and sequestration within plant tissues or polymers rather than being
mineralized to carbon dioxide and nitrogen. Plants transform TNT by reductive
and oxidative mechanisms. A wide range of TNT reductive derivatives, such
as 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2-hydroxylamino-4,
6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene were found in various
plants (Rylott and Bruce 2009). Many of these derivatives accumulate in the roots
where their concentrations usually go beyond that of TNT. Plants are capable of
detoxifying TNT because of presence of a wide range of nitroreductase enzymes,
as found in microbes. Myriophyllum aquaticum, an aquatic plant, transformed
TNT via oxidative mechanisms with the formation of metabolites, such as 2-
amino-4,6-dinitrobenzoate, 2-N-acetoxyamino-4,6-dinitrobenzaldehyde, 2,4-dini-
tro-6-hydroxybenzyl alcohol and 2,4-dinitro-6-hydroxytoluene (Rylott and Bruce
2009). Final two products are result of ring hydroxylation with the release of a
nitro group. The derivatives with less nitro substitutes are more susceptible to
microbial degradation. TNT metabolites are later conjugated with plant-derived
glucose, malonate or glutathione. In recent years, genetic engineering has effi-
ciently improved phytoremediation through a transgenic approach for removal of
TNT from the system. Experiments using tobacco cell-suspension cultures showed
mono- and diglycoside conjugates of 2-HADNT and 4-HADNT (Brentner et al.
2008). 0.025 mM TNT inhibited the growth of wild type tobacco plant but onr
tobacco lines germinated and grew normally at 0.05 mM TNT (Ramos et al.
2005). However, these transgenic plants failed to grow at 0.5 mM TNT, but nfs
lines germinated well at this concentration and removed TNT from hydroponic
media much faster than wild-type plants. Recently, Zhu et al. (2012) demonstrated
the enhanced transformation of TNT by Arabidopsis plants expressing an Old
Yellow Enzyme from Saccharomyces cerevisiae and their results suggested that
the transgenic plants were more useful in bioremediation of TNT. Nano-phyto-
remediation approach by Jiamjitrpanich et al. (2012) is a combination of nano-
technology and phytotechnology for remediation of TNT contaminated
environments. They have investigated the capability of phytoremediation and
nanoscale zero valent iron (nZVI) for removal of TNT from the contaminated soil.
The highest removal efficiency of nano-phytoremediation was found in soil with
the TNT/nZVI ratio of 1/10 (100 mg/kg initial TNT concentration) with the
complete TNT remediation after 60 days.
5 Conclusions
In view of the widespread contamination of soil and groundwater by explosives,

such as TNT, much attention has been paid in understanding its biodegradation
and to develop bioremediation strategies. The potential of anaerobic and aerobic
bacteria to degrade TNT has been well understood. The reductive transformation
of TNT by white rot fungi has been also studied, but the mechanism by which the
fungi mineralize TNT is not well known. The understanding of mechanisms of
reactions and enzymes involved in TNT degradation and their molecular biology
has to be further strengthened by further research. However, a considerable work
has been done on the development of bioremediation technology for TNT con-
tamination sites. Bioreactors and composting systems were also used for decon-
tamination of soils containing high concentration of TNT. No doubt,
phytoremediation has a very high potential of detoxification of TNT explosive-
contaminated soils and water. Composting and bioslurry treated sites were further
subjected to phytoremediation process. Phytoremediation of TNT can be further
improved by the use of transgenic plants and genetically modified microorganisms.
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Phytoremediation of Soil Contaminated
with Explosive Compounds
Katarzyna Panz and Korneliusz Miksch
1 Introduction
Many sites all over the world are today contaminated with explosive substances.
Environmental pollution caused by these compounds is principally associated with
the explosives manufacturing, loading, assembling and exploitation for military
and industrial purposes (Rodgers and Bunce 2001; Pennington and Brannon 2002;
Adamia et al. 2006; Vila et al. 2007a; Rylott and Bruce 2008). The explosives,
which are most often found in the environment, are TNT (2,4,6-trinitrotoluene,
trinitrotoluene), belonging to the nitroaromatic group, two heterocyclic nitramines:
RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine, hexogen) and HMX (octahydro-
1,3,5,7-tetranitro-1,3,5,7-tetrazocine, octogen) and also NG (1,2,3-trinitroxypro-
pane, nitroglycerine) which is nitrate ester (Table 1).
Trinitrotoluene was the main conventional explosive used worldwide by military
forces during the 20th century (van Aken et al. 2004). Due to the stable structure,
TNT persisted in soil for decades (Sens et al. 1999). Concentrations of TNT in
contaminated sites are found extremely heterogeneous, ranging from 0.08 to
87,000 mg/kg (typically range between 4,000 and 10,000 mg/kg) (Clark and
Boopathy 2007; Vila et al. 2008). At present time, the most-widespread conven-
tional explosives are nitramines RDX and HMX, which have higher stability and
detonation power. RDX and HMX concentrations in soil are usually found lower
than that of TNT and range from 800 to 1,900 and 600–900 mg/kg, respectively, but
their respective maximum concentrations detected were 74,000 and 5,700 mg/kg,
respectively (Clark and Boopathy 2007). However, a recently synthesized polycy-
clic nitramine CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane,
hexanitrohexaazaisowurtzitane, HNIW) is today considered as potential replace-
ment for RDX and HMX because of its superior properties as an explosive and
K. Panz (&) K. Miksch

Environmental Biotechnology Department, Silesian University of Technology, Akademicka
2 A Str, Gliwice, Poland
e-mail: katarzyna.panz@polsl.pl

Springer International Publishing Switzerland 2014
236 K. Panz and K. Miksch
Table 1 Chemical structure and basic chemical properties of the most popular explosive
compounds
Compound Chemical group Chemical structure Molecular Log Water
name weight Kow solubility
(g/mol) (mg/l at
25 C)
TNT Nitrocompounds CH3 227.13 0.87 130
O2N 2
RDX Heterocyclic 2 222.12 0.9 56.35

nitramines
N
N N
O2N 2
2
HMX Heterocyclic 2 296.15 0.16 4.46
nitramines N
O2N N N 2
N
2
CL-20 Heterocyclic 438.18 1.92 3.65

nitramines
NG Nitroesters 227.09 1.62 1,800
propellant material (Rocheleau et al. 2008). Its environmental fate and impact were
carefully investigated before its widespread usage. Nitroglycerine is also widely
used for the production of dynamite, gunpowder, and rocket propellants, and in the
pharmaceutical industry, as a vasodilator for the treatment of angina pectoris.
Environmental assessments, conducted at military firing ranges in the United States
and Canada, identified NG as a potential soil contaminant with concentrations in soil
as high as 4,700 mg/kg (Rocheleau et al. 2011).
Seven nitro-substituted explosives, including TNT and RDX, have been listed
as priority pollutants and HMX as a contaminant of concern by U.S.
Phytoremediation of Soil Contaminated with Explosive Compounds 237
Environmental Protection Agency (US EPA). According to studies conducted in

many laboratories, TNT, RDX, HMX, CL-20 and NG are confirmed toxic to most
of organisms including bacteria, algae, plants, earthworms, aquatic invertebrates,
and also to animals, including mammals and human beings (Boyer et al. 2007;
Rocheleau et al. 2011). Moreover, 2,4,6-trinitrotoluene is recognized as a potential
human carcinogen (class C) by the US EPA. Direct contact with TNT can cause
anemia, liver disorder, skin irritation or immune system damage (Sens et al. 1999;
Rodgers and Bunce 2001; Pennington and Brannon 2002; Lewis et al. 2004;
Adamia et al. 2006; Best et al. 2006; Vila et al. 2007a). RDX is also treated as a
possible human carcinogen by US EPA (Vila et al. 2007b).
The adverse impact of explosives on living organisms, their environmental
persistence and low susceptibility to biodegradation clearly indicates that reme-
diation of soil contaminated with explosives is highly essential (Sens et al. 1999;
Pennington and Brannon 2002). A long established practice for remediating
explosive-contaminated soils has been excavation and subsequent incineration.
Despite its effectiveness, this method is not very practical from logistical and
economical perspectives (Vanek et al. 2007; Duringer et al. 2010). Many chemical
processes (adsorption, chemical reduction, advanced oxidation processes) were
also used to remove explosives from contaminated soil and water. These methods
are usually expensive and cause only segregation rather than destruction of the
compounds (adsorption). On the other hand, during the treatment processes
(chemical reduction, advanced oxidation processes), recalcitrant and toxic reaction
by-products may be produced. The main disadvantage of physical and chemical
methods is fact that they can be used only in ex situ conditions (Rodgers and
Bunce 2001). Alternatively, in situ bioremediation technologies can be applied
on-site with minor soil manipulation, have a few health and environmental pre-
cautions to consider and are less expensive in comparison to other methods
(Duringer et al. 2010). Among bioremediation methods, one of the most often
investigated in order to clean explosives contaminated soil is phytoremediation,
which involves the use of green plants to remediate soil and water. It was
confirmed that not only wild-type, but also genetically modified plants can be
useful in explosives decontamination (Vanek et al. 2006).
2 Phytoremediation as a Tool for Explosive-Contaminated

Soil Remediation
Phytoremediation is the set of technologies which use vegetation for in situ

treatment of soil, sediments, groundwater, wastewater and landfill leachates con-
taminated with different types of contaminants. These technologies use wild-type
or genetically modified plants to remove, stabilize, sequester or degrade moder-
ately hydrophobic environmental pollutants (compounds and chemicals, which
logarithm octanol–water coefficientsKOW = 0.5–3.5) (Dietz and Schnoor 2001;
Vila et al. 2007a; Campos et al. 2008). Phytoremediation applications can be

classified based on the mechanisms involved. According to US EPA, basic phy-
toremediation technologies include:
• Phytoextraction (Phytoaccumulation)—uptake and translocation of contami-
nants from soil by plant roots into the aerial parts of the plants;
• Phytodegradation (Phytotransformation)—breakdown of contaminants taken up
by plants through metabolic processes within the plant, or breakdown of con-
taminants external to the plant through the effect of compounds (such as,
enzymes) produced by the plants. Pollutants are degraded and incorporated into
the plant tissues.
• Rhizodegradation—breakdown of contaminants in the soil through microbial
activity that is enhanced by the presence of the rhizosphere. Microorganisms
(yeast, fungi, or bacteria) use root exudates as a source of carbon and energy.
• Phytostabilization—use of certain plant species to immobilize contaminants in
the soil and groundwater through absorption and accumulation by roots,
adsorption onto roots, or precipitation within the root zone.
• Rhizofiltration—adsorption or precipitation onto plant roots or absorption into
the roots of contaminants that are in solution surrounding the root zone.
• Phytovolatilization—uptake and transpiration of a contaminant by a plant with
release of the contaminant or a modified form of the contaminant to the
atmosphere from the plant.
Phytoremediation technologies have many advantages which include low costs,
in situ applicability, minimal environmental disturbance and high public accep-
tance (Vanek et al. 2002, 2007; Panz and Miksch 2012). The main limitation of
these methods is long time requirement for the completion of the process and
adverse environmental factors (like inappropriate temperature, pH, nutrients
content, moisture etc.) which inhibit plant growth and also the toxicity of the
pollutant and its degradation products to the plants (Vanek et al. 2007). Despite
some drawbacks, phytoremediation is a promising technology and can be suc-
cessfully used at many sites contaminated with different pollutants. Many
researches indicate that plants can effectively extract metals and organic com-
pounds from different kinds of soil and accumulate and/or transform them in the
plants tissues. Explosive substances are generally available to plant uptake. The
log Kow value is 0.87 for TNT, 0.9 for RDX (Vila et al. 2007a), 1.92 for CL-20
(Balakrishnan et al. 2004) and 1.62 for nitroglycerine (Rocheleau et al. 2011).
Only HMX is less available to plant uptake because its log Kow was determined to
be in the range of 0.06 and 0.13 (Groom et al. 2002). It was confirmed by many
laboratory and field studies that phytoremediation can be applied for sites con-
taminated with explosives, however the main limitation is high toxicity of these
substances to plants.
3 Phytotoxicity of Explosives
Toxicity of different explosive substances to plants was confirmed in both

screening studies and detailed laboratory experiments. The toxicity is the char-
acteristic of explosive compound and largely depends on its concentration and
plant species (Burken et al. 2000).
Among the common explosives, the most toxic explosive is 2,4,6-trinitrotolu-
ene. It was observed that the seed germination rate and plant growth were decreased
with an increase in TNT concentration (Peterson et al. 1996, 1998; Krishnan et al.
2000; Best et al. 2008; Vila et al. 2008). However, the scale of toxicity differed
significantly for different plants species. Among the investigated plants, the most
sensitive to TNT in the soil was alfalfa (Medicago sativa) which could not grow at a
concentration of 100 mg/kg (Scheidemann et al. 1998). Similarly, seed germination
in cress (Lepidium sativum) and cabbage (Brassica rapa) was inhibited at con-
centration of 200 mg/kg in soil (Gong et al. 1999). The most resistant to TNT were
common bean (Phaseolus vulgaris) (Gong et al. 1999) and rice (Oryza sativa) (Vila
et al. 2008) which could tolerate up to 500 mg/kg while oats (Avena sativa) did not
show any adverse effects even at a concentration of 1,600 mg/kg (Gong et al. 1999).
On the contrary, it was also observed that lower concentrations (5–50 mg/kg) of
TNT can cause even growth stimulation (Gong et al. 1999; Rocheleau et al. 2006).
The lowest threshold concentration (LOAEC) of TNT was found to be 50 mg/kg by
Gong et al. (1999) in the toxicity tests using cress (L. sativum), turnip (B. rapa), oat
(A. sativa) and wheat (Triticum aestivum). EC50 value of 2.34 mg/1 for the effect of
TNT on lettuce root elongation was reported by Rocheleau et al. (2006). It was also
demonstrated that TNT, at sublethal concentrations, can induce morphologically
abnormal development of roots. Root hairs of the plants grown in a TNT-amended
soil were found sparse, short, and abnormal (Peterson et al. 1996; Gong et al. 1999).
Weathering and aging influence on the toxicity changes in soil contaminated
with 2,4,6-trinitrotoluene was also investigated. Research showed that these pro-
cesses contribute to a significant increase in TNT toxicity causing a decrease in
seedling emergence and fresh and dry shoot mass of alfalfa (M. sativa) and rye-
grass (Lolium perenne). Toxicity increase in the weathered and aged soil may
occur due to accumulation of more toxic transformation products than parent
compound freshly introduced into soil (Rocheleau et al. 2006).
In most research conducted with RDX, no adverse effect on seedling emergence
was observed. Best et al. (2006) showed that ryegrass (L. perenne) and alfalfa (M.
sativa) germinated in the soil with RDX up to a concentration of 1,540 mg/kg.
Winfield et al. (2004) investigated 15 higher plants (9 dicotyledonous and 6
monocotyledonous) and found all of them germinated and grew in the soil con-
taminated with RDX up to a concentration of 4,000 mg/kg. However, many
adverse effects of RDX on plants were also observed. Unfavorable changes
occurred in 80 % of investigated plants in at least one organ (root or shoot). Some
changes indicated RDX teratogenicity: atypical symmetry, bifurcated leaves, fused
leaves, irregular leaf margins, curved leaf margins, underdeveloped roots, and
coiled root tips while another changes were related to dysfunctions in the cellular
maintenance and repair mechanisms: necrosis, chlorosis, yellow spot lesions or
aberrations in metabolic or physiological pathways (atypical stem pigmentation,
decreased root exudates). Phytotoxic symptoms like bleaching and necrosis
located on leaf blade margins of wheat (T. aestivum), rice (O. sativa) and soybean
(Glycine max) occurred in plants cultivated in soil contaminated with RDX (Vila
et al. 2007a, b). Changes in the plants growing in RDX and TNT contaminated soil
indicate that RDX was mainly translocated to the aerial parts of the plant whereas
TNT was metabolized in roots (Vila et al. 2007a).
However, little information is available on the phytotoxicity of HMX in plants.
Research conducted so far with higher plants revealed that they usually tolerate
high HMX concentrations. Lettuce (Lactuca sativa) and barley (Hordeum vulgare)
were grown in the concentration up to 3,320 mg/kg in the artificial soil (Robidoux
et al. 2003) while ryegrass (L. perenne) was grown in the higher concentration i.e.
10,000 mg/kg (Rocheleau et al. 2008). However, no phytotoxic symptoms were
observed in the plants cultivated in HMX contaminated soil. However, high bio-
concentration coefficients were established for HMX (similarly like for RDX)
which can cause its bioaccumulation in the food chain (Rocheleau et al. 2008).
Reports about CL-20 phytotoxicity are very scarce and ambiguous. CL-20 and
its possible biotransformation products did not inhibit seed germination and early
seedling (16–19 d) growth of alfalfa (M. sativa) and ryegrass (L. perenne) up to a
concentration of 10,000 mg/kg in a Sassafras sandy loam soil (SSL). It was also
stated that up to 200 mg/kg of CL-20 bioaccumulate in ryegrass shoots when
exposed to 9,832 mg/kg in soil (Gong et al. 2004). In an other research, Strigul
et al. (2006) measured 12 mg/kg in ryegrass leaves and 16 mg/kg in ryegrass roots
exposed to a soil CL-20 concentration of 100 mg/kg. Further, Rocheleau et al.
(2008) confirmed that CL-20 had no adverse effects on the ryegrass growth at a
concentration up to 10,000 mg/kg. Instead, it had a stimulatory effect on ryegrass
shoot growth at the greatest concentration so far tested (9,604 mg/kg soil) after the
21 days exposure. Longer ryegrass exposure to the explosive revealed a significant
inhibition in the root growth. At the same time, it also showed that the accumu-
lation of CL-20 occurred mainly in the root which is in contrary to the results
obtained by other scientists who observed the explosive accumulation in aerial
parts of plants. The contrasting results of these studies show that available data are
insufficient to assess the potential ecological impacts of an accidental release of
CL-20 to the environment (Rocheleau et al. 2008).
Nitroglycerine (NG) is an explosive compound which ecotoxicological potential
was assessed mainly in hydroponic solutions, liquid seed germination media, or cell
culture media (Rocheleau et al. 2011). It was stated that germination of white
mustard (Sinapis alba) was almost completely inhibited in a liquid medium sup-
plemented with 400 mg/l NG while root growth was inhibited by 80 % at a con-
centration of 200 mg/l in comparison to the negative control (Podlipna et al. 2008).
However, this kind of data cannot be applied directly for the development of
ecotoxicological benchmarks for plant exposures in soil. Toxicity of nitroglycerine
to higher plants has been scantly reported. A study was conducted with alfalfa
(M. sativa), barnyard grass (Echinochloa crusgalli), and ryegrass (L. perenne)
exposed to NG in Sassafras sandy loam soil. The concentration of NG, which
caused germination inhibition, ranged from 296 to 325 mg/kg in soil freshly
amended with nitroglycerine, and from 126 to 485 mg/kg in weathered and aged
soil contaminated with NG. However, the plant growth was effected at lower
concentrations: the median effective concentration for adverse impact on shoot
growth ranged from 40 to 231 mg/kg in soil freshly amended with NG, and from 23
to 185 mg/kg in weathered and aged soil contaminated with NG. Weathering and
aging soil with NG did not significantly affect the phytotoxicity (Rocheleau et al.
2011).
4 Uptake and Fate of Explosives in Plants
Many laboratory studies were conducted in order to establish uptake of different

explosive substances and their fate in plants. It was observed that all the explo-
sives, which are commonly found in the environment, can be assimilated by plants,
but the level of uptake is characteristic for the explosive and the kind of plant. For
the particular explosive substance, its fate in the plant was different. Generally,
TNT, after entering the plant, is mainly transformed in the root and final trans-
formation products are deposited in vacuoles and cell wall, NG is transformed into
dinitroglycerine in roots and subsequently translocated into the shoots while
nitramines (RDX, HMX, CL-20) are mainly transported to the aerial parts of the
plants and accumulate mainly in plant leaves. Detailed description of different
explosives uptake and fate in plants is presented in the following subsections.
4.1 Uptake and Fate of TNT by Plants
Most of the researches on the phytoremediation of explosives in soil and water

were concentrated on TNT which is highly toxic and prevalent in the environment.
The majority of studies have focused on the ability of aquatic and terrestrial plants
for the uptake of trinitrotoluene in hydroponic culture and only a few experiments
were conducted with contaminated soil in this context (Table 2).
It was observed that aquatic plant species absorbed most of the TNT from water
in a short period of time. Best et al. (1997) reported 94–100 % removal of 2,4,6-
trinitrotoluene from contaminated water by 10 plant species (submersed and
emergent macrophytes). Two water milfoil species (Myriophyllum aquaticum and
Myriophyllum spicatum) caused complete TNT removal from contaminated water
in 5–7 days time depending on the initial concentration (Hughes et al. 1997;
Pavlostathis et al. 1998; Bhadra et al. 1999; Wang et al. 2003). Helophytes—
perennial marsh plants that bears its over wintering buds in the mud below the
surface—were also able to uptake considerable quantity of TNT from
Table 2 TNT uptake by terrestrial plants in soil

Plant species Initial Incubation TNT TNT uptake References
concentration time (d) concentration by plant
(mg/kg) decrease (%) (mg/g fresh
biomass)
Vetiveria zizanioides 80 12 88 n.a. Das et al.
(2010)
Oryza sativa 500 40 n.a. 0.8 Vila et al.
(2007a)
Abutilon avicennae 120 50 76.8 n.a. Chang et al.
(2003)
Grass mixture 100 40 100 n.a Schnoor
Paspalum notatum, (2011)
Panicum vigratum
Populus deltoides 100 40 100 n.a. Schnoor
x nigra, DN34 (2011)
n.a.—not analyzed
contaminated water. Most of the investigated species (e.g. common reed

(Phragmites australis), blue rush (Juncus glaucus), slim sedge (Carex gracilis),
reed canary grass (Phalaris arundinacea) absorbed over 90 % of TNT from the
liquid medium after 7–10 days incubation time (Best et al. 1999; Nepovim et al.
2005; Vanek et al. 2006). The most efficient was common reed (P. australis) which
took up 98 % of TNT within 10 days (Vanek et al. 2006). Terrestrial plants ability
to absorb TNT from hydroponic culture was also examined (Adamia et al. 2006;
Makris et al. 2007a, b). Adamia et al. (2006) chose 8 plants to determine their
ability to assimilate TNT. Among selected plants, soybean (G. max) showed
maximum uptake (0.21 mg/g fw) while chickpea (Cicer arietinum) and ryegrass
(Lolium multiflorum) absorbed slightly less TNT from the water. The greatest TNT
concentration decrease was observed in samples with ryegrass. After 72 h of
incubation there was a decrease of 94.3 % in TNT. In a study conducted with
vetiver grass (Vetiveria zizanioides), it was observed that this plant was able to
take up all of the TNT from the solution during a period of 8 days. The maximum
uptake of 2,4,6-trinitrotoluene by vetiver grass was 1.03 mg/g fw (Makris et al.
2007a, b). Vetiver grass was also found effective in trinitrotoluene removal from
the contaminated soil. Das et al. (2010) showed that it was able to remove 97 % of
TNT from the soil after 3 days of incubation with an initial concentration of
40 mg/kg. However, removal of TNT was slightly less efficient at higher con-
centrations (80 mg/kg): after 12 days, 88 % of TNT was removed. In another
study, it was confirmed that higher TNT concentrations need longer incubation
time, as phytoremediation process may be slow. Chang et al. (2003) studied phy-
toremediation of TNT from soil by growing Indian mallow (Abutilon avicennae)
in the soil column reactor with trinitrotoluene-contaminated soil (120 mg/kg).
After 50 days, a decrease of 76.8 % in TNT concentration was observed. However,
it cannot be fully attributed to the plant because in the control (without plants)
a decrease of 51.9 % in TNT concentration was also observed. Not only TNT
concentration, but also soil composition, weathering and aging show the impact on
the 2,4,6-trinitrotoluene uptake by plants. Two kinds of soil [one containing mostly
sand (70 %) and the second one containing mainly clay (72.2 %)] were spiked with
100 mg/kg TNT and treated with a grass mixture of bahiagrass (Paspalum nota-
tum), Pensacola and switch grass (Panicum vigratum), Alamo and hybrid poplar
cuttings (Populus deltoides x nigra, DN34). After 40 days, 100 % removal of
extractable TNT was observed in all freshly contaminated soil samples with grass
mixture and hybrid poplar cuttings. No doubt soil aging has also a significant effect
on TNT removal from ‘‘clay’’ soil—it was 85 % (for the samples with grasses
mixture) while in the ‘‘sandy’’ soil, it was only slightly below 100 % (Schnoor
2011).
Based on the previous studies, it can be stated that plants are able to take up
TNT from the environment. After entering the plant, this compound is completely
converted into more polar compounds which is evidenced by the lack of detectable
levels of TNT in the plant extracts. Most of the transformation process occurs in
the roots, which was confirmed in tests using 14C-TNT and autoradiography
analysis (Hughes et al. 1997; Bhadra et al. 1999; Sens et al. 1999; Nepovim et al.
2005; Adamia et al. 2006; Makris et al. 2007b; Vila et al. 2007a, 2008). TNT
transformation products remain mainly in the root, less than 25 % of the taken up
radioactivity is translocated to aerial parts (Vila et al. 2007a, 2008). In another
research, it was reported that roots accumulated 95 % of 14C and remaining 5 %
was detected in leaves (Sens et al. 1999). In previous studies, several workers
reported that the first phytodegradation products were 4-amino-2,6-dinitrotoluene
(4A26DNT) and 2-amino-4,6-dinitrotoluene (2A46DNT). Additionally, many
unidentified polar compounds were also detected (Hughes et al. 1997; Best et al.
1999; Bhadra et al. 1999; Sens et al. 1999; Nepovim et al. 2005; Vila et al. 2007a).
Wang et al. (2003) demonstrated that primary TNT transformation products are
2-hydroxyamino-4,6-dinitrotoluene (2HA46DNT) and 4-hydroxyamino-2,6-dini-
trotoluene (4H26DNT) which was also confirmed by Vila et al. (2005). An
additional metabolite—1,3,5-trinitrobenzen (2,3,5-TNB) was also detected by
Cruz-Uribe and Rorrer (2005) and Makris et al. (2007a, b). The sequential
transformation steps are abiotic oxygenation and azoxy-product formation
(2,20 ,6,60 -tetranitro-4,40 -azoxytoluene and 4,40 ,6,60 -tetranitro-2,20 -azoxytoluene),
followed by a fast metabolic reaction that leads to oxidized products and bound
residue formation or a slower hydroxylamine isomer metabolic reaction preceded
by phytoreduction to amines (4A26DNT and 2A46DNT). The final trinitrotoluene
transformation products are most likely mono- and diglycoside conjugates which
are deposited in vacuoles and cell walls (Sens et al. 1999; Wang et al. 2003; Vila
et al. 2005, 2007a, Vila et al. 2008). Sens et al. (1999) used special cell frac-
tionation method to analyze plant used for TNT phytoremediation and demon-
strated that 14C was partitioned in wheat (T. aestivum) with 43 % in the cytoplasm
and 57 % in the cell wall.
Several studies on gene expression during TNT detoxification by plants were
also conducted. It was observed that the same key groups of genes played a role in
upregulation of TNT response as in the classic transformation, conjugation and
sequestration model of xenobiotic detoxification (Rylott et al. 2011a). Genes are

induced and regulate secretion of enzymes which encode transformation activi-
ties—nitroreductases (Adamia et al. 2006; Makris et al. 2007b; Rylott and Bruce
2008; Rylott et al. 2011a), oxophytodienoate reductases (OPRs), and cytochromes
P450 (Beynon et al. 2009; Rylott et al. 2011a). Another groups of enzymes like
peroxidases, phenol oxidase (Makris et al. 2007b), UDP-glycosyltransferase
(UGT) and glutathione S transferases (GST) also play a role in the formation of
amino derivatives of sugars and glutathione-binding processes (Sens et al. 1999;
Rylott and Bruce 2008; Beynon et al. 2009; Rao et al. 2009; Rylott et al. 2011a).
4.2 Uptake and Fate of Nitramines (RDX, HMX, CL-20)

by Plants
Nitramines structure and properties are different from the characteristics of nit-
rocompounds (Table 1). Therefore, their uptake and fate in plants also occur in the
different ways.
Studies on the RDX phytoremediation, conducted in both soil (Table 3) and
hydroponic culture, revealed that RDX uptake by plants was lower than that
observed for TNT. Best et al. (1997) investigated RDX uptake by 11 species.
Among the selected plants, only reed canary grass (P. arundinacea) showed a
significantly higher decrease (27 %) in hexogen concentration in comparison to
11 % in control (without plants). In other research, Best et al. (1999) demonstrated
Table 3 Nitramines uptake by terrestrial plants in soil

Plant species Initial RDX Incubation Explosive uptake References
concentration time (d) by plant*/
(mg/kg) removal from
the soil**
RDX
Oryza sativa 138 42 3.71* Vila et al. (2007a)
Triticum aestivum 138 42 64.54* Vila et al. (2007a)
Oryza sativa 1,000 40 31.5* Vila et al. (2007b)
Zea mays 100 28 1.21* Chen et al. (2011)
Sorghum sudanense 100 28 0.49* Chen et al. (2011)
Triticum aestivum 100 28 1.4* Chen et al. (2011)
Glycine max 100 28 2.8* Chen et al. (2011)
HMX
Lolium perenne 30 77 8.1** Groom et al. (2002)
Brassica rapa 30 77 5.2** Groom et al. (2002)
CL-20
Phaseolus vulgaris 500 182 100** Strigul et al. (2006)
Lolium perenne 1,000 365 100** Strigul et al. (2006)
*
(mg/g dw)
**
(%)
higher RDX uptake by plants (between 27 and 90 %) which was contributed to

lower RDX concentration and longer incubation time. It was also stated that the
ability of a plant to take up hexogen from solution depends on the plant species;
the highest RDX uptake (89.57 %) was observed in woolgrass (Scirpus cyperinus)
while the lowest (27.17 %) was recorded in water star-grass (Heteranthera dubia).
Parrofeather water milfoil (M. aquaticum) was reported to take up 75 % of RDX
from water solution. In the same study, RDX uptake by axenic hairy root cultures
of Madagascar periwinkle (Catharanthus roseus) was also investigated. In samples
containing C. roseus hairy root culture, there was only 20 % hexogen reduction
after 60 days of incubation (Bhadra et al. 2001). Thompson et al. (1999) also
studied RDX uptake from hydroponic solution by hybrid poplar tree (P. delto-
ides 9 nigra, DN34). Hexogen removal from the medium was 71 % after 7 days
incubation. The uptake of RDX from soil was generally slower than in the
hydroponic systems mainly because of its decreased bioavailability in soil. Bush
beans took up less than 16 % of RDX in soil after 60 days while 60 % was
removed from solution after 7 days by the same plant (Harvey et al. 1991).
14
C-RDX and autoradiography analysis were used by many researchers to estab-
lish the fate of RDX after entering the plant system (Harvey et al. 1991; Thompson
et al. 1999; Vila et al. 2007a, b). Thompson et al. (1999) reported that over 60 % of
radioactivity of 14C-RDX taken up by hybrid poplars was found in the leaf tissues
after 2 days of incubation. Analysis of plant extracts by HPLC equipped with
radiochemical detection indicated that RDX was not significantly transformed
during exposure periods of up to 7 days. Vila et al. (2007a) studied the ability of
corn (Zea mays), soybean (G. max), wheat (T. aestivum) and rice (O. sativa) to take
up RDX from soil which contained 138 mg/kg of soil. During 42 days growth
period, phytotoxic symptoms including chlorosis and bleaching were observed on
the leaf blades. These symptoms indicate RDX accumulation in the leaf tissues
which was confirmed by radio-HPLC. More than 80 % of the assimilated RDX
was translocated to the aerial parts. It was also stated that over 90 % of the soluble
residues was detected as the parent compound. Among the investigated plants,
only wheat, despite displaying phytotoxic symptoms, was able to assimilate and
accumulate high quantities of RDX (65.54 mg/g dw) without showing a decrease
in its growth. In contrast, other plants assimilated lower quantities of hexogen, the
lowest being rice with an assimilation level of only 3.71 mg/g dw. Soybean, when
cultivated in similar conditions, showed necrosis which caused the leaves to fall
off the plant. The observations of Vila et al. (2007a) were further confirmed by
Chen et al. (2011) who investigated the potential of wheat, sorghum, soybean and
corn to take up and accumulate RDX. The highest RDX uptake quantity was
observed for wheat seedling whereas soybean could not survive in a contaminated
environment. In another study conducted by Vila et al. (2007b), the ability of rice
to take up high concentrations of RDX (31.5 mg/g) from the soil (2,000 mg/kg)
was investigated despite the fact that phytotoxicity symptoms (bleaching, necrosis)
appeared. HPLC analysis showed that 89 % of taken up radioactivity was trans-
located to leaves, 90 % of which was detected in leaf extremities. Interestingly,
95 % of the radioactivity was RDX in its parent form, only 5 % detected in leaves
could be of transformation products. Brentner et al. (2010) used the modern

analytical method of phosphor imager autoradiography to assess the RDX uptake
level and compartments in which this substance accumulated in two different
species. Results indicated that the fate of taken up RDX may be different in
different plants species. In both cases, most of the investigated compound was
translocated to the aerial parts, but a disproportionate distribution was more
obvious for poplar (90.9 % in leaves, 3.9 % in roots) than for switchgrass (58.1 %
in leaves, 41.9 % in roots). It was also stated that the strongest 14C activity
appeared around chloroplasts and lignified tissues, indicating translocation of RDX
or its metabolites into chloroplasts or incorporation of these molecules into the
plant structure (i.e. cell wall).
Most of studies conducted to establish the fate of RDX in plants demonstrated
that 80–90 % of assimilated hexogen was accumulated in the aerial parts of the
plants while reports about RDX-degradation products are scarce. Small amounts of
nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) were
detected in extracts from yellow nutsedge (Cyperus esculentus), which was grown
in water containing hexogen (Larson et al. 1999) and in shoots and roots of
ryegrass (L. perenne) raised in RDX contaminated soil (Rocheleau et al. 2008).
RDX transformation was observed when plants were exposed to simulated sun-
light. The primary transformation products were nitrous oxide and 4-nitro-2,4-
diazabutanal, but these products were not found in any other investigations. Plant-
mediated phototransformation of xenobiotic compounds has been termed as
‘‘phytophotolysis’’ (Just and Schnoor 2004). van Aken et al. (2004) observed
complete mineralization of hexogen in poplar (P. deltoides x nigra DN-34) tissue
culture. A three-stage hexogen mineralization process has been suggested: initial
RDX reduction to MNX and DNX in intact plant cells, followed by heterocyclic
ring cleavage yielding formaldehyde and methanol (this stage is light dependent)
and finally transformation via a light-independent step into carbon dioxide. RDX-
degradation products were only observed in tissue culture samples, not in crude
leaf extracts, which indicates that the reaction requires undisturbed plant cells.
As far as HMX phytoremediation is concerned the reports are still scares. This
compound has a similar chemical structure as RDX, but has one more nitro group,
which changes its chemical-physical properties. Lower octanol-water coefficient
and water solubility (only 5 mg/l) of HMX than RDX and TNT limit the ability of
plants to assimilate HMX (Table 3).
The ability of plant to take up octogen from the medium depends on the plant
species. It was stated by Bhadra et al. (2001), who used hydroponic hairy root
cultures of Madagascar periwinkle (C. roseus) and parrot feather milfoil
(M. aquaticum). In the samples with C. roseus, 25 % HMX removal after 60 days
period was observed while the octogen concentration in samples of M. aquaticum
remained unchanged. Groom et al. (2002) analyzed HMX uptake abilities of 11
plants collected from the anti-tank firing-range (located in Alberta, Canada and
contaminated with octogen in a concentration of approximately 30 mg/kg) and
compared with 5 agricultural plants cultivated under controlled conditions. The
biomass accumulated only a small percent of the initial concentration of octogen in
the soil. Plants grown in the laboratory had the higher quantity of HMX than
indigenous plants growing at the anti-tank firing-range. Among the agricultural
plants cultivated under controlled conditions, the highest HMX uptake level was
observed for perennial ryegrass (L. perenne) (8.1 %) and canola (B. rapa) (5.2 %).
However, no direct evidence for plant-mediated biochemical HMX transformation
was obtained; only traces of degradation products were detected in the soil and
plant extracts. A dominant mechanism for HMX translocation and accumulation in
foliar tissue was found to be aqueous transpirational flux and evaporation. The
uptake of heterocyclic nitramines from contaminated soil by ryegrass (L. perenne)
was also investigated by Rocheleau et al. (2008). Lower than 10 % decrease in the
HMX concentration of soil was observed. HPLC analysis of the HMX content in
the soil and plant tissues after 42 days of exposure revealed that octogen, in an
unchanged form, was accumulated mainly in ryegrass shoots. Similar HMX
uptake, accumulation pattern was obtained for the kenaf plant (Hibiscus canna-
binus)—approximately 9 % of the initial octogen added to the soil was taken up by
the plants and accumulated in the aerial parts (Thorne 1999). The most effective in
HMX uptake were poplar seedlings (P. deltoides x nigra, DN-34). These plants
could take up 44.58 % of HMX from the solution after 65 days of incubation.
When the plants were analyzed by radio chromatographic methods, it revealed
70 % translocation of assimilated HMX to the leaves, but no HMX transformation
products (Yoon et al. 2002).
CL-20 is modern explosive superior to HMX and RDX as an explosive and
propellant material. That is why it is considered as a replacement for nitramines
used previously (Rocheleau et al. 2008). There are only a few reports about CL-20
uptake and its fate in plants. Moreover, results obtained by different scientists are
ambiguous. Gong et al. (2004) reported that up to 200 mg/kg CL-20 was accu-
mulated in shoots of ryegrass (L. perenne) cultivated in freshly amended
(9,832 ± 341 mg/kg) Sassafras sandy loam soil (SSL). Despite very good
recoveries (98 ± 3 %) of CL-20 in the limit test, nearly 20 % of the amended CL-
20 was not recovered from the soil at the end of the test. The fate of unrecovered
CL-20 was not known. The possible pathways of CL-20 include biodegradation or
hydrolysis, plant uptake, and binding to soil particles. In a study conducted by
Strigul et al. (2006) complete disappearance of CL-20 from the soil (500 mg/kg)
by bean (P. vulgaris) was observed after 6 months incubation. Similarly, no CL-20
remained in the soil contaminated with 1,000 mg/kg after 12 months vegetative
growth of ryegrass (L. perenne). It was further observed that CL-20 was accu-
mulated more in leaves (15.54 mg/kg) than shoots (12.01 mg/kg) of ryegrass while
in beans tissues, higher accumulation was observed in roots (39.5 mg/kg) than in
leaves (16.35 mg/kg). Rocheleau et al. (2008) also investigated uptake and bio-
accumulation of CL-20 by ryegrass (L. perenne). They observed that CL-20 was
accumulated mainly in roots with only limited translocation to the shoots. In this
study, small amount of mono-nitroso-pentanitro-2,4,8,10,12-hexaazaisowurtzitane
(mononitroso-CL-20), a degradation product of CL-20, was detected in the soils
and in the roots of L. perenne exposed to 9,457 mg/kg soil. This indicates that a
part of the CL-20 was metabolized.
Gene expression studies revealed that in the nitramines detoxification, mainly

involved are: cytochrome P450s, reductases, peroxidases, glutathione S transfer-
ases (GSTs) (Rylott et al. 2011a). There are a few reports available about RDX and
CL-20 metabolism but none about HMX metabolism products in the plant tissues.
This shows that plants have inherently low endogenous abilities to degrade
explosive nitramines. All of the compounds are accumulated in the plant tissues, so
they are bioavailable for herbivores, which increases the risk of biomagnification
across the food chain (Rocheleau et al. 2008; Rylott and Bruce 2008).
4.3 Uptake and Fate of NG by Plants
Nitroglycerine uptake and fate in plants were analyzed mainly in hydroponic

solutions. The feasibility of using yellow nutsedge (Cyperus escalantus), yellow
foxtail (Setaria glauca) and common rush (Juncus effusus) to remove NG from
contaminated water was investigated. About 70–95 % removal of NG by different
grasses was observed after 3 or 5 days. Only a small part of removed nitroglyc-
erine was accumulated in plant tissues of yellow nutsedge (12.2 % of the initial
NG added) and common rush (4.8 % of the initial NG added) while yellow foxtail
did not accumulate nitroglycerine at all. This indicated that NG was mainly
metabolized in grasses tissues by the enzymes (Riefler and Medina 2006).
Podlipna et al. (2008) analyzed degradation of NG in suspension culture of flax
(Linumus itatissimum L. cv. Viola). After 24 h incubation, complete disappearance
of NG from the solution was observed. About 40 % of the initial NG concentration
was transformed into dinitroglycerine isomers: 1,2-DNG (1,2-dinitroglycerine)
and 1,3-DNG (1,3-dinitroglycerine). The only one experiment of NG phyto-
remediation in soil was conducted by Rocheleau et al. (2011) in which Sassafras
sandy loam soil and ryegrass (L. perenne) were used. It was stated that nitro-
glycerine was transformed into dinitroglycerine (DNG) isomers in the soil and/or
roots. DNGs were subsequently translocated into the ryegrass shoots and accu-
mulated. This indicates that NG transformation products, due to their accumulation
in plant tissues, may pose a potential risk for biomagnification within the food
chain.
5 The Use of Transgenic Plants in Explosives

Phytoremediation
It was proved that the most of explosive substances are readily taken up by
different plants species. However, feasibility of the use of phytoremediation
technologies is limited by the toxicity of these substances to plants and lack of
enzymatic mechanisms to metabolize organic compounds effectively which cause
phytoremediation process to be slow and incomplete. In order to overcome these
limitations, genetically modified plants were engineered. Bacterial genes encoding

enzymes able to breakdown explosives were introduced into plants (Table 4). This
kind of genetic modifications enhance the plants abilities to take up and metabolize
explosives compounds in comparison to wild-type plants. Phytoremediation with
the use of transgenic plants have been tested only at the laboratory scale, but
positive results indicate that this technology is considered most suitable to be
applied on a large scale at contaminated sites (Rosser et al. 2001; Eapen et al.
2007; van Aken 2009).
The first plant designed especially for TNT remediation was tobacco (Nicotiana
tabacum) which was modified by introducing the bacterial enzyme pentaerythri-
toltetranitrate (PETN) reductase (belonging to flavonitroreductase group). This
enzyme was derived from Enterobacter cloacae which was previously isolated
from the explosive-contaminated soil. Seeds of the transgenic tobacco plant were
able to germinate and grow in the solution containing 0.05 mM TNT while this
TNT concentration was inhibitory for wild-type plants (French et al. 1999). It was
further observed that together with the flavonitroreductases, many other nitrore-
ductases (including NfsA, NfsB, NfsI and PnrA) (Table 4) could catalyze the
sequential reduction of TNT nitro groups into hydroxyloamino and amino deriv-
atives (Pieper and Reineke 2000; Rosser et al. 2001; van Aken 2009). Another
transgenic tobacco expressing NfsI nitroreductase (NR) derived from E. cloacae
also, tolerated higher TNT concentrations than non-transgenic plant. It was able to
remove all of the TNT from the solution that had an initial concentration of
0.25 mM while, in samples with unmodified plants, TNT removal was of insig-
nificance (Hannink et al. 2001). Using the same genetically modified plants,
Hannink et al. (2007) found that TNT transformation products (4-hydroxyloamino-
2,6-dinitrotoluene) bind more favorably to the plants’ macromolecules in trans-
genic tobacco as compared to wild-type plants. Investigations with nitroreductase
expressing transgenic tobacco conducted in TNT-contaminated soil also revealed
that this plant had increased tolerance to TNT in comparison to unmodified
tobacco. It was also reported that in the rhizosphere of transgenic plant roots in the
contaminated soil, there was an increase in the number of culturable bacteria and
the functional and genetic diversity of the microbial community at high concen-
trations of TNT in contrast to the rhizosphere of wild-type plants cultivated in the
similar conditions (Travis et al. 2007). Another plant, which was modified to
enhance its abilities to survive at high TNT concentrations was Arabidopsis tha-
liana. Nitroreductase NfsA (derived from Escherichia coli) was introduced into
this plant. It was stated that transgenic plant was able to take up 7–8 times more
TNT than unmodified Arabidopsis. The transgenic plant also demonstrated inter-
tissue trinitrotoluene reduction which was not observed in natural plants (Kuru-
mata et al. 2005). In another study, A. thaliana expressing the nfsI gene (derived
from E. cloacae) was also investigated for tolerance against TNT. It was observed
that in the soil experiment with genetically modified plants, the transgenic lines
could grow at TNT concentrations up to 250 mg/kg whereas wild-type plant
growth was severely affected at 50 mg/kg (Strand et al. 2009). Similarly, hybrid
aspen (Populus tremula x tremuloides var. Etropole) was engineered to express a
250
Table 4 Transgenic plants engineered for explosives phytoremediation

Compound Plant species Source Gene/enzyme References
TNT Nicotiana tabacum Enterobacter cloacae Pentaerythritoltetranitrate French et al. (1999)
(PETN) reductase
TNT Nicotiana tabacum Enterobacter cloacae NfsI nitroreductase Hannink et al. (2001)
TNT Arabidopsis thaliana Escherichia coli NfsA nitroreductase Kurumata et al. (2005)
TNT Arabidopsis thaliana Enterobacter cloacae NfsI nitroreductase Strand et al. (2009)
TNT Populustremula x Pseudomonas putida PnrA nitroreductase van Dillewijn et al. (2008)
tremuloides var.
Etropole
NG Nicotiana tabacum Enterobacter cloacae Pentaerythritoltetranitrate French et al. (1999)
(PETN) reductase
RDX Arabidopsis thaliana Rhodococcus rhodochrous XplA Flavodoxin-cytochrome Seth-Smith et al. (2002); Jackson et al.
P450-like enzyme (2007); Strand et al. (2009)
RDX Arabidopsis thaliana Rhodococcus rhodochrous XplB Flavodoxinreductase Jackson et al. (2007)
RDX Arabidopsis thaliana Rhodococcus rhodochrous XplA ? XplB Jackson et al. (2007)
TNT ? RDX Arabidopsis thaliana Rhodococcus rhodochrous and XplA-NR and XplA-XplB-NR Rylott et al. (2011b)
Enterobacter cloacae
K. Panz and K. Miksch
bacterial nitroreductase gene, PnrA, from Pseudomonas putida. Transgenic plant

was shown to tolerate and take up greater amounts of TNT from contaminated
waters and soil as compared to wild-type plants. In hydroponic culture, genetically
modified aspen showed growth even at a TNT concentration 57 mg/l while the
growth of wild-type trees was severely affected at a concentration of 11 mg/l. In
contaminated soil, the growth of wild-type trees was completely inhibited at a
concentration of 500 mg/kg whereas transgenic aspen could survive and grow
even at a concentration of 1,000 mg/kg. It was also observed that an increase in the
uptake level and rate by genetically modified trees in comparison to wild-type
trees was especially significant at high TNT concentrations (higher than 50 mg/l in
hydroponic solutions and 750 and 1,000 mg/kg soil) (van Dillewijn et al. 2008).
Nitroglycerine phytoremediation was also studied with the use of transgenic
plants. Tobacco expressing pentaerythritoltetranitrate (PETN) reductase in com-
parison to wild-type plants showed an enhanced metabolism of NG. Plant seeds
could germinate and grow in the presence of high concentrations of nitroglycerine
(1.0 mM) which caused inhibition of germination and growth in wild-type plants
(van Aken 2009).
Transgenic plants were also designed to improve the plant’s RDX-degradation
abilities. Arabidopsis (A. thaliana) was modified by introducing XplA gene
derived from Rhodococcus rhodochrous which was originally isolated from hex-
ogen-contaminated soil. XplA gene encodes an RDX-degrading fused flavodoxin-
cytochrome P450-like enzyme. Genetically modified Arabidopsis was more
resistant to the toxic effects of hexogen and able to take up higher quantities of
RDX than wild-type plants (32–100 % uptake for modified Arabidopsis versus
10 % uptake for unmodified plants). Moreover, less RDX was accumulated in
genetically modified plants tissues than in the wild-type plants tissues. This
observation, along with the higher uptake level, supports the idea that this is a
more effective route of transformation (Seth-Smith et al. 2002; Rylott et al. 2006).
In another study, two genetically modified Arabidopsis lines expressing XplA
were described, and both removed 100 % RDX from a solution that had an initial
concentration of 180 mM, while the wild-type plants removed only 19 % of RDX
from the medium after 5 days. Transgenic Arabidopsis plants were also used in the
soil experiment. It was demonstrated that this plant was able to grow larger in
contaminated soil than unmodified Arabidopsis (Strand et al. 2009). Jackson et al.
(2007) presented an unusual microbial P450 system able to degrade RDX, con-
sisting of flavodoxin reductase XplB and fused flavodoxin-cytochrome P450 XplA.
Arabidopsis plants expressing both XplA and XplB were also engineered. These
plants were able to remove RDX 30 times faster than XplA lines. While XplB lines
demonstrated uptake rates similar to those of wild-type plants.
RDX and TNT are often found on contaminated sites together. Therefore,
transgenic plants able to tolerate and assimilate high concentrations of both
compounds were engineered. Rylott et al. (2011b) prepared and investigated
Arabidopsis lines expressing XplA-NR and XplA-XplB-NR genes. XplA-NR
plants could remove both RDX and TNT from the liquid culture, but with an
increasing concentrations of TNT, the rate of RDX uptake was decreased. Despite
phytotoxic symptoms (bleaching and biomass reductions) observed during the

experiment, plants were more tolerant to TNT than either XplA-only expressing
plants or wild-type plants. Results obtained for XplA-XplB-NR expressing Ara-
bidopsis were even more promising. This line was prepared with the XplA-XplB
line which had the fastest RDX removal rate (Jackson et al. 2007) with the nfsI
gene. This plant removed all the RDX and TNT from the solution within 3 days
and 17 h, respectively. For TNT, it was considerably quicker than it was observed
for wild-type plants or XplA-XplB lines, while RDX removal by the wild-type or
XplA-XplB line was only two-thirds of this explosive in this time.
However, HMX removal by phytoremediation is less feasible than RDX and
TNT. Due to the similarities between octogen and hexogen structure, there have
been attempts to remove this compound using genetically modified plants
expressing the XplA gene. However, transgenic lines have not assimilated more
HMX than wild-type plants (Rylott and Bruce 2008).
6 Phytoremediation of Explosives: Field Studies
The majority of explosives phytoremediation investigations were conducted at a

laboratory scale, some in the greenhouse, but field-scale application trials were
scarce. Wetland systems for the treatment of explosives were implemented at the
Iowa Army Ammunition Plant in Middletown, Iowa. Their removal results,
obtained in these systems, were below the EPA human health advisory level of
0.002 mg/l (McCutcheon and Schnoor 2003). The only one example of field-scale
in situ application was carried out at Eglin Air Force Base (Eglin AFB) contam-
inated with TNT, RDX and HMX. Bahiagrass (P. notatum) Pensacola was planted
on three 0.4 acre plots and samples were collected twice a year for 18 months. Soil
and plant samples were taken from each plot while the control sample was
unplanted soil. It was observed that TNT was transformed both in the planted and
in the ‘‘control’’ soil while RDX and HMX could not be effectively treated because
they migrated into the deep soil layers and was not bioavailable for plant uptake.
7 Conclusions
Phytoremediation is a ‘‘green’’ technology which can be used for removal of many

xenobiotics from the environment. Plants abilities to uptake and transform dif-
ferent explosive compounds were extensively investigated. Results obtained in
many research revealed that:
• TNT is easily assimilated from contaminated water and soil by many plants.
Subsequently, it is metabolized in the root tissues and transformation products
(mono- and diglycoside conjugates) are deposited in vacuoles and cell walls.
• RDX is taken up by different plants at a high level, but lower than TNT. This
compound generally is not metabolized after entering the plant, but is accu-
mulated in unchanged form mainly (approx 90 % of taken up hexogen) in aerial
parts of the plants.
• HMX uptake by plants is very low (about 10 %) in comparison to TNT and
RDX uptake. Similar to hexogen, octogen is also accumulated in shoots and
leaves.
• CL-20—high amounts of this explosive are assimilated by the plants. However,
its fate in plant is not completely traced out. For some plant species, accumu-
lation was found mainly in roots while for another mostly in leaves. Only small
amount of CL-20 entering the plant tissue is transformed.
• NG is readily taken up by plants, transformed into dinitroglycerine isomers and
subsequently translocated and accumulated in shoots.
Thus, it may be concluded that phytoremediation is a suitable technology for
TNT, RDX, CL-20 and NG removal from contaminated soil while for HMX, more
adequate method is to be worked out. However, plants can be used only, when
explosives concentrations are below their phytotoxic levels. Hence, phytoreme-
diation is not an appropriate technology for highly contaminated sites. In case of
the use of phytoremediation in the areas contaminated with hexogen, hexa-
nitrohexaazaisowurtzitane and nitroglycerine which accumulate in plants tissues,
plant removal after vegetative growth must be planned to avoid entering explosive
substances into the food chain. Application of transgenic plants, which are spe-
cifically designed to degrade selected compounds, seems to be the best practical
solution. Plants with introduced bacterial genes have higher phytoremediation
potential than that of wild-type plants. Transgenic lines are often more resistant to
the toxic effects of explosives and take up higher quantities of explosive com-
pounds and also degrade them more effectively.
Despite the promising results of phytoremediation processes achieved at a
laboratory scale there is a need for more site applications to assess how these
technologies perform in the natural environment.
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Stable Isotope Tools for Tracking In Situ
Degradation Processes of Military
Energetic Compounds
Anat Bernstein, Faina Gelman and Zeev Ronen
1 General Introduction
Military energetic compounds are common environmental pollutants, contami-

nating soils and water worldwide (Juhasz and Naidu 2007; Lima et al. 2011;
Bernstein and Ronen 2012). In situ microbial degradation of these compounds may
be a positive process that reduces their concentration in the environment without
any active human involvement. Nevertheless, assessing the extent of biodegra-
dation processes in situ is challenging, and often cannot be achieved with satis-
factory sensitivity using conventional methods. Isotope methods, on the other
hand, may be a suitable alternative for assessing these processes. This chapter will
focus on the application of compound-specific isotope analysis (CSIA) to study the
fate of military energetic compounds in the subsurface, focusing on nitroaromatics,
nitramines, and perchlorate. CSIA can also be used to provide an intrinsic
mechanistic understanding of the transformation reactions of such compounds and
this aspect will be described as well.
A. Bernstein
Institute for Soil, Water and Environmental Sciences, Agricultural Research Organization,
Volcani Center, 50250 Bet Dagan, Israel
F. Gelman
Geological Survey of Israel, 30 Malkhey Israel Street 95501 Jerusalem, Israel
Z. Ronen (&)
Zuckerberg Institute for Water Research, Department of Environmental Hydrology and
Microbiology, Ben-Gurion University of the Negev, Sede Boqer Campus,
84990 Negev, Israel
e-mail: zeevrone@bgu.ac.il

260 A. Bernstein et al.
2 Compound-Specific Isotope Analysis
2.1 Application for Assessment of Natural Attenuation
Natural attenuation is a remedial approach that reduces the mass, toxicity,

mobility, volume or concentration of contaminants in soil or groundwater without
active human intervention (Wiedemeier et al. 1996). With its clear advantage in
terms of remediation costs (Landis 2000), natural attenuation is often the preferred
remedial strategy. Nevertheless, natural attenuation can only be considered as a
remedial strategy, replacing active methods, when it is shown to be as capable of
attaining site-specific remediation objectives (Rügner et al. 2006). Thus, possible
natural attenuation processes of a target contaminant must be identified and their
ability to reduce the extent of contamination must be quantified (Illman and
Alvarez 2009).
One important natural attenuation process is biodegradation, in which pollu-
tants are transformed by indigenous bacteria (Madsen 1998). The main biodeg-
radation issues for study are its extent, rate, and pathways (Bombach et al. 2010).
These are most frequently assessed by monitoring the decrease in the compound’s
concentration and/or the concentration of its daughter products. However, quan-
tifying biodegradation rates of contaminants based on shifts in concentration is not
always as conclusive as one would like (Bockelmann et al. 2003; Wilson et al.
2004). Since a decrease in a contaminant’s concentration cannot be related to
biodegradation alone, as it may also be due to other natural attenuation processes,
such as dispersion, sorption or volatilization. In addition, different unknown
parameters increase the complexity of solving such problems. In practice, the
number of monitoring wells in the field is often limited, therefore, exact knowl-
edge of a plume’s shape is lacking. Moreover, temporal data on the contaminant’s
release to the environment are normally unknown and variable hydrogeological
conditions are often unavailable. Under such circumstances, an assessment of
biodegradation of the contaminant based on shifts in its concentration may be of
low sensitivity.
In the last 15 years, CSIA has been used as a complementary approach that can
overcome the above difficulties. This methodology is not based on monitoring
shifts in concentration, but rather shifts in isotopic composition. Such shifts in
degrading compounds stem from the fact that light isotopes are converted slightly
more rapidly than heavy isotopes (Elsner et al. 2005; Braeckevelt et al. 2012;
Thullner et al. 2012). Therefore, as biodegradation proceeds, the remaining pool of
pollutants becomes enriched with respect to its isotopic composition. It is well
accepted that shifts in the isotopic composition of a molecule are only little, if at
all, influenced by processes in which the inner bonds remain intact, such as vol-
atilization, sorption, or dilution (Meckenstock et al. 2004; Braeckevelt et al. 2012).
Therefore, isotopic shifts that are observed along a contamination plume are
exclusively related to degradation processes. This enables estimating the degra-
dation extent of a target compound in situ with relatively high sensitivity.
Stable Isotope Tools for Tracking In Situ Degradation Processes 261
Another important feature of CSIA’s application stems from the fact that pri-
mary isotopic effects are apparent when the heavy isotope is directly involved in
the bond that is being cleaved, whereas secondary, often undetectable, isotopic
effects occur when the heavy isotope is adjacent to the atoms in the bond that is
being cleaved. Since secondary isotope effects are commonly at least one order of
magnitude lower than primary isotope effects (Elsner et al. 2005), important
information can be gained on the degradation pathway, i.e., one can conclude
which bonds were involved in the rate-limiting step of the reaction, and thus shed
light on the reaction’s mechanism.
The robustness of the CSIA concept is reflected in a large number of hydro-
geological applications in the last decade. In fact, this method has been accepted as
a tool for delineating natural attenuation in groundwater systems (Hunkeler et al.
2008). To date, most of these applications have concentrated on the most common
environmental pollutants (Thullner et al. 2012), but they have been also applied for
military energetic compounds to some extent.
2.2 Quantitative Interpretation of Isotopic Shifts
The relative abundance of stable isotopes is defined as the ratio (R) between the
absolute abundance (A) of the heavy (H) and light (L) isotopes:
R ¼ H A=L A ð1Þ
Shifts in the ratio of targeted compounds can be correlated to shifts in the extent
of compound transformation, as described by the Rayleigh equation:
ða1Þ
R C
¼ ð2Þ
R0 C0
where R is the isotopic ratio of the compound in a sample taken during a degra-
dation process, R0 is the initial isotopic ratio in the source, C is the compound
concentration in a sample, C0 is the initial compound concentration, and a is
defined as the isotope fractionation factor: a proportionality constant that is spe-
cific for the reaction. Frequently, the term enrichment factor, e, is defined rather
than fractionation factor, where e = (a - 1) 9 1000.
Since isotopic enrichment is measured for the entire molecule and not specif-
ically for the atoms in the reacting position, the enrichment factor for the reacting
position is ‘‘diluted’’ by atoms of that same molecule that do not react. A position-
specific enrichment factor can, therefore, be defined:
ereactiveposition ¼ n ebulk ð3Þ
where n is the number of atoms of the same element in the molecule, and ebulk is
the enrichment factor which is found experimentally using the Rayleigh equation
(Eq. 2).
In mechanistic studies, kinetic isotope effects (KIE) are often defined, as they
represent the difference in reaction rates (k) of the light (L) and heavy (H) iso-
topes: KIE = kL/kH (Elsner et al. 2005; Hofstetter and Berg 2011). If experiments
with labeled compounds are carried out in parallel with non-labeled compounds,
KIE can be quantified directly from the ratio between the rates of the non-labeled
and labeled compounds. In environmental studies, on the other hand, data are
obtained from natural non-labeled compounds. In this case, one can indirectly
calculate intrinsic KIE based on the experimentally derived eeactive position, where:
1
KIE ¼ ereactive position ð4Þ
1þ 1;000
When isotope ratios of a specific contaminant are measured in the field samples,
and when laboratory-derived enrichment factors (e) are available, the Rayleigh
equation allows calculating the extent of biodegradation, C/C0, along the con-
taminant plume.
Having calculated the degradation extent along the plume, one might further
assess the in situ degradation rate of the target compound. If the residence time of
the compound from its release to the environment to its sampling is known, e.g.
using hydrogeological parameters, then assuming that biodegradation follows first-
order kinetics, the rate factor, k, can be calculated for the degradation rate as:
h i
C
C0
k¼ ð5Þ
t
This can be further used to calculate the half life, t1/2, of the substrate:
lnð0:5Þ
t12 ¼ ð6Þ
k
2.3 Analytical Aspects
2.3.1 Explosive Organic Compounds
The increasing application of CSIA to organic compounds in recent years is related

to analytical developments. These developments have enabled the isotopic anal-
ysis of a target compound, even dissolved in heterogeneous aqueous samples.
Extensive application of CSIA in environmental studies has concentrated mainly
on the most common organic groundwater pollutants, such as chlorinated
aliphatics, petroleum hydrocarbons, and gasoline additives (Aelion et al. 2009;
Thullner et al. 2012). It may be anticipated that the number of target compounds in
such studies will increase in the coming years along with analytical developments
and the optimization of methods for other compounds of interest. For example,
proper analytical methods have been developed for pesticides (Penning and Elsner
2007; Meyer et al. 2008; Reinnicke et al. 2010) and controlled degradation studies
were performed (Meyer et al. 2009; Penning et al. 2010; Reinnicke et al. 2012),
but they still have not been extrapolated to the field.
CSIA of hydrogen (2H/1H), carbon (13C/12C), and nitrogen (15N/14N) in organic
compounds is most frequently done by interfacing a gas chromatograph (GC) via a
combustion reactor with an isotope ratio mass spectrometer (IRMS; Fig. 1)
(Schmidt et al. 2004, Schmidt and Jochmann 2012; Elsner et al. 2012). Using this
configuration, the targeted compound is first separated from background compo-
nents by the GC, then converted within the reactor to a simple gas (H2, CO2, or N2
for hydrogen, carbon, and nitrogen isotope analysis, respectively), which is further
isotopically analyzed, relative to a reference gas, by an IRMS. As GC is the first
step in this CSIA concept, compounds that are not amenable to GC require greater
efforts in developing appropriate analytical methods (Elsner et al. 2012).
With the development of a method for d13C and d15N analysis, the first
explosive compound, to which CSIA was applied, was TNT (Miyares et al. 1999).
Analysis of δ13 C and δ 15 N

CO2 /N 2 of the
Target compound target compound
Reference gas
IRMS
GC Combustion isotope measurement

separation ( for δ15 N analysis m/z 28, 29 (15 N14 N/ 14 N 14 N)
also reduction) m/z 44, 45 ( 13 CO2 / 12 CO2)
Analysis of δ 18 O and δ 37Cl

IRMS
Pyrolysis and oxygen Reference gas
conversion to CO
Offline isotope measurement

purification m/z 28, 30 (C18 O/C16 O)
Dual inlet IRMS m/z 50, 52 (CH 3 37 Cl/ CH335 Cl)
Chlorine conversion
to methyl chloride
Fig. 1 Compound-specific isotope analysis techniques for d13C and d15N in organic explosives
(upper panel) and d18O and d37Cl in perchlorate (lower panel)
This work was pioneering not only with respect to CSIA of explosives, but also
being the first published report of CSIA’s application to studying nitrogen isotope
effects during the transformation of any organic compound (Ahmad 2007). The
analytical method for d13C and d15N in TNT was later on improved by introducing
online solid-phase micro-extraction (SPME) as an automated technique for con-
centrating the compound prior analysis (Berg et al. 2007).
Early studies of CSIA’s application to the study of isotopic effects in RDX
faced great difficulties following the observed thermal decomposition of the
compound which was thought to occur within the GC injector and the column and
resulted in a strong isotopic effect (Bernstein et al. 2008). This observation led to
the development of an alternative technique that did not consist of online purifi-
cation by GC. Instead, offline purification was carried out by thin-layer chroma-
tography (TLC) at room temperature as an initial step, followed by introduction of
the purified RDX into an elemental analyzer (EA) coupled with continuous flow
(CF) to an IRMS. This technique had the disadvantages of being labor-intensive
and not applicable to carbon isotopes. Fortunately, this method was later replaced
by the development of a proper GC-IRMS method (Gelman et al. 2011) in which
the difficulties stemming from RDX decomposition in the GC were resolved by
applying (1) programmed temperature vaporization at the injector, and (2) a rapid
temperature ramp during the GC run. This method was shown to be applicable for
carbon and nitrogen isotope analysis in RDX. Technical limitation of this method
was high quantity that had to be injected for carbon measurements and the broad
peaks detected under low-temperature ramps (whereas under high-temperature
ramps, undesirably rapid elution was detected).
To date, HMX has received the least attention in terms of isotopic analysis.
With its similarity to RDX, HMX poses difficulties for GC analysis which have not
yet been resolved. Recently, similar to the early offline method for RDX, HMX
purification was carried out by TLC plates (Neta et al. 2012), but this method was
not proven suitable for tracking isotopic fractionation of HMX during the trans-
formation processes. Due to the disadvantages of TLC purification methods—
mainly the need for high quantities of target compound, its labor intensive nature,
and risk of contamination, development of a suitable online CSIA method for
HMX is highly desirable.
Commercially, instruments that interface a liquid chromatograph (LC) with an
IRMS are available (Krummen et al. 2004), and seem to offer a promising alter-
native analytical strategy for CSIA of non-GC-amenable explosive compounds,
such as HMX. However, despite its great potential, LC-IRMS has not yet been
used in contaminant hydrology studies due to its restriction to use aqueous solvents
only, which limits the separation to mainly ion-exchange chromatography
(Schmidt and Jochmann 2012). Moreover, LC-IRMS is currently restricted to
carbon isotope analyses (Elsner et al. 2012), whereas for RDX and HMX, the
nitrogen isotope composition is perhaps of greatest interest. CSIA of nitramine
explosives may, therefore, benefit from future developments in LC-IRMS.
2.3.2 Analysis of Oxygen (17O/16O, 18

O/16O) and Chlorine (37Cl/35Cl)
in Perchlorate
Compared to organic explosives, analysis of the inorganic perchlorate molecule

requires a completely different analytical concept. Comprised of chlorine and
oxygen and having an anionic, non-GC-amenable nature, GC-IRMS methods are
not suitable for this molecule. The first challenge in the analysis of this compound
is the need to purify it from background components. This is normally done using
highly selective bifunctional anion-exchange resin (Bao and Gu 2004; Böhlke
et al. 2005), followed by the removal of chloride ions by precipitation as AgCl2
(Ader et al. 2001; Coleman et al. 2003). The second challenge is the actual isotopic
analysis of the two elements within the compound. Oxygen isotopes are analyzed
in the purified perchlorate by CF-IRMS after online pyrolysis (Bao and Gu 2004;
Böhlke et al. 2005; Sturchio et al. 2007). Chlorine isotope analysis, on the other
hand, requires further pretreatment since chlorine cannot be converted online to a
simple chlorine-containing gas (Bernstein et al. 2011; Cincinelli et al. 2012).
Therefore, the compound must first be converted in an offline procedure to methyl
chloride (CH35 37
3 Cl and CH3 Cl) (Ader et al. 2001; Sturchio et al. 2003; Böhlke et al.
2005), which is further analyzed in a dual-inlet (DI)-IRMS (Fig. 1, Table 1).
3 Isotope Fractionation in the Transformation of Military

Energetic Compounds
The use of stable isotopes to address transformation processes in energetic com-

pounds was documented in 1977 for the study of KIE and intermediate formation
during alkaline hydrolysis of RDX (Hoffsommer et al. 1977). This very early study
used 2H-labeled RDX. Here, we review the more recent studies based on CSIA
analysis, i.e., analysis of the natural abundance of stable isotopes within the
molecule (Table 2).
3.1 Nitroaromatics
3.1.1 TNT Incubated with Soil
The first study applying CSIA for the identification of isotope fractionation in
nitroaromatics was carried out by Miyares et al. (1999). In their work, TNT was
incubated with soils that were excavated from TNT-contaminated sites, and TNT
reduction and the formation of 2- and 4-aminodinitrotoluene were monitored. The
d13C composition of TNT presented constant values throughout the incubation, as
expected, because the reaction involves the nitro group, with no involvement of
the aromatic ring carbon (Fig. 2).
266
Table 1 Analytical methods for explosive compounds and perchlorate

Compounds Technique Elements Precision (1r, %) Minimal compound quantity required References
measured (lg)
d13C d15N d18O d37Cl d13C d15N d17O, d18O d37Cl
15 14
TNT GC- IRMS N/ N 0.3 0.4 – 2.7 16 – Coffin et al. (2001)
13
C/12C Miyares et al. (1999)
15 14
TNT SPME coupled to N/ N 0.4 0.6 – 1 29 – Berg et al. (2007)
GC-IRMS 13 12
C/ C
15 14
RDX TLC followed by N/ N – 0.13 1.18 – 120 120 Bernstein et al. (2008)
CF-IRMS 18 16
O/ O Sagi-Ben Moshe et al.
(2010)
15
RDX GC- IRMS N/14N 0.4 0.3 – 4 5 – Gelman et al. (2011)
13
C/12C
15 14
HMX TLC followed by N/ N – \0.2a – – 30a – Unpublished data and Neta
CF-IRMS et al. (2012)a
37
Perchlorate DI-IRMS Cl/35Cl 0.05 Details not Ader et al. (2001)
provided
17
Perchlorate CF-IRMS O/16O, 0.1, 0.3 % Details not Böhlke et al. (2005)
18
O/16O provided
a
Determined directly for pure standard with no TLC pretreatment
A. Bernstein et al.
Table 2 Isotope enrichment factor (e) during the transformation of explosives, nitroaromatic compounds, and perchlorate
Compounds Degradation reaction Bacteria ebulk [%] and in brackets ereactive position [%] References
13 15 18 37
C N O Cl
RDX Aerobic denitration Rhodococcus sp. T7 In the range of -2.3 ± 0.8 (-13.8) Bernstein et al.
analytical (2013)
uncertainty
Aerobic denitration Rhodococcus sp. T9 N In the range of -2.3 ± 0.8 (-13.8) Bernstein et al.
analytical (2013)
uncertainty
Aerobic denitration Rhodococcus sp. YH1 In the range of -1.9 ± 0.4 (-11.4) Bernstein et al.
analytical (2013)
uncertainty
Aerobic denitration Rhodococcus sp. YH1 -2.1 ± 0.3 (-12.6) -1.7 ± 0.1 (-10.2) Bernstein et al.
(2008)
Aerobic denitration Rhodococcus sp. 11Y -2.4 to -2.5 (-14.4 Hatzinger (2011)
to -15.0)
Aerobic denitration Rhodococcus sp. DN22 -3.0 to -3.3 (-18.0 Hatzinger (2011)
to -19.8)
Anaerobic reduction Microbial consortium, -5.0 ± 0.3 (-30.0) -5.3 ± 0.5 (-31.8) Bernstein et al.
excavated from (2008)
groundwater
Anaerobic reduction Five microbial consortia, -2.1 to -3.7 (-12.6) Sagi-Ben Moshe et al.
excavated from soils (2010)
Anaerobic reduction Details not provided -5.0 (-30.0) Hatzinger (2011)
Alkaline hydrolysis Abiotic reaction -7.8 ± 0.5 (-23.4) -5.3 ± 0.8 (-31.8) Gelman et al. (2011)
Nitrobenzene Enzymatic oxidation Comamonas sp. strain -3.9 ± 0.09 % -0.75 ± 0.09 % (-0.75) Hofstetter et al.
JS765 (-23.4) (2008b)

Enzymatic reduction Pseudomonas -0.57 ± 0.06 % -26.6 ± 0.7 % Hofstetter et al.
pseudoalcaligenes (-3.42) (-26.6) (2008b)
strain JS45
2-Methyl- Abiotic reduction in Abiotic reaction -31.9 ± 1.0 (-31.9) Hartenbach et al.
nitrobenzene Fe(II) sorbed to (2006)
goethite
suspension
Reduction in Na+ and Abiotic reaction -37.7 ± 2.3 Hartenbach et al.
K+ suspensions (2006)
of reduced
ferruginous
smectite
267
(continued)
Table 2 (continued)
268
13 15 18 37
C N O Cl
4-Methyl- Abiotic reduction in Abiotic reaction -31.3 ± 1.4 (-31.3) Hartenbach et al.
nitrobenzene Fe(II) sorbed to (2006)
goethite
Reduction in Na+ and Abiotic reaction -38.7 ± 1.5 Hofstetter et al.
K+ suspensions (2008a)
of reduced
ferruginous
smectite
2-Cl- nitrobenzene Abiotic reduction in Abiotic reaction -29.2 ± 0.3 (-29.2) Hartenbach et al.
Fe(II) sorbed to (2006)
goethite
suspension
Abiotic reduction in Abiotic reaction -30.2 ± 1.7 (-30.2) Hartenbach et al.
juglone (8- (2006)
hydroxy-1,4-
naphthoquinone)
in the presence of
H2S
of reduced
ferruginous
smectite
3-Cl- nitrobenzene Abiotic reduction by Abiotic reaction -32.9 ± 0.7 to - Tobler et al. (2007)
mineral-bound 41.9 ± 1.1
Fe(II) species in
suspensions of
goethite under
variable
experimental
conditions
(continued)
A. Bernstein et al.
Table 2 (continued)
13 15 18 37
C N O Cl
of reduced
ferruginous
smectite
4-Cl- nitrobenzene Abiotic reduction in Abiotic reaction -29.4 ± 0.8 (-29.4) Hartenbach et al.
Fe(II) sorbed to (2006)
goethite
suspension
Abiotic reduction in Abiotic reaction -28.0 ± 0.8 (-28.0) Hartenbach et al.
juglone (8- (2006)
hydroxy-1,4-
naphthoquinone)
in the presence of
H2S
Abiotic reduction by Abiotic reaction -31.1 ± 1.0 Tobler et al. (2007)
mineral-bound
Fe(II) species in
suspensions of
goethite under
variable
experimental
conditions

of reduced
ferruginous
smectite
1,2- Dinitrobenzene Reduction in Na+ and Abiotic reaction -17.4 ± 0.8 to - Hofstetter et al.
K+ suspensions 18.4 ± 0.7(-34.8 to (2008a)
of reduced -36.8)
ferruginous
smectite
(continued)
269
Table 2 (continued)
270
13 15 18 37
C N O Cl
1,4- Dinitrobenzene Reduction in Na+ and Abiotic reaction -17.3 ± 0.9 to - Hofstetter et al.
K+ suspensions 16.5 ± 0.7 (-34.6 to (2008a)
of reduced -33.0)
ferruginous
smectite
TNT Not defined Incubation with No fractionation Coffin et al. (2001)
contaminated soil detected for Miyares et al. (1999)
carbon with
degradation
extent of down to
C/C0 = 0.14
Perchlorate Reduction with Dechlorosoma suillum -14.8 ± 1.3 % - Coleman et al. (2003)
acetate as an strain PS (Azospira
electron donor suillum strain PS)
Reduction with Dechlorosoma suillum 14.94 ± 0.15 % Ader et al. (2008)
acetate as an strain PS (Azospira
electron donor suillum strain PS)
Reduction with Dechlorosoma suillum -12.1 to -16.6 % Sturchio et al. (2003)
acetate as an JPLRND
electron donor
Reduction with Dechlorosoma sp. FBR2 -29.0 to -36.6 % -11.5 to -14.5 % Sturchio et al. (2007)
acetate as an and Azospira suillum
electron donor JPLRND
In situ reduction by Undefined -8.0 to -9.2 % -3.1 to -4.6 % Hatzinger et al.
indigenous (2009)
aquifer bacteria
A. Bernstein et al.
CH3 CH3 CH3

O 2N NO O 2N NHOH O 2N NO2
CH3
O 2N NO2
NO2 NO2 NH2
CH3 CH3 CH3
NO2 O 2N NO 2 O 2N NO 2 O 2N NH2
NO
NHOH NO 2
2-and 4-
aminodinitrotoluene
Fig. 2 TNT degradation pathway postulated by Miyares et al. (1999)
3.1.2 Abiotic Reduction Experiments for a Variety of Nitroaromatics
In a set of highly controlled abiotic reduction experiments of various nitroaromatic

compounds (all nitrobenzenes) as shown in Fig. 3 (Hartenbach et al. 2006, 2008;
Tobler et al. 2007; Hofstetter et al. 2008a), 15N enrichment was found to be in the
range of -28.0 to -41.9 % for the reacting position. Hofstetter (2011) later made
the generalization that 15N-KIE associated with aromatic nitro group reduction
under environmental conditions is confined to the range of 1.03–1.04 (e of -30 to
-40 % in the reacting position), regardless of compound and reductant, while
13
C-, 15N-, and 2H-KIE during N-atom oxidation, oxidative N dealkylation, and
aromatic ring di- and mono-oxygenation are highly compound-specific and differ
for each degradation pathway.
3.1.3 Biotic Experiments with Nitrobenzene
Biotic experiments with the nitroaromatic compound, like nitrobenzene, were

conducted for dioxygenation and partial reduction reactions (Fig. 4). Nitrobenzene
was analyzed along the two distinct pathways for d13C and d15N fractionation.
Results showed that while the oxidation reaction is accompanied by a primary
NO 2 NO NHOH NH2
R,X R,X R,X R,X
Fig. 3 Abiotic reduction of nitroaromatic compounds associated with 15N enrichment of -28.0
to -41.9 % (Hartenbach et al. 2006, 2008; Hofstetter et al. 2008a; Tobler et al. 2007)
Fig. 4 Dioxygenation NO 2 OH
(upper pathway) and partial OH OH
reduction (lower pathway) by
Comamonas sp. strain JS765 NO 2 OH
and Pseudomonas
pseudoalcaligenes strain OH
JS45, respectively (Hofstetter NO HN NH 2
et al. 2008b) OH
carbon isotope effect and a minor nitrogen effect, the nitro group reduction reac-
tion is accompanied by the opposite isotopic trend (Table 2) (Hofstetter et al.
2008b). This difference in dual-isotope trends provides a tool to differentiate
between the two pathways in complex environments.
3.2 Nitramines
Experiments to identify the isotopic fractionation associated with the transfor-

mation of nitramine explosives have been limited to RDX. These experiments,
however, cover a relatively wide range of reactions, including anaerobic nitro
group reduction, aerobic denitration, and abiotic alkaline hydrolysis.
3.2.1 Anaerobic Nitro Group Reduction
Isotope enrichment during anaerobic reduction of the nitro group in RDX was
studied for undefined microbial consortia excavated from contaminated sediments.
Both 15N and 18O isotope effects were studied. Since this reaction involves the
nitro group in the compound and the formation of nitroso derivatives, it resulted in
15
N enrichment at the reactive position, ranging from -12.6 to -30.0 % (Bern-
stein et al. 2008; Sagi-Ben Moshe et al. 2010; Hatzinger 2011), as well as 18O
enrichment at the reactive position of -31.8 % (Bernstein et al. 2008) (Table 2).
3.2.2 Aerobic Denitration
Enrichment in 13C, 15N, and 18O during RDX aerobic denitration was studied with
distinct Rhodococcus strains (Table 2). It was proposed that N–N bonds were
cleaved in the rate-limiting step of this reaction, resulting in primary 15N isotope
enrichment of -11.4 to -19.8 % at the reactive position (Hatzinger 2011;
Bernstein et al. 2013), but no observable 13C enrichment (Bernstein et al. 2013).
Interestingly, the reaction was also accompanied by 18O enrichment at the reactive
position, of -10.2 %, and it was proposed that internal hydrogen bonds may be
causing this observed effect (Bernstein et al. 2008). This explanation, however,
remained purely speculative.
3.2.3 Abiotic Alkaline Hydrolysis
Finally, isotopic enrichment (13C, 15N) during abiotic alkaline hydrolysis of RDX
showed primary isotope effects at the reactive position of -23.4 % for 13C and
-31.8 % for 15N, suggesting simultaneous loosening of C–H and N–N bonds
during the rate-limiting step.
3.3 Perchlorate
Only a few studies have documented chlorine and oxygen isotope fractionation of
perchlorate during its degradation. With its high reduction potential (ClO4- ?
8H+ Cl- ? 4H2O, E0 = 1.287 V (Urbansky 2002)), perchlorate is an ideal
electron acceptor for microorganisms which reduce it to chloride through chlorate
and chlorite (Coates and Achenbach 2004). Isotope fractionation was documented
for this process with Dechlorosoma (Azospira) strains (Coleman et al. 2003;
Sturchio et al. 2007; Ader et al. 2008). Typical enrichment factors for this reaction
by the above genus were ca. -15 % for chlorine and -29.0 to -36.6 % for
oxygen (Table 2). It should be noted that the values obtained for oxygen are
remarkably high.
An exceptional study that did not use microbial isolates of the genus Dechlo-
rosoma (or Azospira) quantified the enrichment factor for this process in situ, by
the aquifer’s undefined indigenous microbial consortia (Hatzinger et al. 2009).
Here, much lower enrichment factors were found: -8.0 to -9.2 % for oxygen and
-3.1 to -4.6 % for chlorine. The authors explained this difference as a result of
heterogeneity in field studies, which is absent in well mixed laboratory experi-
ments. One can also relate this difference to bioavailability restrictions (Kampara
et al. 2008; Thullner et al. 2008). Alternatively, since the in situ biodegrading
microorganisms were not identified, it might also be the result of different masking
effects of different microorganisms, even if, they share the same enzymatic
reaction (Nijenhuis et al. 2005; Cichocka et al. 2008).
4 Field Applications of CSIA for Contaminated

Environments
To date, studies, aimed at assessing the degradation of military energetic com-

pounds in the environment, have focused on groundwater, although some were
carried out in the unsaturated zone as well. This fits the general trend of CSIA
applications, which normally focus on contaminated groundwater rather than

contaminated soils. The reason for this may be practical due to the difficulties and
high cost of drilling and extracting soil samples along soil profiles, as opposed to
pumping groundwater from monitoring wells. It may also simply reflect a greater
interest in contaminated groundwater than in contaminated soils. It should be
noted that in soils, due to additional processes, such as dissolution and precipi-
tation, the system diverges from being a closed system, putting the applicability of
the Rayleigh equation into question. Divergence from a closed system is also
relevant for saturated porous media (groundwater). However, the influence of this
divergence has been studied to some extent in the latter habitat (Abe and Hunkeler
2006; Van Breukelen 2007; Van Breukelen and Prommer 2008), but never
addressed in unsaturated porous media which may be of even greater complexity.
Nevertheless, CSIA can still be applied to unsaturated soils to assess the extent of
bulk degradation, identify soil layers along the profile with higher degradation
activity, and get an indication of in situ degradation pathways.
4.1 TNT
The first application of CSIA to study explosive degradation in groundwater

(Miyares et al. 1999; Pennington et al. 2001) focused on d13C and d15N mea-
surements in TNT along a contamination plume underneath the Louisiana Army
Ammunition Plant. Although the d13C values of TNT along the studied contam-
ination plume were not considered significantly different, a significant d15N iso-
tope enrichment of 7.7–8.5 % was observed in two different sampling campaigns
from the center of the studied plume outward (Miyares et al. 1999). Following the
clear d15N enrichment trend along this plume, it was suggested that using d15N
measurements of TNT from groundwater might be promising for monitoring its
natural attenuation. Although it seems likely, based on the d15N enrichment and
lack of d13C enrichment, that this trend was the result of nitro group reduction, the
observed spatial enrichment in d15N composition of TNT was not correlated in that
study to a specific process and hence not used to quantify the extent of
degradation.
A second field study was concentrated on d13C in TNT and DNT isomers
contaminating groundwater in Portugal (Amaral et al. 2009). The carbon isotope
signature showed similar values for three different monitoring wells tested, except
for a single, more enriched 2,6-DNT value, which was found in the younger water
body and, therefore, not related to biodegradation, but to initial isotope signature
of the compound. Complementary groundwater dating using 3H-3He method
indicated that the studied contamination was more than 50 years old. It was
consequently concluded that TNT and DNT isomers are very recalcitrant in this
aquifer and expected to persist in the sub-surface for long periods of time.
Although it was analyzed, the d15N composition of the compounds was not
reported in this study since the uncertainties of the measurements were too large to
draw any conclusions.
A single study also focused on TNT degradation in contaminated soils (Miyares
et al. 1999; Pennington et al. 2001). This study was based on 49 soil cores
retrieved from five different locations at depths of 1.5 to 20.5 m. TNT was
detected in most soil cores at concentrations ranging from 0.1 to 1.8 lg/g, and
extractable TNT was analyzed for d15N. However, due to the low concentration of
TNT in the extracts, reliable d15N measurements could not be performed.
4.2 RDX
Some studies had an advantage of the stable isotopes of RDX’s possible end
product nitrate to gain information on RDX transformation in aquifers (DiGnazio
et al. 1998; Bordeleau et al. 2008). Such a strategy, however, is not very sensitive,
since there are various nitrate sources in groundwater whose relative importance is
not known. Isotopic analysis of RDX itself in the groundwater was applied in a
single study aimed at calculating the extent and rate of biodegradation along a ca.
1.3-km long contamination plume, as well as to gain insight into the vertical
distribution of degradation (Bernstein 2008, Bernstein et al. 2010). In this study,
both d15N and d18O isotope analyses were implemented, but the precision of the
latter was too low (Bernstein 2008). Generally, this study showed a d15N
enrichment of 10.8 % along the plume. Based on geochemical data, it was sug-
gested that the aerobic denitration pathway is dominant for RDX biodegradation
along this RDX contamination plume, but anaerobic biodegradation could not be
excluded. Quantitative estimation of the extent of degradation along the plume was
calculated for two extreme scenarios: purely aerobic and purely anaerobic deg-
radation. For the two different scenarios, the calculated half life (Eq. 6) for RDX
biodegradation in the upper 15 m of the aquifer ranged between 4.4 and 12.8 years
assuming purely aerobic biodegradation, and between 10.9 and 31.2 years
assuming purely anaerobic biodegradation. A correlation of degradation extent
with depth was presented as well, by analyzing RDX in a three-well cluster.
Degradation rate was shown to decrease with depth, and it was hypothesized that
the relatively low concentration of dissolved oxygen in the deep sub-surface, or the
expected lower nutrient availability, limit the rate of RDX biodegradation with
depth (Bernstein et al. 2010).
Another study focused on RDX degradation in unsaturated soils. Soil samples
along 46 m of the unsaturated zone were collected at high resolution, extracted and
analyzed for d15N composition in RDX. A non-uniform d15N composition of RDX
was detected along the profile, in the range of 10.6 %. The most enriched value of
d15N in RDX was detected in the uppermost soil layer, in which high organic
content was apparent. The d15N values along the rest of the profile were more
depleted, which was surprising, as it was expected that under steady-state condi-
tions, the deeper the sample, the more enriched it would be. Nevertheless, the
profile is known to have been under transient flow and transport conditions for the
last decade, explaining why the observed enrichment trend could differ from
expectations.
4.3 Perchlorate
Perchlorate degradation is characterized by large enrichment factors (e) which

enable the detection of even very minor levels of degradation. For chlorine, having
a typical enrichment factor of ca. -15 % and an analytical uncertainty of ±0.3 %,
Sturchio et al. (2003) estimated that as little as 2 % degradation can be detected
using CSIA tools. However, it should be noted that the source of the perchlorate
presented some variability in its composition and therefore, small shifts might also
be attributable to the heterogeneity of the source.
Despite of high sensitivity in tracking perchlorate degradation using CSIA
tools, field studies concerning CSIA of perchlorate are almost exclusively related
to environmental forensics, with the aim of identifying sources of contamination
(Bao and Gu 2004; Böhlke et al. 2005, 2009; Jackson et al. 2010; Sturchio et al.
2012). It is possible that perchlorate degradation in all studied plumes was simply
too low to be detected. A single study, in which CSIA was used to assess in situ
perchlorate degradation, was conducted along a ca. 35 m soil profile (Gal 2010).
Based on shifts of ca. 1.6 % along the profile, degradation was calculated to be up
to 10 %.
5 CSIA for Delineating Degradation Pathways In Situ
Data on multiple isotopes in environmental studies are often combined for forensic
applications—to determine the source of the compounds as also demonstrated for
military energetic compounds (Coffin et al. 2001; Bao and Gu 2004; Böhlke et al.
2005; Pierrini et al. 2007; Widory et al. 2009). However, such multi-isotope
information is also a powerful tool for differentiating between degradation path-
ways in situ, since different reactions present typical multi-isotope plots (Elsner
et al. 2005; Elsner 2010; Hofstetter and Berg 2011; Braeckevelt et al. 2012).
Application of the multi-isotope concept in a field study was first demonstrated in
2005 for groundwater contaminated with methyl tert-butyl ether (MTBE) (Zwank
et al. 2005). Since then, this concept has been applied in an increasing number of
studies.
In terms of explosives, three studies have been conducted to differentiate
between degradation pathways in situ (Fig. 5, Table 3). The first focused on
nitrobenzene while the other two were concentrated on RDX.
0 0
Aerobic
50 denitration Aerobic
Reduction
P. pseudoalcaligenes -2 -3
denitration
30 -4 Anaerobic Alkaline
δ15 N
reduction -6 hydrolysis
Oxidation -6
10 Comamonas sp. -9
-8
-10 -10 -12

-35 -25 -15 -5 15 20 25 30 35 -45 -35 -25
δ13 C δ18 O δ13 C
Fig. 5 Dual-isotopic trends for (1) aerobic nitrobenzene oxidation by Comamonas sp. strain
JS765 vs partial reduction by P. pseudoalcaligenes JS45 (left panel) (Hofstetter et al. 2008b);
(2) anaerobic reduction vs aerobic denitration of RDX. Isotopic ratios of sampled groundwater are
projected on the plot, with error bars of ±2r (±0.26 for d15N and ±2.36 for d18O) (middle panel)
(Bernstein 2008); (3) aerobic denitration vs. abiotic alkaline hydrolysis of RDX (right panel)
(Bernstein et al. 2013)
Table 3 Slopes of dual-isotope plots for transformation reactions of explosives and perchlorate
Compounds Reaction Slope of dual-isotope plot References
d15N/d13C d15N/ d18O/
d18O sd37Cl
Nitrobenzene Microbial reduction 46.6 Hofstetter et al.
(2008b)
Microbial oxidation 0.2 Hofstetter et al.
(2008b)
RDX Microbial aerobic No13C 1.2 Bernstein (2008),
denitration enrichment Bernstein
et al. (2013)
Microbial anaerobic 0.9 Bernstein (2008)
reduction
Abiotic alkaline 0.7 Gelman et al.
hydrolysis (2011)
Perchlorate Microbial reduction 2.5 Sturchio et al.
(2007)
5.1 Oxidation Versus Reduction of Nitrobenzene
Two different aerobic nitrobenzene-degradation pathways were studied by Hof-

stetter et al. (2008b): oxidation by Comamonas sp. strain JS765 and partial
reduction by Pseudomonas pseudoalcaligenes strain JS45. Comamonas sp. strain
JS765 mineralizes nitrobenzene following initial dioxygenation at the aromatic
ring. The oxidation reaction is initiated with dearomatization of the benzene ring,
and reflected in large primary 13C enrichment and small 15N enrichment. In
contrast, a reduction is initiated by the partial reduction of nitrobenzene to form
hydroxylaminobenzene. This reaction, which only involves the nitro group, is

accompanied by significant 15N fractionation and only small changes in the 13C
signature of the compound. The two distinct enrichment trends (Fig. 5) enable
differentiation between the two possible reactions in the environment. Neverthe-
less, to date, they have not been tested in situ.
5.2 Aerobic Denitration Versus Anaerobic Reduction

of RDX
An attempt was made to differentiate between aerobic and anaerobic degradation

using a dual-isotope plot of d15N versus d18O enrichment trends (Bernstein 2008).
As shown in Fig. 5, two dimensional isotope curves were plotted using enrichment
factors found in laboratory microcosm studies with indigenous bacteria under
controlled conditions (Bernstein et al. 2008). However, the relative similarity
between the slopes of the two dual-isotope curves, together with the relatively low
precision of the d18O measurements, did not enable to identify the relative
importance of aerobic versus anaerobic biodegradation in situ, in a contaminated
aquifer (Fig. 5).
5.3 Aerobic Denitration Versus Abiotic Alkaline Hydrolysis

of RDX
Combining d13C and d15N enrichment trends, aerobic microbial denitration and
abiotic alkaline hydrolysis of RDX can be differentiated. During aerobic microbial
denitration, 13C enrichment of RDX is lacking and 15N enrichment is observed,
whereas during abiotic alkaline hydrolysis, both 13C and 15N enrichments are
observed (Gelman et al. 2011; Bernstein et al. 2013). This can serve to differentiate
between the two processes in situ.
6 CSIA for Studying Intrinsic Degradation Mechanisms
Owing to the fact that primary isotope effects are detected in atoms that are
directly involved in bond cleavage or formation during the rate-limiting step of the
reaction, whereas smaller and secondary isotope effects are detected in the adja-
cent bonds, one can gain unique insight into the mechanism of the reaction. This
was recently used to reveal the degradation mechanism of RDX denitration by
aerobic Rhodococcus species (Bernstein et al. 2013).
The general question addressed in that study was which atom is being cleaved
in the rate-limiting step of the reaction. Although previous studies had shown that
denitration is the key step in RDX degradation by Rhodococcus species (Fournier
et al. 2002; Bhushan et al. 2003; Jackson et al. 2007), a more recent study sug-
gested that this pathway is initiated by cleavage of an inner ring C–H bond (Halasz
et al. 2010) rather than of a N–N bond. CSIA was chosen to delineate the deg-
radation mechanisms: whereas the cleavage of a C–H bond is expected to mark
isotope enrichment on carbon, but not nitrogen, N–N bond cleavage is expected to
mark isotope enrichment on nitrogen, but not carbon (Fig. 6). Indeed, applying this
tool showed clear d15N enrichment of RDX in incubation studies with Rhodo-
coccus species, while there was no d13C enrichment. Consequently, it was sug-
gested that N–N bond cleavage is the rate-limiting step of the reaction.
(I) H•-abstraction from CH2

(Halasz et al., 2010)
δ15N
•
O2N C• NO2 H• abstraction

N N
N
NO2 δ13C
(II) Single transfer (Bhushan

et al., 2003; Fournier et al.,
2002; Jackson et al., 2007)
δ15N
O2N NO2 O2 N N-N cleavage

N N N N
N Intermediate not defined N

by the authors
NO2 NO2
δ13C
(III) Alkaline hydrolysis

OH-
(Gelman et al., 2011) H
δ 15 N
O2N NO 2 Alkaline
N N
hydrolysis
N
NO2 δ13C
Fig. 6 Different proposed denitration pathways for RDX and hypothetical dual-isotope plot for
the reactions (modified from (Bernstein et al. 2013)
7 Conclusions
Isotope tools provide highly sensitive information on environmental processes

which are otherwise difficult to assess. They enable estimation of degradation
extents in situ, and reveal the governing degradation pathway. Moreover, they
enable identifying the mechanistic steps in reactions. These properties have also
served to study the environmental fate of military energetic compounds, but to
date, such studies have been limited to numerous laboratory investigations and a
few for field applications.
Future work would greatly benefit from further method of optimization for
CSIA of nitramines as well as the development of online analytical schemes for
perchlorate. In particular, the ability to analyze the nitramine explosives (mainly
HMX) directly without offline preparation protocols, would promote studies on
their fate in the environment. This might be achieved in the future by suitable LC-
IRMS methods or other techniques. With respect to perchlorate, it might be
anticipated that non-IRMS instrument, such as a multicollector inductively cou-
pled plasma MS interfaced with ion chromatography, will be found suitable for
direct analysis of 35Cl in aqueous samples.
Acknowledgments This work was supported, in part, by grant 167/2008 from the Israel Science
Foundation.
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Biodegradation of
Hexanitrohexaazaisowurtzitane (CL-20)
Julius Pavlov and Mohammed Sidhoum
1 Introduction
Explosive contamination surveys of military ranges in the United States and

Canada have revealed that residues of energetic materials in soils can be widely
and very unevenly dispersed (Crocker et al. 2006). While the level of explosive
residue contamination is very low in general (up to about 50 lg/kg), but higher
concentrations are found near firing points, detonation sites, and armored targets
(1 mg/kg to above 100 mg/kg). In addition, at such spots, entire lumps of energetic
materials can be encountered (Jenkins et al. 1998, 2001; Pennington et al. 2001,
2005; Walsh et al. 2001; Hewitt 2002). Since military site environmental char-
acterization, outside the US and Canada, has mainly been carried out at facilities in
the UK, Australia, Germany, and Sweden, the available contamination data are
quite sparse (Spain 2000). A Swedish survey of the Alvdalen Shooting Range
reports low levels of explosive and propellant residues in soil except for a few
locations where nitramine concentrations were as high as 6.5 mg/kg for RDX and
4.2 lg/kg for HMX (Wingfors et al. 2006). There is always a possibility that
residues of energetic materials may migrate from the soil surface and sub-surface
to the groundwater (Clausen et al. 2004), which may have a widespread envi-
ronmental impact. This is particularly true for high-solubility energetics, such as
nitroguanidine and nitrotriazolone, which are being increasingly used in novel
explosive formulations. Although the environmental transport of RDX and HMX
is very limited by their low aqueous solubility and high soil adsorption, the US
Environmental Protection Agency (2004) has designated RDX a priority pollutant
and HMX a contaminant of concern.
J. Pavlov M. Sidhoum (&)

Center for Environmental Systems, Stevens Institute of Technology, Hoboken, NJ, USA
e-mail: msidhoum@stevens.edu

286 J. Pavlov and M. Sidhoum
RDX and HMX can be transformed and degraded by numerous microorgan-

isms, including both aerobic and anaerobic bacteria, as well as aerobic fungi
(Crocker et al. 2006; Karakaya et al. 2009). The metabolic pathways involved in
the environmental transformation of RDX and HMX have been described by
Hawari et al. (2000a).
Since then, a lot of research has been conducted into the biological fate of the
relatively new nitramine high explosive 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-
hexaazaisowurtzitane (HNIW or CL-20). Its high manufacturing cost prevents it
from being used as widely as the other two principal nitramine explosives.
However, its superior performance ensures its continual use. Therefore, it may be
viewed as a next-generation energetic material.
The CL-20 molecule is based on the rigid and strained isowurtzitane cage
(Fig. 1) with six alternating carbon atoms substituted by nitramine groups
(=NNO2). The high nitrogen content and steric strain contribute to the high energy
content, which is much higher than that of either RDX or HMX. In fact, CL-20 is
the most powerful chemical explosive thus far known to mankind. In addition,
unlike HMX and RDX, the CL-20 molecule contains three slightly elongated
carbon–carbon bonds (Fig. 2). Of these, C1–C4 bond is elongated to 1.59-1.60 Å
(compared to an ordinary sp3–sp3 C–C bond at 1.54 Å). The C1–C4 bond is longer
than the C5–C6 and C9–C10 bonds, which are about 1.58–1.59 Å. This indicates
that the C1–C4 bond is weaker than the other C–C bonds, in other words, it is
subject to the greatest strain of the three and is the most easily broken (Zhou et al.
2002).
Aerobic soil biotic degradation of CL-20 has been reported by several
researchers (Crocker et al. 2005; Strigul et al. 2006). In addition, Trott et al. (2003)
reported CL-20 degradation by Agrobacterium sp., while Bhushan et al. (2003a)
studied its transformation by the anaerobic denitrifier Pseudomonas sp. FA1 when
Fig. 1 Molecular structures NO2

of HMX, RDX, and CL-20
O2 N NO2 N
O2 N
N N N
N
N NO2
N
NO2 O2 N
RDX HMX
O2 N NO2 NO2
O2 N NO2 N
N N N N
O2 N NO2
N N
N N N
N N O2 N NO2
NO2 NO2
O2 N
HNIW
Biodegradation of Hexanitrohexaazaisowurtzitane (CL-20) 287
Fig. 2 Atomic numbering in 1

C
the CL-20 cage
O 2N
3 4
N C 2 NO 2
N
O 2N 7 8 NO 2
N N
6 5
C C
10 9
C C 11
12 N
N NO 2
O 2N
CL-20 was used as a sole nitrogen source. Trott et al. (2003) reported that CL-20
was readily degraded in soil environments, but did not specify whether it happened
in aerobic or anaerobic conditions. The anaerobic Clostridium sp. EDB2 was also
demonstrated as a HNIW degrader (Bhushan et al. 2004c). CL-20 transformation
by pure cultures of bacteria and fungi has also been reported (Crocker et al. 2006).
In marked contrast to HMX and RDX, CL-20 was found more amenable to abiotic
degradation (Balakrishnan et al. 2003; Monteil-Rivera et al. 2004; Szecsody et al.
2004).
The study of Gong et al. (2004) revealed that CL-20 was nontoxic to the marine
bacterium Vibrio fischeri, the freshwater green alga Selenastrum capricornutum,
terrestrial plants and indigenous soil microorganisms at concentrations of up to
10 g/kg soil. However, Robidoux et al. (2004) demonstrated that CL-20 is lethal to
the earthworm Eisenia andrei at levels even as low as 90.7 mg/kg soil.
In contrast, RDX and HMX showed no detrimental effects on soil microbial
activities even at a concentration up to 12.5 g/kg soil (Gong et al. 2001, 2002).
However, these energetic compounds caused reproductive damage to earthworms
at concentrations higher than 46.7 and 15.8 mg/kg, respectively, without affecting
their survival even at concentrations up to 167.3 and 711.0 mg/kg, respectively
(Robidoux et al. 2002).
Nitramine energetics are typically biodegraded following one or more known
mechanisms. First, one or more nitro groups can be enzymatically reduced to the
corresponding nitroso or hydroxylamino moiety. Second, a N–N bond may be
cleaved homolytically, releasing a nitro group to yield the nitrite ion as the final
product. Third, a direct ring cleavage may occur at a N–N or C–N bond. Fourth, a
hydroxylation or a hydride ion transfer can occur at a carbon atom. Thus, initial
enzymatic reactions destabilize the nitramine ring. Any intermediates that result
from initial ring cleavages are presumed to be too unstable and hence rapidly to
decompose in the aqueous environment. Thus, cyclic nitramines, in general,
degrade in the environment by a combination of biotic and abiotic reactions whose
end products may include nitrite ion, nitrous oxide, ammonia, and formaldehyde
(RDX and HMX) or formic acid and glyoxal (CL-20) (Hawari et al. 2000b, 2001;
Fournier et al. 2002; Bhushan et al. 2003b, 2004a, b, 2005a; Zhao et al. 2004).
Once simple compounds are formed, they can be further metabolized by the same
or other microorganisms (Coleman et al. 1998; Hawari et al. 2000b, 2001; Four-
nier et al. 2002, 2004; Halasz et al. 2002; Seth-Smith et al. 2002; Zhao et al. 2002,
2003; Van Aken et al. 2004; Thompson et al. 2005; Fournier et al. 2006). Ideally,
the ultimate degradation products of a nitramine include carbon dioxide, nitrogen,
and methane. The proportionate yield of metabolites and end products will cer-
tainly depend on the combined action of various microorganism populations in the
local environment, as well as the feasibility of any possible abiotic degradation
processes, such as hydrolysis, photochemical reactions, etc.
Activated sludge was found ineffective to biodegrade CL-20 with or without
supplemental nitrogen or carbon sources (Karakaya et al. 2009). However, in the
same study, activated sludge was reported to mineralize the products of abiotic
alkaline hydrolysis of CL-20, which clearly indicates that a combined approach is
feasible to remove CL-20 contamination. Such knowledge is necessary for the
design of suitable bioremediation processes and environmental monitoring.
2 Aerobic Biodegradation Processes
2.1 Activated Sludge
Aerobic soil bacteria can utilize CL-20 as a nitrogen source for their growth, and
they thereby biodegrade it (Bhushan et al. 2003a; Trott et al. 2003). The only
known anaerobic bacterium capable of effectively degrading CL-20 as well as
RDX and HMX is Clostridium sp. EDB2 (Bhushan et al. 2004c). In addition,
biodegradation of CL-20 was observed in surface and sub-surface soils under both
aerobic and anaerobic conditions (Jenkins et al. 2003; Trott et al. 2003; Crocker
et al. 2005; Strigul et al. 2006).
Under unsaturated conditions and at low CL-20 concentrations, the explosive
degrades very slowly in aerobic soils (Jenkins et al. 2003), with half-life in the
range of 144–686 days. Practically no degradation was observed at concentrations
above 125 mg/kg (Strigul et al. 2006). For biodegradation to be effected at this
high concentration, soils had to be amended with starch or cellulose up to
1,000 mg/kg. The observed fungal growth in the amended soils indicates that CL-
20 is a substrate for fungal metabolism (Strigul et al. 2006).
No significant CL-20 degradation was observed over a period of 16 weeks of
treatment with activated sludge (Karakaya et al. 2009). A minor reduction in CL-
20 concentration was attributed to abiotic transformations, such as hydrolysis (pH
of the medium was above neutral at the end of the incubation period). It may be
concluded that the activated sludge process, common in wastewater treatment
plants, is not a viable option for treatment of CL-20 contaminated effluents.
However, the same researchers observed that activated sludge slowly, but quite
successfully, tackled the aqueous liquor obtained after alkaline-hydrolysis degra-
dation of HNIW (Karakaya et al. 2009). Using 14C-labeled CL-20, it was noted
that 14CO2 release (mineralization) began almost immediately after the start of
incubation, and close to 20 % of the available carbon in the CL-20 hydrolysate
was transformed into 14CO2 within 24 h. After 11 days of incubation, the min-
eralization of the hydrolysate reached 56 % of the initial amount. Thereafter,
14
CO2 production leveled off, and a plateau at 65.5 % mineralization was attained
after 7 weeks of incubation. When the experiment was terminated at that time, the
amount of 14C found in the residual biomass accounted for about 34 % of the
original, thereby bringing the total carbon recovered to 99 +%. This indicates that
the detected 14CO2 was not a by-product of the CL-20 alkaline hydrolysis reaction.
These results suggest that a two-step process involving alkaline hydrolysis fol-
lowed by aerobic biological treatment is a potential option for the treatment and
disposal of CL-20 and its hydrolysate.
2.2 Biodegradation by Phanerochaete chrysosporium
2.2.1 Kinetic Modeling
Fournier et al. (2006) demonstrated that the white rot fungus Phanerochaete
chrysosporium mineralized CL-20 during ligninolytic growth, when it secretes
enzymes necessary for the biodegradation of chemicals in response to nitrogen or
carbon starvation. The ligninolytic system of P. chrysosporium consists of lignin
peroxidases (LiP) and manganese-dependent peroxidases (MnP) as well as
hydrogen-peroxide-generating oxidases (Barr and Aust 1994). In soils amended
with amounts of CL-20 at a maximum of 3 times its water solubility (3.5–10 mg/
kg), the highest rates of biodegradation were observed. First-order kinetics of
biodegradation was observed in biologically active surface soils amended with
glucose, with rate constants in the range 0.068–1.222 d-1 (Crocker et al. 2005). In
soils with high CL-20 concentrations (250 mg/kg), anaerobic biodegradation
prevailed, and starch amendments shortened the lag phase (Strigul et al. 2006).
The above soil studies did not discuss either the specific microorganisms that
biodegraded CL-20, or any intermediates or final products. Presumably, bio-
transformation products were observed as new peaks in HPLC chromatograms
(Strigul et al. 2006), but no attempts were made to identify them. Crocker et al.
(2005) reported only the release of 14CO2 from 14C-labeled CL-20 under aerobic
conditions.
A detailed study of CL-20 biodegradation by P. chrysosporium was carried out
by Karakaya et al. (2009). Substrate disappearance was monitored at initial CL-20
concentrations of up to 500 mg/l. In all experiments, CL-20 biodegradation dis-
played a lag phase of about 2.5 days. Irrespective of the initial concentration
(except for the case of 500 mg/l CL-20), the substrate had completely disappeared
in less than 95 h of incubation. However, in the case of 500 mg/l concentration,
only about 10 % of CL-20 depletion was achieved after 113 h. Very significantly,
it was observed that the biomass production was not affected in any case, i.e.,
fungal growth was not inhibited in the presence of the nitramine. A similar
observation was also made by Stahl et al. (2001) with the biodegradation of RDX
(up to 250 mg/l) by P. chrysosporium. However, the fungus did not degrade RDX
above its solubility limit, unlike CL-20, which was more amenable to biodegra-
dation by P. chrysosporium.
Karakaya et al. (2009) were successful in modeling the experimental biodeg-
radation data. The logistic kinetic model (Schmidt et al. 1985; Alexander 1999)
was applied, because it works well with fungal systems. In its differential form, the
logistic model is represented by Eq. 1.

dX X
¼ rX 1 ð1Þ
dt Xmax
Upon integration, the model is transformed into Eq. 2:
Xmax
X ¼ ð2Þ
Xmax Xo
1þ Xo exprt
where t is time, r is the maximum specific growth rate in a particular environment,

X is the population density at a given time, X0 is the initial population density, and
Xmax is the maximum population density achievable in the same environment. The
substrate degradation rate is related to the biomass growth as reflected by Eq. 3:
dS k S Xmax
¼ ð3Þ
dt 1 þ XmaxXX o
exprt
o
where S is the substrate concentration.

Upon integration of Eq. 3, we obtain
S ¼ So ½/ðexprt 1Þ þ 1k=r ð4Þ

where / ¼ XXmax
o
and k ¼ VmaxKmXmax : Vmax represents the maximum specific reaction
rate, and Km is the half-saturation constant.
After subtraction of the lag time, regression analysis was used to derive the
kinetic parameters for Eq (2) and (4). The values of k and r were found to be 13.5
and 0.145 h-1, respectively. Using these parameters, the general logistic model for
CL-20 biodegradation by P. chrysosporium is written as:
93:1
0:00034 0:145 t

S ¼ S0 exp 1 þ 1 ð5Þ
Xmax
Xmax
X ¼ Xmax 0:00034
ð6Þ
1þ 0:00034 exp0:145 t
Equation 5 fits well CL-20 biodegradation time-concentration profiles over a

wide range of initial CL-20 concentrations (1–100 mg/l).
2.2.2 Effects of Nutrients on the Biodegradation of CL-20

by P. chrysosporium
Karakaya et al. (2009) employed glycerol as a carbon nutrient source and

ammonium sulfate as a nitrogen source in various carbon to nitrogen (C:N) ratios.
It was observed that P. chrysosporium did not require carbon starvation to degrade
CL-20. The explosive was degraded almost completely in both low- and high-
carbon media. The lag phase was longer at high glycerol levels. A high initial
amount of glycerol was demonstrated to result in higher fungal biomass growth.
On the other hand, CL-20 was consumed simultaneously with ammonium sulfate
in growing cultures of the fungus in a high-nitrogen medium (1 g/l ammonium
sulfate). In low N media (0.1 and 0.2 g/l ammonium sulfate), CL-20 degradation
was initiated as soon as the ammonium sulfate nitrogen was depleted, indicating
that secondary metabolism was initiated in response to nitrogen limitation.
The results showed that CL-20 biodegradation by P. chrysosporium does not
necessarily require nitrogen limitation. However, if a high amount of easily
metabolized nitrogen is present in the system, CL-20 biodegradation will be
delayed, but fungal growth will not be adversely affected. Since a high initial
concentration of ammonium sulfate resulted in the enhanced fungal growth, it can
be concluded that such initial ‘‘stimulus’’ of the fungi will result in faster CL-20
degradation, once the ammonium nitrogen is depleted. Thus, the initial amounts of
supplied nitrogen and carbon nutrients are important controlling factors in CL-20
biodegradation by P. chrysosporium. The apparent absence of nitrogen limitation
may be attributed to the dependence of CL-20 degradation on the MnP enzyme,
which is produced in nitrogen sufficient media, rather than to LiP dependence
(Fritsche et al. 2000). This observation is supported by Fournier et al. (2006), who
have suggested a direct degradation of CL-20 by manganese peroxidase. The role
of this enzyme is to oxidize Mn2+ to Mn3+ which binds to an appropriate available
ligand, forming a highly oxidative complex which can then attack the substrate.
Although the precise mechanism of MnPs action on secondary metabolites is not
yet clear, high levels of this ligninolytic enzyme typically correspond to the ability
of white rot fungi to degrade nitro compounds. A high amount of supplied nitrogen
can enhance the MnP attack on explosive substrates (Kapich et al. 2004). In the
latter study, a very low MnP activity was observed in the absence of peptone, but
when that was added, the activity of MnP markedly increased. It was reported that
a high concentration of peptone nitrogen (up to 3–4 g/l of peptone) stimulated
MnP production by P. chrysosporium.
In marked contrast, Stahl et al. (2001) reported that the RDX degradation rate
by growing P. chrysosporium culture was approximately 10 times higher under
nitrogen-limited (ligninolytic) conditions as compared to nitrogen-sufficient (non-
ligninolytic) conditions. This study also confirmed that RDX was degraded by
MnP, not by LiP.
Karakaya et al. (2009) also found that the type of nitrogen source (organic vs.
inorganic) had a considerable effect on CL-20 biotransformation by P. chrysos-
porium. Ammonium sulfate was compared to yeast extract as a nitrogen source. In
both cases, the produced maximum biomass was comparable, but with yeast
extract, the degradation of CL-20 was significantly slower. The slow biodegra-
dation with yeast extract may be attributed to a possible alteration of the lignin-
olytic enzyme system of the fungus. The sensitivity of ligninolytic enzymes to the
presence of various nitrogen compounds has also been reported by other
researchers (Dutta et al. 1998; Vahabzadeh et al. 2004). Karakaya et al. (2009)
demonstrated that P. chrysosporium may also utilize CL-20 as the sole nitrogen
source in nitrogen-deficient environments, but with a lag time of around 3 weeks.
The initial presence of an external source of nitrogen was found critical to fast
fungal growth and enhanced biodegradation of CL-20.
2.2.3 Mineralization of [14C]-CL-20 by P. chrysosporium
Using uniformly 14C-labeled HNIW, Karakaya et al. (2009) showed that CL-20
mineralization began after 2 days of incubation with P. chrysosporium and the
process was completed within 100 h after inoculation in high C-low N medium.
However, the release of 14CO2 did not exceed 8.5 % of the initial 14C amount
within this period. Even after a 46-day incubation period, less than 50 % miner-
alization was achieved. Even with ammonium sulfate concentration at 1 g/l (high
C-high N), CL-20 mineralization was only slightly higher (51.2 %). It can be
concluded from this experiment that after about 4 weeks of incubation, the deg-
radation process was completed, as evidenced by the material balance results:
upon combining the residual 14C in the microcosms with the amount released as
14
CO2, a total 14C recovery in the range of 90.5–102.1 % was obtained.
Fournier et al. (2006) reported a considerably higher maximum mineralization
(80 %) of CL-20 in a nitrogen-limited growth medium containing 1.2 mM
ammonium tartrate. The different nitrogen sources and incubation conditions, the
use of glucose instead of glycerol (10 g/l) as a carbon source and/or tenfold lower
initial CL-20 in Fournier’s medium may account for the observed higher
mineralization.
2.2.4 Role of Mycelial Mass and Extracellular Fluid

in the Biodegradation of CL-20 by P. chrysosporium
Upon exposure to the liquid supernatant and the pellet fractions of P. chrysos-
porium, CL-20 begins to degrade without a lag phase (Karakaya et al. 2009).
Microcosms containing both pellets and a liquid fraction degraded CL-20 com-
pletely in 36 h. Within 48 h, nearly complete degradation of CL-20 was achieved
by using only cell mass (without extracellular enzymes). However, the initial rate
of CL-20 degradation was significantly lower than that observed using both
extracellular enzymes and biomass. This strongly suggests that extracellular

enzymes are operative. Degradation rates increased after 24 h, which clearly
indicates the regeneration of extracellular enzymes. In cultures containing only the
liquid fraction, initial degradation occurred, then slowed markedly and finally
reached a plateau after 24 h at 85 % CL-20 depletion.
2.2.5 Effect of Fungal Culture Age on Biodegradation
The operation of treatment and remediation systems based on P. chrysosporium,

e.g. biofilters, relies on the long-term biodegradation ability of the fungal pellets.
The maintenance of high levels of enzymatic activity for extended periods is
critical for such systems. The pellet growth form of the white rot fungi enables
biomass reuse and the continuity of such processes. Karakaya et al. (2009) eval-
uated the ability of aged P. chrysosporium pellets to degrade CL-20. Cultures aged
up to 18 days were able to fully transform CL-20 following pseudo-first order
kinetics with its half-life of 6 h or less. First-order kinetics for pre-grown cultures
has been suggested to apply to white rot fungi co-metabolism (Barr and Aust
1994). Pseudo-first-order rate constants computed for cultures aged 5, 6, 10 and
18-days were 0.269 (0.023), 0.200 (0.006), 0.126 (0.038), and 0.112 (0.027) h-1,
respectively, with correlation coefficients of 0.9967, 0.9995, 0.9470 and 0.9628,
respectively (the values in parentheses are standard errors).
3 Biodegradation Pathways
CL-20 biodegradation follows three main pathways. All three pathways generate
common end products. Pathway 1 involves an initial denitration and opening of the
isowurtzitane cage at the weakest (C1-C4) carbon–carbon bond (Fig. 3).
This reaction is favorable according to computational chemistry (Okovytyy
et al. 2005). The enzymes salicylate 1-monooxygenase (Bhushan et al. 2004b),
nitroreductase (Bhushan et al. 2004a), and dehydrogenase (Bhushan et al. 2005a)
facilitate the transfer of an electron to the substrate molecule to form a radical-
anion which subsequently loses a nitrite ion. Since molecular oxygen quenches the
reaction by scavenging free electrons, the reaction can only proceed in anaerobic
conditions (Bhushan et al. 2004a). The initial loss of a nitro group (Okovytyy et al.
2005) destabilizes the isowurtzitane cage, leading to a spontaneous cleavage of the
C1–C4 bond. The same enzymes then facilitate a second single-electron transfer to
the denitrated intermediate to eliminate a second nitro group from it. This leads to
the formation of two isomeric intermediates (I and II; Fig. 3) containing two C=N
bonds (Bhushan et al. 2004a, b). Enzyme assays using both ring and nitro-labeled
[15N]-CL-20 lend support to the hypothesis of formation of such intermediates
(Bhushan et al. 2004b, 2005a). This mechanism has also been discussed in relation
to CL-20 biodegradation by Pseudomonas sp. FA1 (Bhushan et al. 2003a), and by
CL-20
O2N NO2 NO2
Pathway 1 Pathway 3
Salicylate monooxygenase Dehydrogenase
Nitroreductase O2N NO2 Diaphorase
NO2
Dehydrogenase (Clostridium sp. EDB2)
Manganese peroxidase
Dehydrogenase
Pathway 2 (Clostridium sp. EDB2)
NO2 NO2 O2N NO2 NO 2
N N N N N N
O2N NO2 NO2

N N N N N N
N N N
O2N ON NO2
NO2 I NO2
N N
OR
N IV
H NO2
NO2
O2N NO2 NO 2 III

N N N
N N N
NO2
II
- -
HCOO, (CHO) 2 , N 2O, NO 2
Fig. 3 A reaction scheme outlining the three possible pathways of biodegradation of CL-20
abiotic degradation using zero-valent iron (Balakrishnan et al. 2004), alkaline

hydrolysis (Balakrishnan et al. 2003; Qasim et al. 2004), as well as photodegra-
dation (Hawari et al. 2004; Qasim et al. 2005).
Two hypothetical and contrasting reaction pathways have been proposed for the
subsequent fate of isomeric intermediates I and II. First, Hawari’s work under
abiotic and biotic conditions, the latter with in vitro enzyme assays, proposes that
these intermediates are unstable in water and will hydrolyze to yield species such
as nitrite (2–5 mol), nitrous oxide (0–0.3 mol), ammonium (1.4–2 mol), formate
(2–5.3 mol), and traces of glyoxal (Balakrishnan et al. 2003, 2004; Hawari et al.
2004). Under biotic conditions, end products obtained included 1.5–2.3 mol of
nitrite, 1.5–3.3 mol of nitrous oxide, 0–1.3 mol of ammonium, 0–1.7 mol of for-
mate and 0–1 mol of glyoxal per mole of substrate (Bhushan et al. 2003a, 2004a,
b, 2005a). Some secondary metabolites produced in trace amounts from the
spontaneous decomposition of I and II were also postulated to hydrolyze sponta-
neously. More factual data are needed to remove the speculation from this
mechanism.
The second possible pathway is based in part on computational stability pre-
dictions related to molecular structures of intermediates of alkaline hydrolysis and
photodegradation of CL-20 (Qasim et al. 2004, 2005; Okovytyy et al. 2005). Some
supporting UV/vis and FTIR spectral data are provided. In this pathway, the loss of
nitro groups would continue without further ring cleavages, resulting in the con-
jugation of p-bonds on the three rings to yield a stable pyrazolo-pyrazine aromatic
molecule. However, direct evidence for the formation of such a molecule has not
been found, either in biotic or abiotic degradation of CL-20. The validity of this
mechanism as a viable alternative in the degradation of CL-20 is still unclear.
In the second main pathway for biotransformation of CL-20 (Fig. 3, Pathway
2), a hydride transfer occurs across a N–N bond to release nitrite and generate the
denitrohydrogenated intermediate III. This pathway is mediated by a dehydroge-
nase enzyme from Clostridium sp. EDB2 (Bhushan et al. 2005a) and a purified
diaphorase (Bhushan et al. 2005b).
In the third pathway (Fig. 3, Pathway 3), a nitro group is reduced to yield the
mononitroso derivative of CL-20 (IV). This pathway is also facilitated by the
dehydrogenase enzyme of strain EDB2 (Bhushan et al. 2005a). The fate of
intermediates III and IV was not explored due to their transient nature and very
low levels of their presence.
Again, in pathways 2 and 3, the intermediates obtained after the initial enzy-
matic steps were postulated to destabilize the isowurtzitane cage and cause its
cleavage, to ultimately yield a mixture of similar end products as shown in
pathway 1 (Bhushan et al. 2005a, b). The biotransformation of CL-20 by the
dehydrogenase enzyme from strain EDB2 was postulated to follow primarily
pathway 1 with possible minor contributions from pathways 2 and 3 (Bhushan
et al. 2005a).
CL-20 mineralization by the white rot fungus P. chrysosporium has been
postulated to occur via the action of the highly oxidative extracellular enzyme
manganese peroxidase (MnP) (Fournier et al. 2006). Notably, a non-specific
enzymatic system of white rot fungi enables them to degrade a number of nitro
explosives, including HMX and RDX (Pal and Christodoulatos 1995; Hawari et al.
1999; Sheramata and Hawari 2000; Fournier et al. 2004). Nitrous oxide (45 % of
N balance) and carbon dioxide ([80 % of C balance) were the only end-products
observed during ligninolytic growth of P. chrysosporium. A doubly denitrated
metabolite, possibly I/II, was produced by MnP in an enzyme assay. Nitrite,
glyoxal, and small amounts of nitrous oxide and nitrate were also observed. It
appears that nitrite was further metabolized for the purpose of growth, and glyoxal
was mineralized by P. chrysosporium, but not degraded by MnP.
4 Conclusions
Activated sludge is ineffective in transforming CL-20 either in the presence or

absence of supplemental nitrogen and carbon sources. However, activated sludge
is able to mineralize significantly the base hydrolysis products of CL-20. There-
fore, alkaline hydrolysis coupled with aerobic microbial treatment is a technically
feasible option for the degradation of CL-20 and its intermediate products from
waste streams.
The white rot fungus P. chrysosporium is capable of degrading CL-20 in the

presence of supplementary carbon and nitrogen sources. External nitrogen and
carbon sources are important factors controlling the CL-20 biodegradation in
growing cultures. P. chrysosporium was able to degrade CL-20 up to concentra-
tions of 100 mg/l, and the explosive did not inhibit the growth of the fungus up to
concentrations of 500 mg/l. CL-20 biodegradation by P. chrysosporium follows
the logistic kinetic growth model.
Although CL-20 biodegradation follows three main possible pathways, they
generate similar end products.
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Pathways of 2,4,6-Trinitrotoluene
Transformation by Aerobic Yeasts
Ayrat M. Ziganshin and Robin Gerlach
1 Introduction
The production and use of various highly persistent synthetic compounds lead to
environmental pollution. Among such compounds, 2,4,6-trinitrotoluene (TNT) is
the one which is commonly used as an explosive. Synthesis and wide use of TNT
in ammunition have resulted in the contamination of soil, air, surface water, and
groundwater. TNT and its nitro group reduction products are highly toxic,
potentially mutagenic and persistent contaminants which can persist in the envi-
ronment for a long time (Spain et al. 2000; Stenuit et al. 2005; Smets et al. 2007;
Singh et al. 2012). The U.S. Environmental Protection Agency has classified TNT
as one of the most dangerous pollutants in the biosphere. Hence, remediation of
TNT-contaminated sites is urgently warranted at places of its production and use
(Keith and Telliard 1979; Fiorella and Spain 1997).
Human exposure to TNT or its nitro group reduction metabolites can lead to the
development of diseases, such as aplastic anemia, cataracts, impaired liver func-
tion and the formation of tumors in the urinary tract (Hathaway 1985; Yinon 1990;
Leung et al. 1995). Hence, it is inevitable to work out strategies targeting the
degradation of TNT.
Decontamination of sites contaminated with explosives, especially with TNT, is
possible with application of various physical, chemical, and biological methods.
The main advantages of bioremediation are environmental friendliness and
involvement of low cost (Rodgers and Bunce 2001).
A. M. Ziganshin (&)
Department of Microbiology, Kazan (Volga Region) Federal University, ul. Kremlyovskaya
18, Kazan, The Republic of Tatarstan, Russia, 420008,
e-mail: a.ziganshin06@fulbrightmail.org
R. Gerlach
Center for Biofilm Engineering and Department of Chemical and Biological Engineering,
Montana State University, Bozeman, MT 59717, USA

302 A. M. Ziganshin and R. Gerlach
Since 1980, a wide range of TNT transformation pathways have been worked
out. The reductive transformation of the nitro groups appears to be the most
commonly observed biologically mediated transformation of TNT (Michels and
Gottschalk 1994; Naumov et al. 1999; Huang et al. 2000; Borch et al. 2005). This
transformation proceeds via nitroso-dinitrotoluenes (NSDNTs) and hydroxyla-
mino-dinitrotoluenes (HADNTs) with latter being a meta-stable and easily
detectable intermediate on the path to complete nitro group to amino group
reduction. However, the school of Prof. H.J. Knackmuss focused on the discovery
of alternative TNT biotransformation pathways (Vorbeck et al. 1994, 1998). Some
microorganisms were found to perform TNT transformation via hydride ion
addition to the aromatic ring. This TNT transformation pathway was first descri-
bed by Vorbeck et al. (1994) for Mycobacterium sp. strain HL 4-NT-1 which leads
to the formation of a C-3 monohydride-Meisenheimer complex of TNT (3-H-–
TNT). Further transformation of 3-H-TNT leads to the accumulation of a C-3,C-5
dihydride-Meisenheimer complex of TNT (3,5-2H-–TNT), which can be pro-
tonated to form 3,5-2H-TNT.H+. Three different isomers of 3,5-2H-–TNT.H+
were identified. Subsequent studies have demonstrated that other microorganisms
are also able to reduce the aromatic ring of TNT by hydride ion attack (French
et al. 1998; Kim and Song 2000; Pak et al. 2000; Zaripov et al. 2002; Jain et al.
2004; Wittich et al. 2008; Ziganshin et al. 2010a, b).
TNT nitro group reduction by microbial enzyme systems can lead to the
accumulation of highly toxic nitroso- and hydroxylamino-dinitrotoluenes (Leung
et al. 1995; Zaripov et al. 2002). TNT aromatic ring attack by hydride ions can
cause transformation of TNT with the release of nitrogen in the form of NO2-. In
several works, denitration was suggested to be the result of the destruction of
TNT-hydride complexes (French et al. 1998; Jain et al. 2004; Williams et al. 2004;
Ziganshin et al. 2007, 2010a, b). Other workers suggest that the elimination of a
nitro group can occur not only via the hydride pathway, but also during reactions
(including abiotic reactions) of HADNTs with Meisenheimer dihydride com-
plexes, producing amino-dimethyl-tetranitrobiphenyls or secondary diarylamines
(Pak et al. 2000; Wittich et al. 2008).
Obviously, the use of microorganisms for TNT reduction via hydride ion
addition and subsequent degradation of the formed complexes is highly promising
from a viewpoint of bioremediation of TNT-contaminated sites.
The ability to detect metabolites of TNT transformation via alternative path-
ways is very important from the viewpoint of evaluating the effectiveness of TNT-
contaminated site remediation. Improvement in methods for detecting TNT
transformation intermediates certainly contributes to our understanding of trans-
formation mechanisms and thus helps in better controlling TNT degradation. The
use of improved HPLC–MS methods for separation of TNT transformation
products helped in the identification of carbon containing metabolites produced via
nitro group reduction as well as via aromatic ring reduction in a single HPLC run,
therefore minimizing the potential for changes in sample composition after sam-
pling (Ziganshin et al. 2007, 2010a, b). This had not been possible in most of the
previous works investigating the aromatic ring reduction of TNT by
Pathways of 2,4,6-Trinitrotoluene Transformation by Aerobic Yeasts 303
microorganisms (French et al. 1998; Pak et al. 2000; Jain et al. 2004; Williams
et al. 2004; Wittich et al. 2008).
As a result, new insights into the mechanism of TNT transformation by Yarrowia
lipolytica AN-L15 and Geotrichum sp. AN-Z4 (closely related to Geotrichum
candidum) have been made which indicate interesting interactions and interplays of
biological, chemical and physical parameters affecting TNT transformation.
Aromatic ring as well as nitro group reduction products were monitored and
detected along with nitrite, nitrate and nitric oxide as transformation products. An
improved understanding and control of the transformation pathway should
enhance the effectiveness of technologies for the bioremediation of TNT-con-
taminated areas in future.
2 Pathways of TNT Degradation
The lack of a complete understanding of the exact mechanisms of TNT reduction

mediated by hydride ions and denitration of the formed metabolites prompted us to
study this process in more detail. Two newly isolated yeasts were found capable of
TNT transformation via hydride ion addition to the TNT aromatic ring (Ziganshin
et al. 2007, 2010b). We also established analytical methods which enabled us to
study TNT transformation via hydride complexes initiated by these yeasts (Borch
and Gerlach 2004; Ziganshin et al. 2007).
The two yeast strains were identified as Yarrowia lipolytica AN-L15 (Ziganshin
et al. 2007) and Geotrichum sp. AN-Z4 [closely related to Geotrichum candidum;
Ziganshin et al. (2010b)]. Direct aromatic ring reduction via hydride Meisenhei-
mer complex formation was observed as the dominant pathway of TNT trans-
formation by these yeasts along with some TNT nitro group reduction occurring
simultaneously.
The aromatic ring reduction of TNT allows the fission of the aromatic rings
which opens up the possibility of TNT mineralization. However, only a few strains
of bacteria (French et al. 1998; Vorbeck et al. 1998; Pak et al. 2000; Kim et al.
2002; van Dillewijn et al. 2008; Wittich et al. 2008) and fungi (Kim and Song
2000; Zaripov et al. 2002; Jain et al. 2004) were demonstrated to be capable of
TNT transformation via aromatic ring reduction.
Since the effectiveness of remediation technologies for TNT-contaminated areas
depends on the effective control of biological and abiotic transformations of TNT,
HPLC-based methods were developed for the separation and identification of key
intermediates formed during TNT conversion (Borch and Gerlach 2004; Borch
et al. 2005; Ziganshin et al. 2007). These methods utilize reversed phase high
performance liquid chromatography (HPLC) separation combined with diode-array
(DAD) and atmospheric pressure chemical ionization mass spectrometric
(APCI-MS) detection. In particular, varying the temperature and composition
of chromatographic eluents, the isomers, 2-hydroxylamino-4,6-dinitrotoluene
(2-HADNT) and 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT) were separated
by HPLC. It was a great challenge in previous works (e.g., Michels and Gottschalk
1994; Vorbeck et al. 1998; Hawari et al. 1999; Naumov et al. 1999).
After isolation of the two yeast strains (AN-L15 and AN-Z4), the HPLC
methods were used to reliably separate many of the TNT aromatic ring transfor-
mation products as well. Hence, these methods were applied to explore the
mechanism of TNT aromatic ring reduction by these yeast strains.
The dominant pathway of TNT conversion by the yeasts is based on the
addition of hydride ions to the aromatic ring. In this pathway, we demonstrated
that the reduction of TNT by hydride ions was associated with the formation of at
least eight different mono- and dihydride-Meisenheimer complexes (Figs. 1, 2,
Table 1) (Ziganshin et al. 2007, 2010a, b). In earlier studies, researchers had only
been able to demonstrate the formation of only five hydride forms of TNT, in
particular 3-H--TNT, 3,5-2H--TNT and three isomers of 3,5-2H--TNT.H+
(Vorbeck et al. 1998; Pak et al. 2000; Williams et al. 2004).
Our work detected eight metabolites which were characterized by us as TNT-
hydride complexes based on (1) their UV–visible absorbance spectra, which show
strong absorbance maxima between 440 and 500 nm, (2) their mass spectra
(Table 1), and (3) observed biotic and abiotic conversion reactions (Ziganshin
et al. 2007).
Molecular ions and mass fragments, observed during negative mode APCI-MS
mass spectrometry of 3-H--TNT, were similar to those obtained in earlier work
(Yinon et al. 1995) with dominant fragments of m/z = 197, 210, and 227. Mass
spectrometric analysis of other TNT-hydride complexes after HPLC-based puri-
fication enabled us to divide them into three groups (Table 1):
1. compounds with a molecular ion at m/z 227 (compounds 1, 5, 7, and 8),
presumably TNT-monohydride complexes;
2. a compound with a molecular ion at m/z 228 (compound 4), presumably a TNT-
dihydride complex;
3. compounds with a molecular ion at m/z 230 (compounds 2, 3, and 6), pre-
sumably protonated TNT-dihydride complexes.
The existence of three complexes with a molecular mass of 230 was also earlier
reported (Vorbeck et al. 1998; Pak et al. 2000) and characterized as 3,5-2H--
TNT.H+ isomers. Compounds 1 and 5 appear to be additional isomers of 3-H--
TNT (compound 7) and were not described before. They were characterized by us
in 2007 (Ziganshin et al. 2007). Besides, we also observed and characterized an
apparent C-1 Meisenheimer monohydride complex (1-H--TNT, compound 8),
which was produced by the yeasts to a much lesser extent than the 3-H--TNT
complexes and did not interconvert into any of the other seven detected TNT-
hydride complexes.
The HPLC-method was also utilized in our works for separating 3,5-2H--TNT.
Previously, the existence of this metabolite was only proposed by Vorbeck et al.
(1998). However, we were able to separate it from the 3,5-2H--TNT.H+ isomers
and obtain its mass and UV–visible absorbance spectra.
Fig. 1 a Growth (A600) of Y. lipolytica AN-L15 and subsequent changes in culture medium pH
(initial pH 7.0). Symbols: filled square, growth (A600) and filled triangle, pH change in the
absence of TNT; square, growth (A600) and triangle, pH change in the presence of TNT.
b Accumulation of metabolites during TNT transformation by strain AN-L15. Symbols: filled
square, TNT (lM); triangle, 3-H--TNT (lM); circle, 1-H-TNT (peak area at 476 nm); filled
circle, sum of Meisenheimer complexes related to 3-H–-TNT (compounds 1–6; peak area at
476 nm; c filled circle, 2-HADNT (lM); circle, 4-HADNT (lM); diamond, NO2- (lM); filled
diamond, NO3- (lM); square, 2,4-DNT (lM); triangle, 4-ADNT (lM). Error bars indicate the
standard deviation of triplicate experiments (Ziganshin et al. 2010a). TNT transformation by
Geotrichum sp. AN-Z4 was similar to TNT transformation by Y. lipolytica AN-L15 and is not
shown. Reprinted with permission from Ziganshin et al.(2010a)
Fig. 2 Proposed pathways of TNT transformation in the presence of Y. lipolytica AN-L15 and
Geotrichum sp. AN-Z4. Data obtained during our research did not allow structural distinction
between the different 3-H–-TNT or 3,5-2H--TNT.H+ isomers
The ability to separate and purify these TNT aromatic ring reduction products
allowed us to explore the stability and conversion of individual TNT-hydride
complexes under various physicochemical conditions. Such studies also enabled us
to estimate the relative importance of biologically and physico-chemically dom-
inated transformation reactions of these compounds.
Previously TNT was detected as the only product of the spontaneous abiotic
transformation of (chemically synthesized) 3-H-–TNT (Vorbeck et al. 1998; Pak
et al. 2000). However, our work demonstrated the possibility of 3-H-–TNT to be
abiotically converted into its isomers (compounds 1 and 5), TNT as well as 3,5-
2H--TNT (compound 4). Besides, we also showed the possibility of five more
TNT-hydride complexes (except for compound 2, a 3,5-2H--TNT.H+ isomer, and
1-H–-TNT) to be interconverted. All these abiotic reactions proceed without the
elimination of nitro groups.
Enzymatic production of 1-H--TNT from TNT was earlier proposed. French
et al. (1998) suggested the production of 1-H--TNT by pentaerythritol tetranitrate
reductase isolated from Enterobacter cloacae PB. They observed predominantly 3-
H--TNT as the product of the initial hydride-ion mediated attack on TNT. Besides,
Table 1 Metabolites formed during transformation of TNT by yeasts under various cultivation
conditions
Sl no. Compound Molecular iona RT at 36 and 50 °C, minb kmax, nmc
1 3-H--TNT isomer 227 4.8/4.7 261, 445
2 3,5-2H--TNT.H+ isomer 230 5.2/4.9 266, 426
3 3,5-2H--TNT.H+ isomer 230 5.3/5.0 263, 478
4 3,5-2H--TNT 228 5.7/5.4 325, 512
5 3-H-–TNT isomer 227 5.9/5.5 262, 465
6 3,5-2H--TNT.H+ isomer 230 6.7/6.2 263, 491
7 3-H--TNT isomer 227 9.9/8.5 256, 480, 550
8 1-H--TNT 227 12.3/10.3 251, 478, 551
9 2-HADNT 212 14.8/12.4 228, 265, 356
10 TNT 227 15.4/13.3 230
11 4-HADNT 212 16.0/13.3 232, 350
12 2,4-DNT 181 17.7/15.1 250
13 2-ADNT 196 17.0/14.1 225, 270, 375
14 4-ADNT 196 17.6/14.7 235, 362
15–16 HAMNT 167 10.9-11.1/– –
a
Molecular ion detected during negative mode APCI-MS analysis
b
HPLC retention times observed at two different separation temperatures
c
UV-visible absorbance maxima
indication of another metabolite with similarspectral characteristics was described.

This second compound was suggested to be 1-H-–TNT, but its production could
not be confirmed in the subsequent work (Williams et al. 2004). Based on the
UV-visible absorbance and mass spectra along with abiotic interconversion studies,
it was concluded that compound 8 must be 1-H--TNT (Ziganshin et al. 2007).
As previously described (Kim and Song 2000; Jain et al. 2004; Williams et al.
2004), the reduction of the aromatic ring of TNT by yeast strains also led to the
release of inorganic nitrogen containing compounds. This pathway is very
important in view of TNT biodegradation efficiency. Lesser nitrated compounds,
such as dinitrotoluenes, are more susceptible to aromatic ring fission than the more
stable TNT.
According to our findings, there are at least two different pathways of TNT
biodegradation via TNT-hydride complexes (Ziganshin et al. 2007, 2010a, b). One
of the pathways leads to the elimination of one nitro group from 3-H–-TNT with
simultaneous accumulation of nitrite and 2,4-dinitrotoluene (2,4-DNT). The
accumulation of the latter metabolite occurred only during the phase of 3-H–-TNT
disappearance (Figs. 1, 2). However, production of 2,4-DNT was dependent on the
pH of the growth medium, which changed throughout the experiment because of
the production of organic acids by the yeasts (Ziganshin et al. 2010a, b).
Yeast strain AN-L15 produced and excreted citrate and pyruvate into the cul-
ture medium, while strain AN-Z4 produced citrate, succinate and isocitrate which
caused acidification of the medium during their growth. This acidification of the
medium also initiated the oxidation of the released nitrite to nitrate.
The production of dinitrotoluenes from TNT results in a class of compounds,

which can more easily undergo aromatic ring fission due to their increased sus-
ceptibility to oxidative attack of the aromatic ring compared to TNT (Spanggord
et al. 1991).
As reflected in Fig. 1, depending on the culture conditions, a small part of 2,4-
DNT formed was also converted to hydroxylamino-mononitrotoluenes (HAMNTs)
in our experiments, indicating the possibility of nitro group reduction of DNTs by
these yeast strains (Ziganshin et al. 2010a).
The second possible pathway of TNT biodegradation by strains AN-L15 and
AN-Z4 seems to proceed via transformation of one of the isomers of 3,5-2H–-
TNT.H+ (apparently compound 2 in our scheme; Ziganshin et al. 2007). The
release of nitrite through this pathway started during the early stages of TNT
transformation, even without detectable 2,4-DNT production (Fig. 1). Continued
aerobic cultivation of the yeasts led to a decrease in medium pH and to the
oxidation of nitrite into nitrate. The nitrite to nitrate conversion occurred enzy-
matically as well as abiotically once a low enough pH was attained. Nitric oxide
(NO) production was also observed and attributed to an abiotically occurring
disproportionation reaction, which converts nitrite to nitrate and nitric oxide
(Ziganshin et al. 2010a; Khilyas et al. 2013).
Williams et al. (2004), in their experiments with pentaerythritol tetranitrate
reductase of E. cloacae PB, suggested that the source of nitrite is one of the
isomers of 3,5-2H--TNT.H+ (possibly compound 2 in our scheme). The suggestion
of Pak et al. (2000) regarding the possible role of isomers of amino-dimethyl-
tetranitrobiphenyls in nitrite release could not be confirmed by us, since these
compounds were not detected in any of our experiments. Furthermore, diaryl-
amines, which were formed through condensation of 3,5-2H–-TNT with HADNTs
according to Wittich et al. (2008), were not identified either. Instead, the pro-
duction of nitrite indicates the possibility of aromatic ring fission and therefore, the
possibility of TNT mineralization.
However, as mentioned above, TNT transformation by the yeast strains
Y. lipolytica AN-L15 and Geotrichum sp. AN-Z4 did not solely proceed via
TNT-hydride complexes, but also led to the reduction of nitro groups resulting in
the formation of HADNTs and ADNTs (Figs. 1, 2, Table 1). Retention times,
mass and UV-visible spectra of these nitro group reduction products are consistent
with those obtained during the analysis of chemical standards. However, the extent
of nitro group reduction by strains AN-L15 and AN-Z4 was less common relative
to the aromatic ring reduction pathway described above. Nevertheless, as it has
been observed previously (e.g. Hawari et al. 1998; Borch et al. 2005), nitro group
reduction preferentially (though not exclusively) occurred in the para position
leading to higher concentrations of 4-HADNT and 4-ADNT than of 2-HADNT
and 2-ADNT.
3 Conclusions
Despite several studies on the mechanisms of TNT transformation by organisms of

different evolutionary levels, the understanding of TNT biotransformation is still
far from complete. An improved understanding of the degradation pathways
depends on improved methods for the detection of intermediates formed during
transformation.
The application of a new HPLC-diode array/mass spectrometric detection
method has over the past years helped in improved separation of different peaks
which facilitated the identification of novel intermediates of TNT transformation.
The obligate aerobic yeast strains Y. lipolytica AN-L15 and Geotrichum sp. AN-
Z4 transform TNT via two principally different pathways: (1) aromatic ring
reduction as the primary transformation pathway resulting in TNT-hydride com-
plexes as intermediates and ultimately leading to the elimination of nitro groups
from the aromatic systems as well as the possible destruction of the TNT aromatic
backbone; and (2) nitro group reduction leading to the production of hydroxyla-
mino- and amino-dinitrotoluenes. Eight different TNT-hydride complexes were
identified and characterized as follows: (1) Compounds with molecular ions with
mass to charge ratios (m/z) of 227 are TNT-mono-hydride complexes (1-H--TNT
and 3-H--TNT, of which we identified three different isomers); (2) a compound
with m/z = 228 is a TNT-dihydride complex (3,5-2H–-TNT); and (3) compounds
with m/z = 230 are protonated TNT-dihydride complexes (three isomers of 3,5-
2H--TNT.H+ detected by us).
Our studies not only demonstrated the ability of these yeast strains to produce
TNT-hydride-Meisenheimer complexes, but also to facilitate the elimination of
nitro groups in the form of nitrite and nitrate.
There appear to be at least two principally different pathways of nitro group
elimination from the produced Meisenheimer complexes: one pathway is based on
the decomposition of 3-H--TNT with the formation of 2,4-DNT. Another pathway
is based on the degradation of one of the isomers of 3,5-2H--TNT.H+. It is also
possible that a third pathway exists that involves 1-H--TNT as an intermediate,
but due to the low amounts of 1-H--TNT produced by the yeasts, it has not been
possible to purify sufficient amounts of 1-H--TNT to demonstrate the possible
pathway.
Each one of these pathways results in the release of the nitro-groups from the
carbon skeleton of TNT, accompanied by the appearance of nitrite, which can be
further converted enzymatically to nitrate and abiotically to nitrate and NO.
The yeasts Y. lipolytica AN-L15 and Geotrichum sp. AN-Z4 were isolated from
oil-polluted peat bog and petrochemical wastes, respectively. Their ability to
survive under such extreme conditions, combined with their fairly unique mech-
anism of TNT degradation, makes these microorganisms promising for the bio-
remediation of soils and industrial wastes contaminated with explosives and
potentially other (nitro)aromatic compounds.
Acknowledgments This chapter is dedicated to John Neuman ( Feb 20, 2011), Laboratory
Manager at the Center for Biofilm Engineering at Montana State University from 1994 to 2008.
His indispensable help with all aspects of analytical chemistry, proper laboratory techniques,
safety and ‘life in general’ is gratefully acknowledged and will be forever remembered.
This work was supported by the Fulbright Program. Partial financial support was provided by
the US Department of Defense, Army Research Office, Grant No. DAAD19-03-C-0103 and the
Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG-02-09ER64758.
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Ziganshin AM, Gerlach R, Naumenko EA, Naumova RP (2010b) Aerobic degradation of 2,4,6-
(Russia)
In Situ Degradation and Remediation
of Energetics TNT, RDX, HMX,
and CL-20 and a Byproduct NDMA
in the Sub-Surface Environment
Jim E. Szecsody, Steve Comfort, Herb L. Fredrickson,

Robert E. Riley, Fiona Crocker, Patrick Shea, Jim P. McKinley,
Amy P. Gamerdinger, Hardiljeet K. Boparai, Don C. Girvin,
Jessa V. Moser, Karen Thompson, Tom Resch, Brooks J. DeVary,
Lisa Durkin and Andrew T. Breshears
1 Background
Energetics, such as, RDX, HMX and in some systems CL-20, show low sorption
and natural degradation. They can infiltrate through the vadose zone to ground-
water and move rapidly to the groundwater. If there are no physical pumping
limitations in an aquifer, pump and treat may be an economical option for the
remediation. However, many contaminants, after decades of contact with sub-
surface sediments, have advected/diffused into low permeability materials, hence
conventional pump and treat methods are less effective, as water is generally
pumped through only the higher permeability porous media. In these cases, an
in situ remediation may be a more viable alternative. It should be noted that
degradation of an energetic to an equally or more toxic intermediate is not a viable
remediation strategy. Degradation to non-toxic intermediates or complete degra-
dation to carbon dioxide (and nitrate/nitrite for N mass) is sought to lower the
toxicity risk. For some compounds, this may involve sequential geochemical
environments, such as an up gradient reduced zone and down gradient oxic zone.
Alternatively, if an energetic shows strong sorption to shallow sediments and
natural degradation to toxic recalcitrant intermediates (as in the case of TNT), then
J. E. Szecsody (&) R. E. Riley J. P. McKinley A. P. Gamerdinger D. C. Girvin

J. V. Moser T. Resch B. J. DeVary L. Durkin A. T. Breshears
Pacific Northwest National Laboratory, Richland, WA 99352, USA
e-mail: jim.szecsody@pnnl.gov
S. Comfort P. Shea H. K. Boparai
School of Natural Resources, University of Nebraska, Lincoln, NE 68583, USA
H. L. Fredrickson F. Crocker K. Thompson
Environmental Laboratory at Waterways Experiment Station,
Vicksburg, MS 39180-6199, USA

314 J. E. Szecsody et al.
sediment removal or in situ remediation is needed to permanently immobilize or

degrade intermediates to non-toxic compounds. Field scale bioremediation of
energetics commonly occurs through carbon addition to create a bioreduced zone.
Although many of these systems are successful, but it is to be noted that abiotic
reactions are also occurring (Heijman et al. 1995) that can make predictions of
energetic movement or degradation difficult without an understanding of these
processes. Iron reducing conditions may be created microbially or abiotically
(Tratnyek et al. 2013). Energetic sorption onto sediment is very small and even-
tually, reaches a steady state. It will temporarily reduce mass of a small ground-
water plume, but not degrade the mass (Haderlein et al. 2000). Energetic sorption
is generally reversible, however, some TNT intermediates irreversibly sorb.
Energetic sorption onto biomass, which is increases in a biostimulated zone, may
be a significant process in temporarily removing some energetic mass from the
aqueous solution. Finally, while bioreduced zones are directly biodegrading
energetics, a fraction of the microbial population may be reducing iron and
manganese oxides, which may be indirectly degrading energetics, although at a
different rate than direct bioreduction. Energetic degradation and mineralization
can also occur abiotically (Hofstetter et al. 1999; Szecsody et al. 2007) and cou-
pled abiotic/biotic (parallel or sequential) can also degrade contaminants (Morkin
et al. 2000; Wildman and Alvarez 2001; Bell et al. 2003). An understanding of the
actual processes, that are influencing energetic movement in the sub-surface and
degrading energetic mass, is needed to accurately predict a long-term clean-up
process.
1.1 Natural and Enhanced Biodegradation of Energetics
Most explosives contain multiple electron-withdrawing nitro groups on either

aromatic or heterocyclic rings (e.g. HMX, RDX, and TNT) or cages (e.g. CL-20).
CL-20 is chemically 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-Hexaazaisowurzitane
(Nielsen et al. 1989, 1994, 1998). These nitro moieties cause the explosive to resist
electrophilic attack by oxygenases under aerobic conditions and usually result in
the incomplete mineralization. Complete degradation (i.e. mineralization) is nec-
essary to achieve full risk reduction of energetic compounds in the sub-surface.
The cause for incomplete mineralization of energetics during natural biodegra-
dation is poorly understood and varies on a case to case basis. In some cases, the
limiting factors have been identified which include: (a) lack of major or minor
nutrients, (b) specific microbes required for a degradation step may not be present,
and (c) the geochemical conditions (i.e. aerobic, anaerobic, specific reducing
conditions) required for a degradation step may not occur in the system even
though microbes and nutrients are present.
Currently, the most common methods of enhanced biodegradation of energetics
are nutrient addition, sequential anaerobic–aerobic treatment, and less commonly
coupled abiotic/biotic treatment. Although many researchers have attempted to
In Situ Degradation and Remediation of Energetics 315
improve bioremediation rates by adding carbon sources to promote co-metabolism,

but many reaction intermediates still remain recalcitrant and can be more toxic to
microbes than the parent compounds. Complete degradation (i.e. mineralization) is
necessary to achieve full risk reduction of energetic compounds in the sub-surface.
Biodegradation of TNT occurs through several different metabolic pathways
(McCormick et al. 1976). Lenke et al. (1998) have examined microbial transfor-
mation of TNT and its derivatives. Although the initial steps of TNT degradation
appear to occur under both aerobic (Boopathy et al. 1994) and anaerobic condi-
tions (Funk et al. 1993), but to enhance degradation, TNT is first subjected to
anaerobic conditions, followed by aerobic conditions (Bruns-Nagel et al. 1998).
Since this type of coupled anaerobic–aerobic condition is unlikely to occur nat-
urally, TNT degradation remains incomplete resulting in the accumulation of
reaction intermediates (Comfort et al. 1995). As the mono-amino intermediates of
TNT biodegradation are more toxic than TNT itself, complete mineralization is
essential needed to lower toxicological risks.
Natural biodegradation (i.e. transformation of parent compound) can be
enhanced by addition of a carbon source, such as molasses or lactate, but this does
not necessarily increase mineralization rates. For example, augmented TNT bio-
degradation at the field scale required 182 h for half-life, but complete minerali-
zation required 620 h. As the result, when TNT is flowing through a bioreactive
zone, there may not be enough resident time for mineralization to occur. A similar
problem does occur with HMX. Its rapid degradation (55 h half-life) was
observed, but with much slower mineralization rates (466 h half-life).
RDX takes a longer mineralization time and as a consequence, complete RDX
mineralization has not been observed in the natural systems. However, enhanced
biodegradation of RDX is widely reported (McCormick et al. 1981; Binks et al.
1995; Freedman and Sutherland 1998; Ronen et al. 1998; Sheremata and Hawari
2000; Waisner et al. 2002) and is typically orders of magnitude slower than TNT
(Adam et al. 2005, Brannon et al. 1992). The general consensus is that RDX is
biodegraded cometabolically faster under anaerobic rather than aerobic conditions
(Coleman et al. 1998; Hawari 2000).
1.2 Abiotic Degradation of Energetics
Abiotic technologies, such as zero valent iron and chemically reducing natural iron
in sediment, were found very successful in initially degrading energetics (Agrawal
and Tratnyek 1996; Singh et al. 1998b; Szecsody et al. 2001, 2004b), but in some
cases, mineralization does not occur. For example, a purely abiotic mineralization
pathway does not occur for RDX (Hawari et al. 2000a), so either sequential
anaerobic/aerobic pathways or coupled abiotic/biotic processes are needed to
achieve mineralization of such explosives. In most cases, where energetics have
been tested, abiotic degradation rates have greatly exceeded the enhanced bio-
degradation rates. Abiotic reduction of energetics, such as nitroaromatic pesticides
(Tratnyek and Macalady 1989) and polyhalogenated methanes (Pecher et al. 2002)
is reported to proceed rapidly.
The abiotic reduction of TNT has not been directly compared to bioaugmen-
tation in the same natural sediment system, but abiotic rates are found as fast as
bioaugmentation rates. The use of a chemical reductant to reduce structural iron in
clays and iron oxides has resulted in rapid TNT degradation is fairly rapid
(Amonette et al. 2000). Nearly equimolar concentrations of Fe(II) and TNT were
used in that experiment. Rates of mineralization in reduced natural sediments are
not known. The abiotic reduction of RDX (hexahydro-1,3,5-trinitro-1,3,5 triazine)
by chemically-reduced natural sediments was 12 times faster than the bioaug-
mentation reduction rate (Szecsody et al. 2001). The initial reduction pathway was
the same as the biotic pathway: RDX ? MNX ? DNX ? TNX (McCormick
et al. 1981; Freedman and Sutherland 1998).
1.3 Coupled Biodegradation and Abiotic Degradation
The remediation of energetics has much to gain in taking advantage of the

apparent rapid abiotic degradation rates of energetics by differing reducing tech-
nologies (ferrous iron, zero valent iron) in combination with biomineralization of
intermediates. Where abiotic degradation rates of energetic compounds to inter-
mediates are considerably faster than biodegradation, coupled with biodegradation
of intermediates, have been proven to effectively increase the rate of mineraliza-
tion (Autenrieth et al. 1999; Hofstetter et al. 1999; Singh et al. 1999; Wildman and
Alvarez 2001).
For example, coupled abiotic/biotic degradation of RDX with a mixture of zero-
valent iron and sediment has shown a 10 times faster mineralization rate than
natural biodegradation (Singh et al. 1998b). Although the coupled biotic/abiotic
mineralization of other explosives by chemically-reduced sediment has not been
investigated, the rapid rates of initial abiotic degradation (relative to biodegrada-
tion rates) indicate that coupled processes work well with RDX. It is likely that
abiotic and biotic processes are geochemically coupled, creating a much more
efficient electron shuttling (i.e. reducing) system than an abiotic or biotic system
alone. The mechanism of this efficiency involves both coupled abiotic and biotic
redox reactions as well as nutrient enrichment.
In one coupled system, the abiotic reduction of nitroaromatic compounds by
surface-bound ferrous iron was rapid because of the presence of iron-reducing
bacteria, which re-reduced the ferric to ferrous iron i.e. parallel biotic/abiotic redox
reactions (Heijman et al. 1995). In this study, the observed rate of nitroaromatic
reduction was not actually controlled by the abiotic reduction rate, but by the
microbial regeneration of the reactive surface sites (i.e. iron reduction). In a dif-
ferent coupled system, RDX was abiotically reduced by zero valent iron and then it
was most likely mineralized biotically by the microbial colony in the natural
sediment through redox reactions in series (Singh et al. 1998b). In fact,
remediation of energetics should take an advantage of the apparent rapid abiotic

degradation rates of energetics by differing reducing technologies (ferrous iron,
zero valent iron), as well as the biomineralization of intermediate compounds.
Although it is not clear why biomineralization rates of energetics are faster for
coupled abiotic/biotic systems than purely biotic systems, but, one hypothesis is
that intermediates offer a more available nitrogen source than the parent energetic
compound. A common method of enhancing bioremediation of explosives is to
introduce a carbon source, thus forcing the microbes to utilize the nitroaromatic as
a nitrogen source.
1.4 Sub-Surface Sediment with Reducing Conditions:

Natural, Biologically, or Chemically Reduced
Reduced zones in the natural environment are invariably created by the microbes.
In contaminated aquifers, iron (or other) reducing conditions can be advantageous
for degradation of some energetics (i.e. RDX, HMX, and NDMA not TNT or CL-
20). Iron reducing conditions can be created by enhancing in situ microbial bio-
reduction (i.e. adding a carbon source), or injecting zero valent iron (either as a
wall, micron or nano-sized particulates) or chemically reducing in situ iron (III)
oxides, as described below. A comprehensive description of in situ chemical
reduction technologies has been outlined by Tratnyek et al. (2013).
A technology for in situ chemical reduction utilizes existing iron in the aquifer
sediment which is chemically treated with a reductant (sodium dithionite buffered
at high pH) by injection for a short time into the contaminated sediment (for
24–60 h), to reduce mineral Fe(III)-oxides (Chilikapati et al. 2000; Vermeul et al.
2002; Szecsody et al. 2004b, 2005a, b). The product Fe(II) may reside in reduced
phases or may be solubilized and reside as adsorbed species on mineral surfaces.
The reduction process results in the chemically reducing groundwater conditions
and the disappearance of dissolved oxygen. The chemically produced reduced iron
phases in the sediment behave similarly to zero-valent permeable iron walls for
some reactions, such as TCE dechlorination (Szecsody et al. 2004b), chromate
reduction (Fruchter et al. 2000), herbicide transformation (Boparai et al. 2006),
and energetic degradation (Boparai et al. 2010). The similarity of reduced sedi-
ment to zero-valent barriers is due to their operational equivalence. Zero-valent
barriers rely not on the oxidation of metallic Fe(0), but on the oxidation of Fe(0) to
Fe(II). Ferrous iron is the reactive compound that is oxidized to ferric iron, either
from adsorbed Fe(II) or from Fe(II) minerals, such as green rust (Genin et al.
1998), to reductively remediate chlorinated aliphatic contaminants (Balko and
Tratnyek 1998; Johnson et al. 1998) or reduction of metals, such as chromate
(Blowes et al. 1997; Buerge and Hug 1997). Aqueous Fe(II) can reduce chromate
(Eary and Rai 1988), while Fe(II), either as a structural mineral component or
adsorbed to an Fe(III)-oxide, clay surface, or zero valent iron surface, is necessary
for the dechlorination reactions (Hofstetter et al. 2003). However, the role of the
surface in this reaction is not clearly understood.
The dithionite chemical treatment dissolves and reduces amorphous and some
crystalline Fe(III) oxides. Although adsorbed Fe(II) appears to be the dominant
Fe(II) component, there may be production of other Fe(II) mineral phases
including Fe(II)-carbonate (siderite), FeS (iron sulfite), and others. Although more
than one Fe(III) phase is reduced in a natural sediment, a simple chemical model
can generally describe experimental and field observations (Szecsody et al. 2005a).
Once the sediment is reduced, subsequent oxidation of adsorbed and structural
ferrous iron in the sediments of the permeable redox barrier occurs naturally by the
inflow of dissolved oxygen through the barrier and additionally by energetics and
other electron acceptors present. In most sub-surface systems, dissolved oxygen in
water is the dominant oxidant of reduced iron species, as contaminants are gen-
erally present at lower molar concentrations relative to dissolved oxygen.
Experimental evidence indicates that the oxidation of Fe(II) in solutions (pH [ 5)
is generally found to be of first order with respect to Fe(II) and O2 concentration
and second order with respect to OH-. Although half life of the rate of oxidation
of aqueous Fe2+ by oxygen at pH 8 is only a few minutes (Eary and Rai 1988;
Buerge and Hug 1997), the oxidation rate observed in natural sediments was found
to be 0.3–1.1 h (Szecsody et al. 2004b). The total reductive capacity of the reduced
sediment can be measured by slow oxidation using air-saturated water. This redox
capacity can be related to the specific field system by knowing the average aquifer
concentrations of dissolved oxygen and other electron acceptors to estimate the
longevity of the reduced zone. It should be also noted that the rate of energetic
abiotic degradation is dependent on the reductive potential in the aquifer, and as
the system is oxidized, the rate of energetic degradation will decrease.
2 Energetic Aqueous Stability
Energetics RDX, HMX, and TNT are stable in most sub-surface aqueous envi-
ronments, as shown by aquifer concentrations of these compounds remaining
stable for years to decades with no evidence of degradation of intermediates. Since
energetics RDX, HMX (Heilmann et al. 1996), TNT and CL-20 are degraded by
alkaline hydrolysis, they are unstable at pH [ 9.5 or 10.
2.1 Methylene Dinitramine as Degradation Intermediate

of RDX and HMX Degradation
Methylene dinitramine (MDNA) as a degradation intermediate of both RDX and

HMX is reported to undergo acidic hydrolosis in water (Hawari et al. 2000a, b,
(a) MDNA initial conc. 10 mg/l

10
pH = 11.0
MDNA conc. (mg/l)

10.0
3.5
5 8.1
8.9
6.9
0
1 10 100 1000
Time (h)
(b) MDNA initial conc. 20 mg/l

1.00
(fin. conc.) (ini. conc.)
reduced sediment, pH 9.5

MDNA / MDNA
0.75
0.50 oxic sediment, pH 6.8
oxic water
pH 6.5
0.25
sediment/water = 0.16 g/ml

0.00
0.01 0.1 1 10 10 2
Time (h)
Fig. 1 Degradation of methylene dinitramine in: a aqueous solutions at different pH, and
b degradation in oxic or reduced sediment. Initial concentration in (a) was 10 mg/l and in (b) was
20 mg/l
2002; Halasz et al. 2002) in anaerobic sludge. In aquifer water, methylene di-
nitramine was found to be stable in the alkaline water (Lopez et al. 1996), but less
stable at neutral pH (Fig. 1a). Degradation of MDNA in oxic sediments (Fig. 1b)
shows the most rapid degradation in oxic water (no sediment, half-life about
0.5 h), but slower degradation in contact with oxic or anoxic sediment (with or
without continuous UV light treatment). The pH of this untreated sediment is 7.2.
It may be concluded that: (a) methylene dinitramine is (abiotically) degraded in
aqueous solution at neutral pH, (b) presence or absence of oxygen in water has no
influence on degradation and (c) presence of oxic sediment slows degradation,
possibly due to adsorption.
MDNA degradation is pH dependent in aqueous solutions, and in porous media,

is also dependent on the soil/water ratio (Boparai et al. 2010). Since MDNA
degradation is not eH driven, it is stable in iron-reduced sediments (at alkaline
pH), wherein it could potentially reach elevated concentrations. This could make
MDNA degradation an important rate-controlling step in the overall mineralization
of RDX. An experiment was conducted in which RDX degradation was occurring
in the reduced sediment, and methylene dinitramine was specifically analyzed.
After 300 h experiment, there was no measurable methylene dinitramine in this
batch experiment at a low sediment/water ratio (0.5 g/ml). In contrast, for deg-
radation in porous media at field scale, the sediment/water ratio was 5–10 g/ml,
and with significantly less water, methylene dinitramine built up in concentration,
which might be a rate-limiting step for overall RDX or HMX mineralization.
2.2 TNT and Degradation Intermediates
Experiments have shown that TNT degradation rate was also a function of pH and
was degraded more rapidly by alkaline hydrolysis at pH 11 and 12 (half-life 18 and
8 h, respectively, Fig. 2). TNT was degraded by oxic sediment at pH 10, but at pH
11 and 12, it was degraded more rapidly by alkaline hydrolysis, as degradation
rates with and without the sediment were the same.
Triaminotoluene (TAT) a degradation intermediate of TNT, was investigated
for aqueous stability as a function of pH and dissolved oxygen. Triaminotoluene is
considered unstable in the presence of dissolved oxygen. TAT undergoes rapid
acidic hydrolysis (Fig. 3a). With no pH buffer, dissolving 100 mg/l TAT in
deionized water gives a pH of 3.3, which has a degradation half-life of 6.4 h. At
pH 2.5, the TAT degradation rate is more rapid (half-life 3.8 h), but at neutral pH,
Fig. 2 TNT alkaline

hydrolysis at different pH
TNT / TNT
(a)

TAT / TAT
(b)
TAT / TAT
(c)
TAT / TAT
Fig. 3 Aqueous stability of triaminotoluene (TAT): a at different pH (oxic, no light), b anoxic or

oxic water at pH 3.3 with no light, and c influence of UV light at pH 3.3 in anaerobic water
the TAT degradation rate is very slow (half-life 88 h), but not stable. Under
alkaline conditions, TAT is somewhat more stable with a degradation half-life of
171 h at pH 8.8, and 306 h at pH 12. Due to lack of aqueous stability over a wide
pH range, TAT was most likely degraded in the TNT abiotic/biotic sediment
systems. At pH 3.3, the presence of dissolved oxygen increased the TAT degra-
dation rate to some extent.
(a) 4.0
3.0
CL-20 (mg/l)
2.0
1.0
0.0
4 5 6 7 8 9 10 11 12
pH
(b) 16
Conc. ( µ mol/l)
12 Nitrite
8
9.5
Formate
4
10
Nitrate
0
0.01 0.1 1 10 100
(h)
Fig. 4 Aqueous degradation rate of CL-20: (a) by 24 h at different pH and (b) at pH 9.5 (solid
lines) and pH 10 (dashed line) over time
2.3 CL-20
CL-20 has low aqueous solubility (Holtz et al. 1994; Monteil-Rivera et al. 2004)
and is degraded by alkaline hydrolysis in the homogeneous solution at a pH [ 8.5
(Fig. 3, Hawari et al. 2004), although degradation in sediment–water systems was
observed at all pHs examined. Some CL-20 nitroso-functional groups were
removed, as evidenced by aqueous nitrite (Fig. 4b). One partial CL-20 degradation
pathway is shown in Fig. 24.
2.4 NDMA
The aqueous stability of NDMA at a concentration of 2.3 mg/l was investigated at

pH 2–14 (Fig. 5). At low pH (pH 2.5), NDMA degraded minimally after 27 h,
while at other pH, NDMA was not degraded after 700 h. NDMA degradation rate
at pH 2 showed a 2,540 h half-life. The experiments were repeated again under
(a) 2.5
2.0
NDMA (mg/l) 1.5
2h
1.0
27h
140h
0.5
NDMA initial conc.2.30 mg/L
0.0
2 4 6 8 10 12 14
pH
(b) 1.0
NDMA / NDMA
0.8
0.6 X95,
7.0 pH 6.95
X96,
8.0 pH 8.05
0.4 X97,
9.0 pH 8.97
X98,
10.0pH 9.99
0.2
X99,
11.0pH 11
NDMA initial conc.2.50 mg/L

0.0
1 10 100 1000
(h)
Fig. 5 NDMA aqueous stability: a with pH at different times, and b in alkaline conditions over
time
only alkaline conditions, as these are the conditions of the reduced sediment
(Fig. 4b). NDMA was stable with \2 % degradation in 1,000 h.
In other solutions, NDMA was held at varying aqueous reducing conditions
(i.e. -597, -310, -230, and +100 mV) and for varying periods up to 700 h.
NDMA degradation was not observed even under the highest reducing conditions
(-600 mV fixed by 0.1 M dithionite).
3 Sorption
Sorption of energetics to various sediment components (i.e. organic matter, min-

eral surfaces) from aqueous systems involves both solvation energy and the solute/
surface interaction energy. The solvent effect includes a decrease in water structure
upon solute removal from solution (positive entropy) and an increase in hydrogen
bonding between water molecules (negative enthalpy). For hydrophobic

compounds, the solvent effect is a major driving force retaining compounds on
surfaces (i.e. not specific solute-surface interactions) and so hydrophobic com-
pounds ‘‘partition’’ to the surface. For example, the cage structure of the energetic
CL-20 has low water solubility (\5 mg/l; Holtz et al. 1994). In comparison, RDX
is a significantly smaller molecule (N-heterocyclic ring) with three nitro-groups
and has a moderate water solubility (42–55 mg/l at 25 °C, range dependent on
ionic strength). The retention of an energetic on sediments at equilibrium is
defined by the distribution coefficient, Kd:
Kd or Kp ¼ S (mol=g)=C (mol=cm3 Þ ð1Þ

where S is the concentration of CL-20 sorbed and C is the concentration of the
energetic in the equilibrium solution. Although Kd is not chemical mechanism
specific, Kd measurements are useful because it can describe the overall transport
and attenuation. Assuming a hydrophobic organic sorbs only to soil organic
matter, then this carbon referenced sorption is defined as:
Kp ¼ f oc Koc ð2Þ
where foc is the fraction carbon by weight and Koc is the partition co-efficient and a
hypothetical soil that is 100 % organic carbon. Carbon referenced sorption is
applicable to hydrophobic solutes at concentrations less than half their solubility
and deviation from this linear relationship caused multiple sorption mechanisms
and non-equilibrium behavior. For many compounds, Koc is related to the octanol–
water partition co-efficient (Kow) with a linear free energy relationship
log Koc ¼ x log Kow þ y ð3Þ
where x and y are empirical constants (0.4, 0.29 for solvents; 0.524, 0.618 for
pesticides Karickhoff et al. 1979; Karickhoff 1984). Compounds with low Kow
values partition weakly to sediments, as in the case with RDX (calculated log
Kow = 0.78, which is fairly low Field studies confirm this fact, as RDX is fairly
mobile with low Kd values of 1.0–5.0 (Singh et al. 1999). In contrast, CL-20
should partition strongly to sediment organic matter (calculated log Kow = 3.2).
Carbon-referenced sorption model applies when foc [ 0.001 ([0.1 % carbon),
below which sorption onto mineral surfaces is important. In this case, a weak
dependence on the surface area of the soil is observed:
log Kp ¼ 0:16 log Kow þ log Sa =200 ð4Þ
where Sa is the mineral surface area (25 solutes onto silica; McCormick et al.
1981).
3.1 RDX
Studies were conducted in which RDX was sorbed to a variety of sediments to

determine if sorption was correlated to natural organic matter, clay content, or iron
oxide content (Szecsody et al. 2004b, 2005a). No RDX degradation was observed
with the oxic soils. This is in agreement with the observations of Singh et al.
(1998a) for a Sharpsburg montmorillontic surface soil. RDX sorption by these
sediments showed no depend-ence on sediment organic carbon content or DCB-
extractable Fe (Fig. 6). There was, however, a fair correlation with fraction clay in
the sediment (Fig. 6b, r2 = 0.46), which is likely due to higher total sediment
surface area with a higher fraction clay content. Kd values for RDX were similar to
observations made by others, such as Kd value of 1.2 cm3/g on montmorillonite
(a) 101
Kd (cm /g)
3
0
10
-1 O N B C K W I
10
0 1 2 3 4
Fraction organic carbon
(b) 101
Kd (cm /g)
3
100
B C NW O K
10-1
0 10 20 30 40
Clay (%)
1
(c) 10
Kd (cm3/g)
0
10
O N C B K W
10-1
0 50 100 150 200
II+III
DCB-extractable Fe (umol/g)
Fig. 6 RDX sorption as a function of: a fraction organic carbon, b clay content, and c total
DCB-extractable iron content. Letters denote sediment: O Ocala, N Norborne, C China Lake,
B Burbank, K Kenoma, W Westmoreland, I Iron Springs (characterization described in Szecsody
et al. 2004a)
(Haderlein et al. 1996) and 0.97 cm3/g on Sharpsburg montmorillonite surface soil
(Singh et al. 1998b).
RDX sorption to an energetic-contaminated aquifer sediment (Ft Lewis, WA,
USA) at different concentration showed a nearly linear isotherm (not shown), so
prediction of the transport of a plume could use a model with a constant Kd. Given
the RDX sorption, with Kd = 0.26 cm3/g, the RDX retardation factor at the sed-
iment/water ratio at field scale should be 2.1, so movement of a plume (with no
degradation) would be lagged by two pore volumes due to sorption.
3.2 TNT and Intermediates
In another study, the TNT sorption to an aquifer sediment from a US army base
was characterized. The specific sediment sample used does not have energetic
contamination, but other zones in the sub-surface at this site do have energetic
contamination. The TNT sorption rate is rapid (0.17–0.28 h half-life, Fig. 7a) with
an average Kd = 0.90 ± 0.28 cm3/g for 5 experiments conducted at different soil/
water ratios. TNT sorption was reversible, as an acetonitrile extraction removed
90–100 % of the sorbed TNT (Table 1). TNT long-term interactions with even
oxic sediment showed some degradation after about 24 h (Fig. 7b).
TNT is stable in most natural aquifer waters, but is degraded by alkaline
hydrolysis at a pH [ 10 (Fig. 2). TNT was stable for 500 h at pH 10, but had a
hydrolysis degradation half-life of 20 h at pH 11 and 5 h at pH 12. The TNT
degradation rate in reduced sediment varied with sediment reduction from 1 to
800 h half-life, depending on the amount of sediment reduction. In oxic sediment,
the degradation rate was slow at pH 10 with the degradation half-life was 50 h and
was related to the sediment and not the pH (Fig. 7b). TNT degraded more rapidly
in reduced sediment as a direct function of the amount of ferrous iron present.
3.2.1 2-Amino- and 4-Aminodinitrotoluene Sorption
Sorption rate, sorption mass, and sorption reversibility experiments were con-
ducted with 2–aminodinitrotoluene and 4-aminodinitrotoluene. These compounds
are the first degradation products of TNT. The sorption rate of 2-aminodinitro-
toluene (0.22/h) and 4-aminodinitrotoluene (0.16/h) was rapid (0.1–0.2 h half-
life). The sorption mass of 2-ADNT (0.476 ± 0.22 cm3/g) and 4-ADNT
(0.393 ± 0.24 cm3/g) was similar (Fig. 8). However, solvent extractions showed
that 100 % of the sorbed 4-ADNT could be removed from the surface, but only
22–32 % of the 2-ADNT could be removed from the surface. Therefore, 2-ADNT
sorption was considered only partially reversible (Table 1). The extraction med-
ium contained 50 % methanol and 50 % water sonicated for 24 h.
(a)1.0

0.8
TNT / TNT 0.6
0.4
0.2
sorption rate: 5.8/h
first-order fit (line)
0.0
0.01 0.1 1 10
Time (h)
6
(b)
5 no sediment
4
TNT (mg/l)
3 with sediment
0
0.01 0.1 1 10 100 1000
Time (h)
Fig. 7 TNT sorption rate to oxic Ft. Lewis sediment a within hours, and b slow degradation at
pH 10 by the reduced sediment
Table 1 Sorption mass, rate, and reversibility for TNT and amino-intermediates
Compound Kd Reversible* Rate (1/h)
TNT 0.900 ± 0.28 Yes 0.24
2-ADNT 0.476 ± 0.22 Partial 0.22
4-ADNT 0.393 ± 0.24 Yes 0.16
2,4-DANT 0.301 ± 0.26 No 0.62
2.6-DANT 0.480 ± 0.16 No 0.31
TAT 1.25 ± 0.24 No 0.53
3.2.2 2,4-Diamino- and 2,6-Diaminodinitrotoluene Sorption
2,4-DANT sorption mass averaged 0.301 ± 0.257 cm3/g with a sorption rate of
0.62 h (half-life), whereas 2,6-DANT sorption mass averaged 0.480 ± 0.155 cm3/
g with a sorption rate of 0.31 h (half-life, Fig. 9). Both 2,4-DANT and 2,6-DANT
(a)
1.00
(fin. conc.) ( ini. conc.)

2-ADNT / 2-ADNT 0.75 Oxic aquifer
aqueous
sediments
0.50
0.25
0.00
0 1 2 3 4 5
Time (h)
(b)
1.00
4-ADNT / 4-ADNT
0.75 aqueous
Oxic aquifer
sediments
0.50
0.25
0.00
0 1 2 3 4 5
Time (h)
Fig. 8 Sorption rate and mass for: a 2-amino dinitrotoluene (2-ADNT), and b 4-aminodinitro-
toluene (4-ADNT) on oxic aquifer sediment
had *0 % recovery with a methanol extraction of sorbed mass, indicating that

sorption was not reversible (Table 4). A considerable amount of research has been
conducted to understand the binding of DANT compounds (Weiss et al. 2004). For
many sediments tested, covalent bonds were formed with all the DANTs. These
results are consistent with the hypothesis that the aminotoluenes irreversibly bind
to one or more surface phases on the sediment as the number of amino groups
increases (Achtnich et al. 1999).
3.2.3 Triaminotoluene Sorption
Triamino toluene (TAT) sorption to sediments was slightly greater than TNT
(Table 1), but triaminotoluene sorption was not reversible, as sediment extractions
using methanol or 100 % acetonitrile failed to remove any TAT. As described
earlier, triaminotoluene is not stable in acidic and neutral aqueous solutions, which
made the sorption rate difficult to characterize over a time scale longer than a few
hours. The TAT sorption rate appeared to be rapid (0.53/h), about the same as
TNT, amino-intermediates, and diamino-intermediates (Table 1).
(a)
2,4-DANT / 2,4-DANT
1.00

Oxic aquifer
0.75 aqueous
sediment
0.50
0.25
0.00
0 1 2 3 4 5
Time (h)
(b)
2,6-DANT / 2,6-DANT
1.00
Oxic aquifer
aqueous
sediment
0.75
0.50
0.25
0.00
0 1 2 3 4 5
Time (h)
Fig. 9 Sorption rate and mass for: a 2,4-diaminonitrotoluene (2,4-DANT), and b 2,6-diamino-
nitrotoluene (2,6-DANT) on oxic aquifer sediment
3.3 CL-20
CL-20 was sorbed to a variety of sediments in a study to determine if sorption was

correlated to natural organic matter, clay content, or iron oxide content (Szecsody
et al. 2004b, 2005a). CL-20 sorption to these sediments after 2 h is small, with Kd
values ranging from 0.22 to 3.8 cm3/g. The Kd was correlated to foc (R2 = 0.90)
(Fig. 10a, solid diamonds), but was about an order of magnitude greater than the
estimated from the CL-20 octanol–water partition coefficient, Kow, and the carbon-
reference model. To a first approximation, carbon-reference model (Karickhoff
1984) assumes that sorption of hydrophobic compounds with water solubilities
\10-3 M is dominated by the organic carbon (humin-kerogen) in sediments and
soils and that the sorption Kd varies linearly with foc in soils or sediments. That is,
Kd = Koc foc, where Koc is the carbon-referenced partition coefficient and is
constant for a wide range of soils or sediments for which foc [ 0.001. The
empirical linear regression, log Koc = 0.82 log Kow ? 0.14, derived for a wide
class of neutral organic compounds (Karickhoff 1984), was used here, along with
the measured value of Kow (log Kow = 1.92, Monteil-Rivera et al. 2004), to
estimate a value of Koc = 51.8 (Fig. 10a). Under-prediction of Kd versus foc
indicates that most ([90 %) of the CL-20 sorption on these sediments is domi-
nated by mineral surfaces and not by organic carbon. While CL-20 sorption after
2 h was not related to the total clay fraction in the sediments (Fig. 10b), measured
Kd values for individual clays varied from 0.32 cm3/g (kaolinite) to 0.85 cm3/g
(biotite) to 1.2 cm3/g (illite), indicating a preference for specific clays (Table 2).
Sorption Kds after 2 h showed a linear dependence on the mass of DCB-extract-
able Fe (R2 = 0.81, Fig. 10c). Although this data is limited, this may suggest that
DCB-extractable Fe (i.e. iron oxides) is a key CL-20 sorption phase. For 2 h
sediment experiments, no relationship was observed between Kd and extractable
FeII, or between CL-20 degradation and either total clay, DCB-extractable Fe or
extractable FeII.
For 24 h experiments, CL-20 sorption (with degradation) was not related to
sediment foc (Fig. 10a, open diamonds), total clay fraction (Fig. 10b), or DCB-
extractable Fe (Fig. 10c). The CL-20 abiotic degradation varied significantly
among sediments, accounting for \5 % of the mass for the Kenoma sediment (K)
(a) 101
Kd, Ka (cm g )
-1
0
10
3
-1
10 calculated CL-20 Kd from Kow
O N B C K W
10-2
0 0.5 1 1.5 2
Fraction organic carbon
(b) 10
1
Kd, Ka (cm g )
-1
3
0
10
-1 B C NW O K
10
0 10 20 30 40
Clay (%)
(c) 101
Kd, Ka (cm g )
-1
3
0
10
O N C B K W
-1
10
0 50 100 150 200
II+III
DCB-extractable Fe (umol/g)
Fig. 10 Correlation of CL-20 and RDX sorption (Kd, +) and CL-20 sorption and degradation
(Ka) on natural sediments with: a fraction organic matter, b clay content, and c DCB-extractable
iron content. Letters denote sediment: O Ocala, N Norborne, C China Lake, B Burbank,
K Kenoma, W Westmoreland (characterization described in Szecsody et al. 2004a)
Table 2 CL-20 sorption to minerals

Mineral CL-20 apparent Ka (cm3 g-1) Deg. Extent* FeIItot
(lmol g-1)
Oxides
Silica (accusand) 0.013 S 0
Sand (#70, 1 % biotic) 0.65 M 182
Goethite coated sand 0.022 N 0
Ferrihydrite coated sand 0.002 S 0
Magnetite 1.6 L 22,000
a-Fe(OH)3 0.52 S 0
Al2O3 0.62 S 0
MnO2 1.5 L 0
Albite 0.07 S 0
Phyllosilicates
1:1 Kaolinite 0.04 S 0
2:1 Vermiculite 0.8 S 0
2:1 Smectite clay group
Hectorite [10 L 0.2
Montmorillonite 15 L 2300
Nontronite [10 L 141,000
2:1 Mica group
Illite 6.9 L 19,000
Muscovite 0.43 M 0
Biotite 1.5 L 16,700
Chlorite 1.8 L 24,400
*CL-20 24 h loss: L ([50 %), M(10–50 %), S(\10 %), N (none)
*Calculated from structural substitution for entire volume 0.5 M HCl extraction
after 24 h and for 100 % of the mass for the Ocala sediment (O) after 2 h.
Although these results show significant degradation with some sediments, the
general properties of total clay or iron content are insufficient for the prediction of
abiotic degradation.
4 Abiotic, Biotic and Coupled Degradation in Sediments
4.1 RDX
In aquifer sediments, initial transformation of RDX to MNX to DNX to TNX and

to methylene dinitramine appears to be abiotic steps that occur fairly rapidly. To
determine whether the initial RDX transformation steps are abiotic, parallel
experiments of RDX degradation in reduced sediment without and with bacteri-
cides were compared. These initial intermediates are similar to biodegradation of
RDX (Bhushan et al. 2003b; Binks et al. 1995). The bactericide should not
influence a purely abiotic reaction, as bactericides neither oxidize the reduced

sediment nor degrade/desorb the energetic compounds. A comparison of two RDX
degradation experiments with dithionite-reduced sediment and four different
bactericides showed that: (a) gluteraldehyde and sodium 2-bromoethanesulfonate
had no influence on the degradation rate, (b) HgCl2 slowed and stopped RDX
degradation, and (c) ammonium molybdate appeared to increase the RDX deg-
radation rate (Fig. 11a). It was noted that HgCl2 oxidized the sediment (black
sediment turned grey). In a second series of experiments, gluteraldehyde addition
to reduced sediment had no influence on RDX, MNX, DNX, and TNX transfor-
mation rates (Fig. 11b). Therefore, it appears that these first four steps are abiotic
(Fig. 12).
A comparison of RDX mineralization rates in the aquifer sediments with no
treatment, biostimulation only (7 different carbon additions), chemical reduction,
and chemical reduction and biostimulation dithionite-reduced sediment showed
that RDX mineralization rate and extent was increased mainly by the abiotic
chemical reduction (or zero valent iron addition) and with some additional
(a) 50
40
Conc. ( µ mol/l)
30
reduced sediment with:
red sed*
groundwater
20 100Hg*
mg/L HgCl2
100glut*
mM gluteraldehyde
red sed
groundwater
10 molyammonium molybdate
2 mM
sulf Na 2-bromoethanesulfonate
6 mM
0
0.01 0.1 1 10 100
Time (h)
(b) 100 reduced sed. reduced sed. + gluteraldehyde

RDX RDX
MNX MNX
Conc. ( µ mol/l)
DNX DNX
TNX TNX
RDX
TNX
10
DNX
MNX
0.01 0.1 1 10 10
Time (h)
Fig. 11 Effect of bactericides on the coupled RDX transformation in reduced sediment: a RDX
degradation rate only with addition of differing bactericides (sed/water = 0.02 g/ml), and b RDX,
MNX, and DNX transformation rate with addition of gluteraldehyde (sed/water = 0.2 g/ml).
Bactericide addition kills significant portion of the sediment microbial population, so degradation
in system with bactericide addition denotes the reaction is abiotic
Fig. 12 RDX degradation

pathway in dithionite-reduced
sediment with or without
biostimulation [adapted from
McCormick et al. (1981),
Hawari (2000) with
intermediates between
hydroxylmethylnitramine and
formaldehyde left out]
improvement with nutrient/carbon addition (Fig. 13b). HMX is mineralized more

rapidly than RDX in the reduced sediment. Given that HMX and RDX degradation
steps are similar (nitroso-groups first attacked), it is not surprising that these
sequential abiotic/biotic reactions in the reduced sediment also show similar
results.
RDX mineralization increases significantly with the dithionite treatment
(Fig. 13a). This indicates that sub-surface sediment remediation by in situ
chemical reduction of sediment has to be highly effective. The influence of
dithionite treatment in promoting RDX mineralization was far greater than bi-
ostimulation alone (i.e. either a carbon source or trace nutrients added, or with pre-
stimulation). More specifically, RDX mineralization with untreated sediment had a
31,000 h half-life, whereas anoxic biostimulation with lactate addition had a half-
life of 9,900 h, and biostimulation with trace nutrient addition had a half-life of
14,400 h, and anoxic pre-stimulation with trace nutrient and carbon source addi-
tion (5,600 h half-life). All oxic biostimulation studies showed slower RDX
Fig. 13 RDX mineralization (a) 1.0

rate in: a varying amount of
Fraction mineralized
ferrous iron in sediment by 0.8 2*di/Fe = 28
chemical reduction (moles of
reductant, sodium dithionite 26
0.6
added/moles of reducible iron
in sediment is 2*di/Fe), 3.0
b amount of extractable 0.4

ferrous iron versus chemical
reduction, and c RDX 0.2 0.00
mineralization with chemical 0.08
reduction and biostimulation. 0.0
Reductive capacity in (b) is 1 10 100 1000
calculated from oxygen Time (h)
consumption by sediment
over a 3-week period (b) 120 0.5M HCl extr., column
Column
0.5M
BatchHCl extr., batch
100 0.5M HCl extr.,
Untreated Sed.untreated
Freductive capacity
Fe (µmol/g)
80
60
II
40
20
0
0.01 0.1 1 10 100
II
2*Dith./reducible Fe (mol e-/mol e-)
(c) 1.0
0.8
Fe reduction + trace nutrients
Fe reduction + glucose
0.6
Fe reduction
(by dithionite)
0.4
0.2
glucose
none
0.0
10 100 1000
Time (h)
mineralization rates compared to anoxic systems. In contrast, RDX mineralization

with dithionite treatment (315 h half-life) or dithionite treatment with trace
nutrients (112 h half-life) was 50–300 times more rapid than without any treat-
ment. Addition of 5-micron zero valent iron (0.04–0.4 %—same weight percent-
age as ferrous iron in dithionite-reduced sediment) to sediment achieved nearly the
same RDX mineralization rates (373–540 h half-life) as dithionite-treated
sediment.
A few steps of degradation pathway for RDX transformation to carbon dioxide

in reduced sediment was determined experimentally. The first transformation steps
(RDX ? MNX ? DNX ? TNX ? methylene dinitramine) were determined to
be abiotic, as the addition of a bactericide to the reduced sediment did not slow the
transformation rates. The abiotic transformation steps are rapid (5 min to 4.5 h
half-life, Fig. 14), hence not rate limiting (average half-life 32 min). The average
DNX to TNX degradation rate was 7.28 ± 11.0 9 10-6 mol/g/day (average half-
life 1.8 h). Finally, the average TNX to MDNA degradation rate was
4.48 ± 6.32 9 10-6 mol/g/day (average half-life 4.5 h). These rates were quan-
tified by a sequential reaction fit.
Methylene dinitramine (a toxic intermediate, Buckley et al. 1985) transfor-
mation in reduced sediments was most rapid by acid hydrolysis (i.e. aqueous
degradation reaction) with an average transformation rate of 8.91 ± 2.42 9 10-6
mol/g/day (average half-life 55 h). Although the MDNA concentration does not
build up in the batch experiments of RDX degradation in dithionite-reduced
sediments, it was measured in some 1-D column experiments at low to moderate
concentrations. This observation is consistent with aqueous MDNA degradation,
as the *100 times lower soil/water ratio in batch experiments leads to greater
MDNA degradation. As the range of observed MDNA degradation rates in this
study only varied between 0.5 and 250 h (half-life), it is not the rate-limiting step
in RDX mineralization, where the half-life decreases from 31,000 to 315 h upon
increasing sediment reduction. However, the slowest MDNA degradation rates
were observed in the column studies (high soil/water ratios). Therefore, it is likely
that at the field scale, MDNA degradation could be slowing the overall RDX
mineralization rate. This can be confirmed by any MDNA build-up over a time
period.
Fig. 14 Transformation
rates of RDX and 10 4 formate -> CO2
intermediates in reduced
sediments as a function of the
10 3
ratio of ferrous iron to overall rate:
untreated
reactant (rates ±20 %). Rates RDX -> CO2
Degradation half-life (h)
2
sediment
are calculated based on the 10
TNX -> MDNA
appearance of the next
MDNA ->
degradation product. The 10 1
overall RDX to CO2 rate is
calculated on the rate of CO2 DNX -> TNX
10 0
appearance
MNX -> DNX
10 -1
RDX -> MNX

10 -2
10 -3
0.1 1 10 100 1000
Fe(II)/RDX ratio (mol/mol)
The final RDX mineralization step is the transformation of formate to carbon

dioxide. This reaction can occur biotically, but in iron-reduced (dithionite-treated)
sediments, experiments demonstrated that this was a coupled abiotic/biotic reac-
tion. The importance of the biotic component was determined by introducing a
bactericide, which stopped mineralization, showing the importance in the presence
of microbes in the formate mineralization (Fig. 15a). Alternatively, the strong
abiotic influence was illustrated by addition of ferrous to the system (by dithionite
treatment), which increased the rate of formate mineralization (270x; Fig. 15b).
Formate mineralization was quite slow in the untreated sediment (7,400 h half-
life) with similarity to RDX mineralization (31,000 h half-life), and rapid in
reduced sediment (60 h half-life) again similar to RDX mineralization (315 h half-
life). It is likely that this reaction is the rate-limiting step in RDX mineralization in
dithionite-reduced sediments. Therefore, the apparent strong abiotic control of
RDX mineralization (i.e. 270 time increase in rate in direct proportion to the
amount of dithionite treatment to produce ferrous iron surface phases, Fig. 14) is
not actually abiotic control, but increases the rate of coupled formate minerali-
zation reaction (which also requires microbes). Abiotic transformation of RDX by
adsorbed ferrous iron on magnetite was also earlier observed (Gregory et al. 2004).
4.2 HMX
The initial degradation reaction of HMX to mononitroso HMX is an abiotic reac-

tion, as the addition of a bactericide did not slow HMX degradation in the reduced
sediment (Fig. 16a). The HMX degradation pathway in reduced sediment is very
similar to RDX with initial attack of the nitroso- groups and then ring cleavage
forming methylene dinitramine (Fig. 17). The first five degradation products were
identified by LC–MS (Dr. Steve Comfort, University of Nebraska, Lincoln) as
mono-, di-, tri, and tetra-nitroso HMX and methylene dinitramine. In addition,
greater sediment reduction (more ferrous iron) increased the rate of the initial
reaction (Fig. 16b). Highly reduced sediment resulted in HMX degradation with
half life of 2.1 h in 1-D columns compared to a half life of 200 h for sediment with a
small amount of ferrous iron present. The activation energy for initial HMX deg-
radation reaction by dithionite-reduced sediment was calculated to be 37.5 kJ/mol,
based on 1-D column experiments conducted at differing temperature. This reaction
is actually exothermic and hence, more rapid at colder temperatures.
The degradation of mono-, di-, tri, and tetra-nitroso HMX was not investigated
further to determine if these degradation reactions were abiotic or biotic. As
reflected in Fig. 1a, the fifth degradation product i.e. methylene dinitramine, is
degraded abiotically by acidic hydrolysis.
HMX mineralization in the reduced sediments was predominantly a function of
the amount of sediment reduction (Fig. 18) with a smaller function of biostimu-
lation (trace nutrient or carbon addition, Fig. 17b), as in the case of RDX. In
untreated sediment, the HMX mineralization rate was very slow (half-life
Fig. 15 Formate (a) 1.0

mineralization: a with and
without bactericides or
Fraction mineralization
0.8
chemical reduction or
nutrient addition, and b in Red. Sed.; 2*di/Fe = 28
w/o bacteriacides
sediments with differing 0.6
sediment iron reduction
untr. sed.
0.4 + trace nutrients
+ glucose
0.2 Untr. Sediment

w or w/o bacteriacides
+ Red. Sed. w/bacteriacides
0.0
0.1 1 10 100 1000
Time (h)
(b) 1.0
0.8
Fraction mineralization
2*di/Fe max% half-life Fe/for

28 57.3 59.5 45
3 19.3 239 21
1.6 10.4 545 10 2*di/Fe = 28
0.6 0.08 3.7 2500 2.1
0.00 1.3 7400
w/bact. 0.33 3.8E5
0.4
3.0
0.2
1.6
0.08
0.0 0
0.1 1 10 100 1000
Time (h)
7,800 h), whereas, in dithionite-reduced sediments, HMX mineralization was 48

times more rapid (162 h half-life). Mineralization in reduced sediments was high
as much as 66.4 %. HMX mineralization did not occur, if a bactericide was added
because coupled formate mineralization requires viable microbes (Fig. 15).
The average HMX transformation rate is 1.6 9 10-6 mol/g/day (average half-
life 48 min) at 22 °C in packed porous media. This rate is still two to three orders
of magnitude more rapid than the overall HMX mineralization rate, so it is not rate
limiting. A few HMX intermediates were investigated as in the case of degradation
of methylene dinitramine (half-life 8 h in reduced sediment) which indicates that it
is also not the rate limiting step. The coupled mineralization of formate, which is
very slow in the untreated sediment (7,400 h half-life) and rapid in reduced sed-
iment (60 h half-life), could be the rate-limiting step for HMX mineralization,
where the mineralization half-life changes from 7,800 h in untreated sediment to
162 h in the reduced sediment.
(a) 6
red. sed. +
gluteraldehyde
4
HMX (mg/l) red. sed.
soil/water = 0.1
2 5.0 mg/L HMX
deg rate = 0.088/hr
first-order fit (solid line)
0
0.01 0.1 1 10 100
Time (h)
(b) 6
HMX (mg/l)
4
soil/water = 0.4 0.2 0.1
0
0.01 0.1 1 10 100
Time (h)
Fig. 16 HMX degradation rate: a with and without bactericide, and b at different sediment/water
ratios. Solid line in (a) is a first-order simulation fit to the reduced sediment data
4.3 TNT
Our experiments have shown that trinitrotoluene degradation increased with an

increasing amount of ferrous iron in the sediment, indicating that there may be an
abiotic component for the initial TNT degradation (Fig. 19). Although the TNT
degradation rate was very rapid, mineralization of TNT was very slow and limited
in the extent (untreated sediment 1.3 % CO2 or dithionite-reduced sediments
2.7 % CO2 by 1,400 h). Thus, TNT mineralization rates were about the same in
both untreated and dithionite treated sediments with mineralization half-lives of
28,000–55,000 h. It was inferred that the most rapid TNT mineralization rate may
occur by initial degradation in a reducing environment, followed by oxic bio-
degradation of intermediates. Batch experiments conducted with these sequential
treatments yielded no insignificant increase in the TNT mineralization rate.
Co-metabolic degradation of TNT was investigated which included glucose to
simulate metabolic pathways that can also degrade TNT (Boopathy et al. 1994).
TNT co-metabolic degradation was conducted in the aquifer sediment in both oxic
and anoxic conditions and iron reducing conditions in the presence of additional O2
to evaluate O2 impact on TNT and its intermediate degradation rate. Trinitrotoluene
was degraded to triaminotoluene (TAT) and possibly further by a co-metabolic
process at a moderate rate with glucose addition (primary treatment) and sediment
reduction (secondary treatment) (Fig. 20). Degradation of TNT to triaminotoluene
in untreated sediment (half-life of 55,000 h) was found 7 times more rapid in
Fig. 17 HMX degradation

pathway in reduced
sediments [adapted from
Hawari (2000) with
intermediates between
hydroxylmethylnitramine and
formaldehyde left out]
glucose-amended sediment (half-life 8,050 h, Fig. 21a), and an additional 13 times

more rapid in dithionite-reduced sediment that amended with glucose (half-life
610 h, Fig. 21c). Interestingly, TNT and intermediate degradation rates were the
slowest in the anoxic sediment (Fig. 21b). The final product (triaminotoluene) is
Fig. 18 HMX (a) 1.00

mineralization rate in reduced
sediments: a as a function of
the amount of reduction and dithionite/
0.75 ferrous iron = 28
b with nutrients
0.50
1.6
0.25
0.08
untreated
0.00
1 10 100 1000
Time (h)
(b) 0.40
both: dithionite/
ferrous iron = 1.6
0.30
with trace nutrients
and glucose
0.20
no additions
0.10
0.00
1 10 100 1000
Time (h)
difficult to be measured due to irreversible sorption and rapid aqueous degradation.

This co-metabolic process was earlier reported by various workers (Daun et al.
1998; Achtnich et al. 1999; Elovitz and Weber 1999; Weiss et al. 2004) for the
treatment of surface soils which contained bacteria, daphnids, algae, cress plants,
and earthworms, but not reported in the sub-surface sediment containing only
bacteria. This amino-degradation pathway is a viable sub-surface remediation
technology as it produces dinitroaminotoluene and triaminotoluene products that
irreversibly sorb. Weiss et al. (2004) demonstrated that DANT compounds formed
covalent bonds with sediment components.
Studies conducted in our group on increase in TNT/glucose co-metabolic
degradation in reduced sediments is probably caused by the more rapid abiotic
degradation of TNT intermediates (2- and 4-ADNT and 2,4, and 2,6-DANT) while
TNT degradation is most rapidly degraded biotically. Both 2-ADNT and 4-ADNT
degradation in the reduced sediment is a function of the amount of sediment
30
soil/water (g/ml)
(g/mL)==
TNT (mg/l)
20 0.008
0.02
10
TNT initial
conc. 25 mg/L 0.067
0
0.1 1 10 100
Time (h)
Fig. 19 TNT degradation in reduced sediment at differing soil/water ratio
Fig. 20 TNT cometabolic

reduction pathway in the
presence of glucose
fermentation [adapted from
Daun et al. (1998)]
reduction. With highly reduced sediment, the degradation half-life for 2-ADNT
was found to be 1.3–2.0 h for 4-ADNT. In partially reduced sediment, the deg-
radation half-life for 2-ADNT was 110 h while for 4-ADNT, it was 100 h. Both
2,4-DANT and 2,6-DANT are degraded in reduced sediment more rapidly with the
amount of sediment reduction (available ferrous iron). In highly reduced sediment,
2,4-DANT degradation half-life was 3.0 h and for 2,6-DANT degradation, it was
1.5 h. However, partially reduced sediment, half-life was 100 h, while for 2,4-
DANT degradation and for 2,6-DNT degradation, it was 65 h. There is no report
for degradation of 2,4-DANT or 2,6-DANT in the unreduced sediments.
Fig. 21 TNT/glucose co- (a)1.0

metabolic degradation in
Mass fraction (mol/mol TNT)

a oxic, b anoxic, and 0.8
TNT (aq)
TNT. (sorb) reversible
c abiotically reduced ADNT (aq) sorption
sediment ADNT (sorb)
measured sorption
0.6 DANT (aq)
DANT (sorb) irreversible
TAT. (aq) sorption
0.4 TAT. (sorb)
0.2
0.0
1 24 48 96 200 300 400 600 1000 1600 2300 2400
Time (h)
(b) 1.0
0.8
0.6
0.4
0.2
0.0
1 24 48 96 200 300 400 600 1000 1600 2300 2400
Time (h)
(c) 1.0
0.8
0.6
0.4
0.2
0.0
1 24 48 96 200 300 400 600 1000 1600 2300 2400
Time (h)
TNT mineralization, in parallel co-metabolic TNT/glucose experiments, was

also quantified in aerobic, anaerobic, and reducing systems. Reduced sediment
systems included partially reduced sediment (treated with dithionite/iron ratios of
1.5, and 26), and the addition of trace nutrients (Table 4). Since TAT is formed in
these systems which irreversibly binds, it is assured that there will be little TNT
Fig. 22 TNT/glucose (a) 10

-2
cometabolic mineralization in
X51, oxic
oxic
sequentially reduced, then X52, anoxic
anoxic
oxic sediments: a oxic, X53, red
reduced, 1.6 1.5
anaerobic, and reduced X54, red
reduced, 36 36
X55 red36+tr
reduced,36,tr nut.
sediments, and b subsequent X55anonosed.
control, sed
oxidation after 1,600 h 10
-3
TNT initial
conc 10 mg/l
mg/L
-4
10
10 100 1000
Time (h)
0
10
(b) oxic
51 aq.,51a
oxic 62%
anoxic
52 aq.,52a
anoxic 75%
reduced,
53 1.6 aq.,53a
red., 73%
aqueous
-1
reduced,
54 36 aq.,54a
red., 29%
10 reduced,36,tr
55 nut. 55a
aq., red., 46%
55a no sed.
control, aq.,55aa
no sed. 99%
gasWC trap, red.
-2
10
CO2 trap
-3
10
gas phase
carbon trap
-4
10
10 100 1000
Time (h)
mineralization. Experimental results in oxic, anaerobic, and reducing systems

showed that there was less than 1 % mineralization of TNT in 1,600 h (Fig. 22a).
Thus, there was no significant difference in TNT mineralization in these systems.
The subsequent oxidation of all these experimental systems should lead to
greater mineralization for the reduced systems. It was assumed that processes that
occurred in sequential anaerobic/aerobic sludge (Bruns-Nagel et al. 1998; Acht-
nich et al. 1999; Elovitz and Webber 1999) also occurred in the sub-surface
sediment. Oxidation of anaerobic and reduced experimental systems did not, in
fact, show any additional mineralization in 1,000 h of oxidation after the initial
1,600 h of anaerobic or reducing conditions (Fig. 22b). The mineralization extent
in the first 1,600 h of anaerobic or reducing conditions and in the subsequent
1,000 h of oxidation was \1 %, indicating that rates were extremely slow
(\3 9 10-11 mol/g/day).
4.4 CL-20
Oxic batch experiments were conducted for 24 h with aqueous CL-20 and separate
oxides and phyllosilicates to determine which phases could either react with CL-20
or catalyze its reaction with other species (Szecsody et al. 2004a). In general, the
2:1 clays, the ferrous-iron containing minerals (e.g. magnetite and biotite) and
MnO2 showed the greatest amount of abiotic CL-20 degradation (indicated by ‘L’
in Table 2). However, other minerals caused less degradation [e.g. silica, a-
Fe(OH)3]. The amount of CL-20 abiotic degradation observed for the clays varies
significantly (Table 2), with only minor (S) degradation for kaolinite and nearly
complete (L) degradation for montmorillonite (Table 2), at the solid/solution ratio
of 0.5 g cm-3. Degradation was the greatest for the smectite group of clays, with
total degradation of CL-20 occurring for hectorite and nontronite. The 2 h sorp-
tion-degradation experiments (Table 2) were in good agreement with 24 h results
(Fig. 10).
The moles of CL-20 lost from solution in contact with sediment and mineral
phases for 24 h showed a distinct increase with the mass of solid present (Fig. 23).
This is most pronounced for the 2:1 phyllosilicates (montmorillonite, hectorite,
biotite, illite), MnO2, and magnetite. Kaolinite and nontronite showed the smallest
loss and the slowest CL-20 degradation (Table 2). This suggests that the degra-
dation capacity under oxic conditions is limited and increases with the quantity of
sediment or mineral phase in 24 h batch experiments. Therefore, while abiotic CL-
20 degradation is not related to the total clay content of sediments, but to the mass
of specific clays (Fig. 23a).
Our studies also investigated the role that reactive ferrous iron may have in
contributing to the degradation capacity, was examined. For oxic conditions, no
relationship was observed between CL-20 degradation and 0.5 M HCl-extractable
ferrous iron content of sediments (Fig. 23b, open squares) or discrete mineral
phases (Fig. 23b, crosses). However, when the Norborne sediment was chemically
reduced with sodium dithionite, CL-20 loss was clearly dependent upon the mass
of sediment and thus the mass of extractable FeII present (Fig. 23, solid squares).
This chemical reduction technique (Szecsody et al. 2000, 2004b; Vermeul et al.
2006) dissolves and reduces iron oxides, and also some of the structural ferric iron
in clays. Other explosives (RDX, TNT) are rapidly reduced abiotically with iron-
reducing surfaces (Klausen et al. 1995; Hofstetter et al. 1999; Szecsody et al.
2001). Considering the oxic minerals and sediments and the reduced Norborne
sediments together (Fig. 23b), CL-20 degradation begins to show a strong expo-
nential dependence on FeII (R2 = 0.98) as the molar ratio of FeII to CL-20 exceeds
1:100. This apparent threshold may simply reflect a decrease in Eh that accom-
modates the persistence of higher concentrations of reduced iron in the reduced
sediment.
While degradation is greatly enhanced under reducing conditions, the main
reactive phases promoting CL-20 abiotic degradation under oxic conditions were
specific 2:1 phyllosilicates. Significant degradation occurred in the presence of 2:1
(a) 0.012 biotite

hectorite
0.010
CL-20 loss ( µ mol) biotite
mag CL Mn-oxide
hectorite
bio, CL
0.008 illite
mont CL
montmorillonite
non CL
Mn-oxide
ill CL
0.006 chlorite
Chl CL
musc CL
magnetite magnetite
kao CL
vermiculite
0.004 verm CL
muscovite
hec CL
nontronite
0.002 Mn CL
kaolinite
0.000
0.001 0.01 0.1 1
Mineral mass (g)
(b) 10
2
1
10 reduced
sediments
CL-20 loss ( µ mol)
0
10
-1 oxic sediments
10
-2
10
-3 oxic minerals
10
-4
10
0.01 0.1 1 10 100 1000 10 4
Ferrous iron (µ mol)
Fig. 23 CL-20 degradation after 24 h as a function of: a mineral mass and b mass of 0.5 M HCl-
extractable ferrous iron. Data points represent several solid to solution ratios for individual solids
clays and micas (Table 2), even though no apparent dependence on FeII was
observed (Fig. 23b, crosses). In addition, some oxic sediments promoted signifi-
cant CL-20 degradation within 2 h (Cloudland and Westmoreland sediments,
Table 3). Slow, but steady degradation occurred for some oxic minerals that
contained no ferrous iron or clay (albite, goethite, aluminum oxide, ferrihydrite-
coated sand, silica sand). Acid-cleaned silica sand showed some CL-20 degrada-
tion within 24 h which was consistent with slow CL-20 degradation with glass
(Fig. 2). However, CL-20 was rapidly degraded by hectorite, which contained only
trace amounts of ferrous iron, This suggests that other reactive constituents may
promote CL-20 degradation. Clearly, FeII is not the only reactive species pro-
moting abiotic CL-20 degradation.
Intermediates and end products were first identified by Hawari et al. (2004) that
include compounds 3 and 5 (Fig. 24), glyoxal, nitrate, nitrite, N2O, NH3, and
formate. The current proposed CL-20 degradation pathway for hydrolysis and zero
valent iron involves removal of two NO2 groups before the roof C–C bond breaks
Table 3 CL-20 sorption mass and degradation rates in 1-D columns (Szecsody et al. 2004a, b)
Sediment Exp. Residence time Kd CL-20 degradation
(h) (cm3 g-1)
Mass loss Half-life Rate
(%) (h) (mol h-1 g-1)
Norborne C PD 0.016 0.48 0.32 3.6 5.5E-07
PC 0.11 0.44 2.3 3.2 6.3E-07
PG 0.35 0.54 3.6 6.6 3.1E-07
PB 0.55 0.72 7.4 4.9 4.1E-07
PA 1.8 1.01 17.2 6.7 3.0E-07
PK 3.9 0.26 11.3 22.4 9.0E-08
PP 42 0.19 22.1 118 1.7E-08
Westmoreland PH 0.36 3.21 9.0 1.2 1.7E-06
PL 4.0 3.26 35.3 6.4 3.2E-07
PQ 34 – 93.2 8.8 2.3E-07
Burbank PI 0.38 2.21 2.21 2.2 9.1E-07
PM 3.04 2.14 20.1 9.4 2.1E-07
PR 31 1.43 54.4 27.4 7.4E-08
Ocala PS 32 – 100 – –
China Lake PV 2.0 0.32 0.25 550 3.7E-09
PW 2.5 0.16 0.38 450 4.5E-09
PX 1.8 0.27 0.16 780 2.6E-09
Ferrihydrite PF 0.13 0.47 2.5 3.6 5.6E-07
sand PE 2.0 0.58 15 8.6 2.3E-07
(Fig. 24). Carbon and nitrogen mass balance is useful in demonstrating that only
part of the pathway has been identified, and further identification is needed
(Fig. 25). The CL-20 molecule contains six carbon atoms and 12 nitrogen atoms.
Carbon mass balance of intermediates that include formate, glyoxal, and glycolic
acid (Fig. 25a) in the \100 h time frame account for \50 % of the C mass,
although mass balance is actually better than shown since high molecular weight
degradation products (Fig. 24, compounds 3, 5, 6, 7) are not quantified. Bio-
transformation of CL-20 occurs in a variety of aerobic and reducing systems
(Bhushan et al. 2003a; Trott et al. 2003).
Beyond 100 h, the carbon mass balance increases to a high of 79 % (by
1,600 h), as the main product is carbon dioxide. All of these mineralization
experiments involve microbes. The remaining carbon mass may be incorporated
into the microbial biomass or form other compounds, such as methane. It is clear
that abiotic processes rapidly degrade CL-20 by 10 to 100 times faster than biotic
processes and result in only minor (\30 %) of the CL-20 carbon mass becoming
smaller molecular weight C products. Microbial degradation of initial CL-20 or
intermediates is apparently necessary to break some of the bonds.
Nitrogen mass balance is slightly better than carbon, with as high as 80 % mass
balance at [100 h for the reduced sediment and hydrolysis (Fig. 25). With carbon
mass balance, low N balance at early times (\100 h) does not include high
molecular weight degradation products, which contain considerable N mass. Low
Fig. 24 CL-20 degradation

pathway in highly acidic
conditions [adapted Nedelko
et al. (2000)]
molecular weight N products include nitrate, nitrite and nitrous oxide. Further
work, using a combination of N-15 labeled CL-20 and ring only N-15 labeled
CL-20 molecules, could help distinguish that early time scale N products may be
from nitroso functional groups, whereas late time scale N products are hypothe-
sized from the heterocyclic ring intermediates.
Another set of experiments conducted in our grouop demonstrated the impor-
tance of abiotic and biotic processes to CL-20 mineralization. In oxic sediment
with no bactericide, abiotic component CL-20 biodegradation can occur. CL-20
oxic mineralization occurred in a system with the addition of only trace nutrients
(no C or N, Fig. 26a) with a half-life of 230 h. In anoxic sediment with only C
addition (glucose), the CL-20 mineralization rate was more rapid than in oxic
sediment (Crocker et al. 2005). Its 70 % mineralization has been observed in 670 h
(Fig. 26b). This is the same pathway as with oxic sediments (i.e. N-reducing
bacteria), and the slightly more rapid rate because of removal of the oxygen
electron acceptor; nitrate is being used as the electron acceptor. The specific
proportion of glucose added was with a C/N ratio of 20/1 (considering all 12 N of
(a) 6 moles of C in CL-20

6.0
Product C/CL-20 (mol/mol) anoxic Sed.

oxic Sed.
4.0
reduced sed.
* from Monteil-Rivera et al., 2004 oxic sed.
Fe (0)*
2.0
biotrans.* oxic
sed.
pH=9.5
aq., pH=9.5 pH=8*
0.0
0.1 1 10 100 1000
Time (h)
(b) 12 moles of N in CL-20

12
* from Monteil-Rivera et al., 2004 reduced

sediment
10
Product N/CL-20 (mol/mol)
Hectorite
8
aq.,pH=8*
6 biotrans.* Fe (0)*
Oxic Sed.
4
(pH=9.5)
2 aq., pH=9.5
Oxic Sed.
0
0.1 1 10 100 1000
Time (h)
Fig. 25 CL-20 degradation products showing: a total carbon and b total nitrogen mass balance
[adapted from Szecsody et al. (2005c) with some data (*) from Monteil-Rivera et al. (2004)]
CL-20). In an attempt to stimulate sulfate reducers, an additional anoxic experi-

ment was conducted in which glucose and sulfate were added (Fig. 26b). This
actually had a slower CL-20 mineralization rate than oxic and anoxic sediment
experiments with glucose alone.
In reduced aquifer sediment, up to 48 % mineralization was observed (Fig. 26c)
after 670 h, but the initial mineralization was more rapid than oxic systems. It is
(a) 1.0
nutrients, N, C
total aq.
Mole fraction
0.8
0.6
trace
0.4 nutrients
(no N, C)
0.2
mineralized
0.0
1 10 100 1000
Time (h)
1.0
(b)
Mole fraction
B
anoxic Norborne sed.+ glucose
B Norborne sed.+glucose+SO4
anoxic
0.5
0.0
1 10 100 1000
Time (h)
(c) 1.0
Mole fraction
B
reduced Norborne (di/Fe=26) + glucose
B
reduced Norborne (di/Fe=1.6) + glucose
0.5 B
reduced Norborne (di/Fe=1.6) +
glucose + tr.nutr.+ SO4
0.0
1 10 100 1000
Time (h)
Fig. 26 CL-20 mineralization in: a oxic sediment, b anoxic sediment, and c reduced sediment
not clear if the products are the same as zero valent iron (i.e. initial nitrite, but final
nitrous oxide and ammonia). Ferrous iron sorbed on iron oxide and clay systems
appears to act as an electron donor and catalyst that causes very rapid CL-20
transformation, but it is not known, if these intermediates are more rapidly min-
eralized. These results simply indicate that initial abiotic CL-20 transformation can
promote more rapid CL-20 mineralization for some time, but microbes are rate
limiting for CL-20 and intermediate transformation reactions. The addition of
sulfate in reduced sediments marginally increased the rate of CL-20
mineralization.
NDMA
H3 C CH 3 DMA
N abiotic
H3 C CH 3 + NO (ZVI, magnetite)
Fe-reducing N
N
+ N O (alkaline
2 conditions)
O
UDMH + NH
4
H3 C CH3 microbial?
N (reported in acidic
H3 C CH2OH conditions)
NH 2
N
formaldehyde methylamine
hydroxymethyl
N nitrosamine CH2 O + CH 3 NH 2
microbial
O
HCOOH
microbial microbial or
(rapid oxic) coupled (red.)
CO 2
Fig. 27 NDMA degradation pathways [from Odziemkowski et al. (2000), Szecsody et al.
(2008)]
4.5 NDMA
NDMA is relatively stable in aqueous solution and sorbed minimally (sorption Kd

in a NDMA-contaminated aquifer sediment is 0.12 ml/g). NDMA gets degraded
abiotically by zero valent iron or magnetite under alkaline pH conditions to DMA
or UDMH under acidic conditions (Gui et al. 2000; Odziemkowski et al. 2000). It
will be biotically degraded by a separate pathway (Kaplan and Kaplan 1985;
Bradley et al. 2005; Arienzo et al. 2006; Fournier et al. 2006, Fig. 27). NDMA is
photosensitive in strong UV light and so it can be treated ex situ (Gunnison et al.
2000). In sub-surface sediments, NDMA is rapidly abiotically degraded to DMA
as a direct function of the mass of adsorbed ferrous iron to the sediment (Fig. 28a).
This degradation reaction was abiotic (Szecsody et al. 2008), as the addition of a
bactericide (gluteraldehyde) did not change the NDMA degradation rate
(Fig. 28b). The reduced sediment contains multiple ferrous iron phases, including
adsorbed ferrous iron, ferrous oxides, carbonates, and sulfides. Dithionite reduc-
tion also results in the reduction of some structural Fe(III) in 2:1 smectite clays
(Stucki et al. 1984).
An evidence for the strong role of adsorbed ferrous iron for NDMA degradation
was evident by removal of adsorbed ferrous iron from the sediment by ion
exchange with Ca2+ (Chao and Zhou 1983; Heron et al. 1994) which slowed down
the NDMA degradation rate significantly. With 30 and 70 lmol removal of
adsorbed ferrous iron from the batch system, the NDMA degradation half-life was
increased to 122 and 310 h, respectively (triangles, Fig. 28a). However, addition
of ferrous iron increased the NDMA degradation rate (diamonds and circles,
(a)

1.0
NDMA / NDMA
0.8
0.6 X13
red. sed.
X148 Fe2+
+ 10umol
0.4 X150 Fe2+
+ 50umol
X152 Fe2+
-- 30umol
0.2 X153 Fe2+
-- 70umol
X110
oxic, sterile sed.
0.0
0.1 1 10 100 1000
Time (h)
(b)
1.0
NDMA / NDMA
0.8
0.6
0.4
live
killed
0.2
0.0
1 10 100 1000
Time (h)
(c)
0.4
0.3
0.2 killed
0.1
live
0.0
1 10 100 1000
Time (h)
Fig. 28 NDMA degradation in anaerobic, reduced sediment with: a additions or removal of

ferrous iron, b in reduced sediment with or without a bactericide (gluteraldehyde), and c NDMA
mineralization with or without the bactericide gluteraldehyde in reduced sediment
Fig. 28a), but not to the extent that ferrous iron removal influenced the rate.
Mineralization of NDMA, under iron-reducing conditions, also appears to be an
abiotic reaction and mineralized \20 % of the NDMA (Fig. 28c). In contrast,
NDMA mineralization in the same sediment under oxic conditions was biotic and
mineralized as much as 55 % of the NDMA (Fig. 29a). NDMA mineralization in
the oxic sediment is primarily a biotic process, as demonstrated by the addition of
a bactericide stopping nearly all of the mineralization. The NDMA mineralization
rate in the sterilized system ([50,000 h half-life, \2 % after 2,000 h) was very
slow. Thus, the presence of oxygen has significantly increased NDMA minerali-
zation (Fig. 29c).
(a) 0.6
0.5
Frac. mineralized
0.4
0.3 live
0.2
0.1 killed
0.0
1 10 100 1000
Time (h)
(b) 0.6
no S116
additions
Frac. mineralized
mol propane/mol O2
S123
0.04
S124
0.40
0.4
3.8S125
0.2 oxic Aerojet sed.

250 ppb NDMA
1632 h prestimulation
0.0
1 10 100 1000
Time (h)
(c) 0.6
oxic2.5 ppm
Frac. mineralized
0.5 G
anoxic
H
killed
0.4 I
red.(0.3)
red.J (1.5)
0.3 K (30)
red.
0.2
0.1
0.0
1 10 100 1000
Time (h)
Fig. 29 NDMA mineralization in: a in oxic Aerojet sediment with the presence or absence of a
bactericide gluteraldehyde, b in oxic Aerojet sediment with additions of propane and oxygen in
different proportions, and c inoxic, anerobic, or reduced Aerojet sediment
NDMA mineralization in the oxic systems was probably through a monooxy-

genase enzyme pathway (Mitch et al. 2003; Sharp et al. 2005, 2007). Propane
addition at specific oxygen/propane ratios did increase the mineralization rate to a
small extent (Fig. 29b). However, additions of methane, toluene, and acetylene
had no influence on NDMA mineralization. Likewise, other carbon additions
(yeast, humic acid, glucose) also did not influence NDMA mineralization.
Numerous attempts were made to simulate microbes in the reduced sediment
with additions of humic acid, yeast, methane, propane, toluene, acetylene, TCE,
nitrate, and glucose, but none of the additions showed any impact on the NDMA
mineralization rate (data not shown). Similarly, monooxygenase enzyme and
carbon additions (i.e. propane, methane, toluene) were not expected to show any
influence, as oxygen is also needed (monooxygenase pathway cannot utilize nitrate
as the electron acceptor). On the other hand, microbial biomass was actually
decreased in the reduced sediment over 1,900 h.
As NDMA can be rapidly degraded to intermediates (half-life 2–10 h) in the
reduced sediment (abiotic process), a sequential treatment system of a reducing
environment, followed by a downgradient oxic, biostimulation may be the most
rapid treatment process. Sequential reduced and then oxic batch experiments
showed little influence of sequential reactions on the NDMA mineralization rate.
More field-realistic sequential reduced-oxic systems were conducted in the 1-D
columns, with addition of propane/air between upgradient reduced column and
downgradient oxic sediment column (Fig. 30). NDMA degradation and mineral-
ization in the sequential reduced/oxic column systems were characterized in nine
experiments with a range of residence times and a range of differences in residence
time between the reduced and oxic sediment columns.
Fig. 30 NDMA degradation (a) 103

and mineralization rates in
NDMA degradation half-life (h)
reduced, oxic, and sequential

reduced then oxic 1-D
columns showing: a NDMA
degradation rate as a function 102
of residence time, and
b NDMA mineralization as a
function of residence time
1
10
B
Sequential Red./Oxic Columns
D
Reduced Column (abiotic)
F Column (biotic)
Oxic
100
4
1 10 100 1000 10
Residence time in 1-D column (h)
(b) 104
NDMA mineralization half-life (h)
103
Sequential
B Red./Oxic Columns
Reduced
D Column (abiotic)
Oxic
F Column (biotic)
2
10
1 10 100 1000 104
Residence time in 1-D column (h)
NDMA degradation in the sequential reduced-oxic sediment systems (Fig. 30a)

was two times more rapid (17.2 ± 4.5 h half-life) than reduced sediment alone
(32.1 ± 4.2 h), followed by slightly more NDMA degradation in the downgradient
oxic sediment column (Szecsody et al. 2008). NDMA mineralization rates in the
sequential reduced-oxic sediment systems (3180 ± 1094 h) were 40 % slower
than in the oxic columns (2293 ± 1866 h; Fig. 30b). Both these data sets indicate
sequential degradation is inefficient, caused either by intermediate of NDMA
degradation from the upgradient reduced column not being biodegraded as easily
as NDMA itself and/or removal of dissolved oxygen from the water that is injected
into the down gradient oxic column (with air/propane) was not efficiently main-
taining an oxic environment.
5 Reactive Transport with Abiotic/Biotic Degradation
While at a molecular scale, solute-surface reactions are the same in non-flowing

and flowing systems, field scale systems frequently exhibit differing behavior from
batch (non flowing) systems due to: a) much higher sediment/water ratio in flow
through porous media, b) advection of aqueous reaction components away from
immobile or surface phase reaction components, c) solute-surface reactions occur
in series (sequentially) in a flowing system rather than in parallel in a batch system,
d) spatial heterogeneities (if present) at a particle to lithologic unit scale influence
on reactions, and e) coupled geochemical-physical reaction effects.
5.1 RDX
In two column experiments, RDX was injected into the dithionite-reduced sedi-
ment column at a flow rate to achieve a residence time of 4.4 and 0.44 h in the
column (i.e. reaction time), which is likely to be 1–2 orders of magnitude faster
than it is found naturally in the contaminated aquifer. Rapid flow rates were used
to ensure accurate measurement of the rates of RDX and intermediate degradation.
In both experiments, RDX, MNX, DNX, TNX, and methylene dinitramine were
detected, even though prior batch studies (at a 10x lower sediment/water ratio, but
under similar geochemical conditions) did not show a build up of methylene
dinitramine (Fig. 1a). As shown in Fig. 31a, although the methylene dinitramine
concentration is low, it could be advected away from the reduced zone, thus
limiting degradation to further products. At a substantially slower flow rate
(resulting in a residence time of 89 h/pore volume), 14-C labeled RDX was
injected, and effluent samples were collected in sealed vials with headspace and a
CO2-trap. Analysis of the effluent RDX showed a decreasing effluent concentration
(to 50 % of influent, Fig. 31b) and CO2 concentration in the effluent sample
headspace was equivalent to 42 % mineralization of RDX. Therefore, rates for the
(a) Pore volumes

100 120 140 160 180 200
2
10
Conc. ( µ mol/l) 1
influent RDX 10 mg/L, 45 umol/L
RDX
10 MNX
0 DNX
10 TNX
-1 MDNA
10
-2
10
400 410 420 430 440 450
Time (h)
Pore volumes
(b) 0 1 2 3 4 5
100
80
60
aqueous species
40
20 carbon dioxide 5.0E-3/h 6.0E-3/h
0
0 100 200 300 400
Time (h)
Fig. 31 RDX degradation and mineralization in 1-D columns with chemically-reduced sediment
at: a a rapid flow rate of 0.44 h/pore volume (RDX and the first four degradation intermediate
concentrations shown), and b at a slow flow rate of 89 h/pore volume showing total aqueous
species and fraction mineralized. RDX mineralization rates were calculated at two points in (b).
Columns have a high sediment/water ratio of 5.6 g/ml
multistep coupled abiotic/biotic processes involved in RDX mineralization are

difficult to predict just from batch studies at what rates that would occur at a field
scale, and a minimum of 1-D column studies at multiple scales (which still do not
incorporate physical/chemical heterogeneities found at field scale) are needed.
5.2 HMX
HMX degrades and mineralizes more rapidly with greater ferrous iron present, as
shown in batch experiments (Fig. 16). Initial reaction was shown to be an abiotic
reaction. At the high sediment/water ratios in columns (and in aquifers), the HMX
degradation half-life was fairly rapid, with 2.1 h in highly reduced sediment
(Fig. 32). HMX degradation half-life was increased with lower reduction to 50 h
with partially reduced sediment. Sorption of HMX to the sediment was calculated
from the initial breakthrough and averaged 0.095 ± 0.013 cm3/g, or an average
retardation factor of 1.4. At colder temperature, the HMX degradation rates (at the

1.0
HMX / HMX
0.5
di/Fe half-life Kd (L/Kg)
Norm HMX
22 2.10 h 0.08
Norm
4.1 HMX
3.46 h 0.10
Norm HMX
1.6 16.4 h 0.11
Norm HMX
0.67 49.8 h 0.09
HMX initial conc. = 5.65 mg/L
0.0
0 5 10 15 20 25
Pore volumes
Fig. 32 HMX sorption and degradation in 1-D columns at differing amounts of abiotic reduction
at 22 °C
same amount of reduction), were more rapid, so the reaction was exothermic. The
HMX degradation rates, even in partially reduced sediment (i.e. slowest 1.8 h half-
life) were very viable to be taken up at field scale.
5.3 TNT
When TNT was injected at different concentrations in 24 columns of varying

reduced sediment conditions and temperatures (10–62 °C), an increase in sorption
and degradation rates with an increase in sediment reduction and decrease in
temperature was similar to results of HMX degradation studies. At 22 °C, TNT
sorption was significantly greater than HMX (Comfort et al. 1995), with a cal-
culated Kd of 1.55 ± 0.67 cm3/g or an average retardation factor of 8.0 (Fig. 33).
As the steady state TNT concentration reflecting the degradation rate was mainly
desired in some experiments, effluent sampling was then not carried out for the
first 10 pore volumes of the experiment. The TNT degradation rate at 22 °C was a
1.0
di/Fe half-life Kd(L/Kg)

0.67
Norm 2.8
TNTh 1.7
1.6
Norm 2.7
TNTh 0.32
TNT / TNT
Norm TNT
4.1 1.7 h 2.0
Norm 0.96
22 TNT h 0.98
TNT initial conc. = 3.40 mg/L
0.5
0.0
0 5 10 15 20 25
Pore volumes
Fig. 33 TNT sorption and degradation in 1-D columns at differing amounts of abiotic reduction
at 22 °C
weak function of the amount of sediment reduction. However, at 10 °C, sediment

reduction had a considerable effect on the TNT degradation rate, showing 37 times
more rapid rate for highly reduced sediment compared to partially reduced sedi-
ment. At 35 °C, a change in TNT degradation rate with temperature was much
smaller (5 times) between highly reduced and partially reduced sediment. Simi-
larly, at 49 °C, the TNT degradation rate change between highly and partially
reduced sediment was also small (4.3 times). Even at 62 °C, the TNT degradation
rate change between highly and partially reduced sediment was also low (2.8
times). Therefore, exothermic reaction (i.e. more reactive at colder temperature)
appears to be less effective at higher temperature.
5.4 CL-20
5.4.1 Water-Saturated Transport
The relative velocity and mass of CL-20 transported through sub-surface sedi-
ments depend on the influence of sorption and degradation relative to the transport
time-scale. The relatively small Kds for CL-20 in 2 h batch experiments suggests
nearly unretarded transport (Table 2). However, since degradation varied from
near zero for the Kenoma sediment to 100 % for Ocala, degradation may ulti-
mately limit the extent of sub-surface contamination. Reactive transport experi-
ments were conducted to study CL-20 sorption and degradation behavior at the
high sediment/water ratios of natural sub-surface systems (10–1000 times greater
than the sediment/water ratio in batch studies). As reflected in Fig. 34, the column
experiments for CL-20 showed a wide range of behaviors with differences in
sorption (i.e. lag relative to the tracer, Szecsody et al. 2004a) and rate of abiotic
degradation (i.e. final steady-state CL-20 concentration, Table 3). Tracer break-
through, which averaged 0.991 ± 0.048 pore volumes for all column experiments,
1.0
CL-20 / CL-20
0.5
conservative
G tracer
China Lake CL-20
Normalized sed.
ferrihydrite-coated sand
Normalized CL-20
Norborne sed.
Normalized CL-20
Westmoreland sed.
Normalized CL-20
L
Burbank sed.
Normalized
Ocala sed. CL-20
0.0
0 5 10 15 20
Pore volumes
Fig. 34 Reactive transport of CL-20 through oxic sediments showing differing sorption or lag
relative to a tracer
agreed well with the calculated porosity from dry and water-saturated sediment
weight in the column. The CL-20 degradation rates for these oxic sediments
ranged from a few hours to 780 h, as defined by a peudo-first-order reaction
(Table 3). The China Lake sediments showed the slowest CL-20 degradation rates
(450–780 h half-life), and low sorption (Kd averaged 0.25 cm3/g) CL-20 could
move long distances in the sub-surface. The average CL-20 degradation rates were
faster for Burbank sediment (13 ± 13 h half-life), Westmoreland sediment
(5.5 ± 3.9 h), Norborne sediment (5.0 ± 1.6 h) and ferrihydrite-coated sand
(6.1 ± 3.5 h). The average CL-20 Kd for all sediments tested in this study was
2.3 cm3/g. For this study, an average Kd for RDX was 2.0 cm3/g. RDX is a typical
groundwater contaminant due to its low sorption and low oxic degradation and
migrates unretarded in some groundwater systems (Spalding and Fulton 1988).
Considering that CL-20 sorption is, on average, only 10 % greater than RDX,
CL-20 has a high risk of becoming a groundwater contaminant, particularly for
sediments with low degradation rates, such as the China Lake sediment.
5.4.2 Low Water Saturation
Under unsaturated conditions, regions of stagnant or immobile water develop in

the soils. At very low water contents (10–20 % moisture), solutes can be excluded
from a fraction of the pore space due to ‘‘isolated’’ water, or immobile regions
where solutes cannot diffuse freely between the mobile and isolated domain. We
hypothesize that under these hydrodynamic conditions that develop at very low
water contents, sorptive solutes may experience decreased accessibility to some
sorptive surfaces, and therefore, a corresponding change in sorption will be
observed. In addition, there may be also changes in the sorption/desorption rate
and abiotic degradation rate. The magnitude of the change in apparent distribution
coefficient (Kd) was greater than a factor of 5 for both RDX and CL-20 (CL-20
shown in Fig. 35). Although these results are for controlled laboratory systems,
they should be considered while estimating solute transport in the vadose zone.
The implications are most significant for arid soils where water contents approach
10–20 % moisture saturation. It is important to consider that hydrodynamic con-
ditions at low water contents may affect sorption behavior. Slower CL-20 degra-
dation in low-water content field sediments was also earlier reported by Jenkins
et al. (2003).
5.5 Influence of Sediment-Energetic Aging on Reactive

Transport
As energetics can sorb then degrade with sediments over a considerable period of
time, the influence of prolonged exposure to sediments on sorption and degrada-
tion predictions was investigated. Aging was investigated by using stop-flow
1.00
Kd/Kd max
1.51
Kd(norm) = 0.00103(sat)
0.10
0.01
0 20 40 60 80 100
Water saturation (%)
Fig. 35 Influence of water saturation on CL-20, based on low water content column experiments
conducted in a centrifuge. Shown is the distribution coefficient (Kd) divided by the Kd in water-
saturated conditions
column experi-ments, where desorption mass, desorption rate, and degradation

were investigated as a function of contact time before desorp-tion. Two energetics
were compared: (a) RDX, which, in oxic sediment, exhibits sorption but no
degradation, and (b) CL-20, which shows both sorption and degradation. Two
sediments were used, one with a high fraction organic carbon and another with
very low fraction organic carbon. It is hypothesized that: (a) additional binding to
organic matter could occur leading to a desorption-resistant fraction (aging
hypothesis for organic carbon); (b) energetic diffusion into microfractures in low
organic carbon sediment will also lead to a desorption-resistant fraction (aging
hypothesis for minerals; and (c) the degradation rate may be apparently slower due
to no mixing with advection. A series of 24 column experiments were conducted to
address CL-20 and RDX aging with two different sediments. Each column aging
experiment consists of a sorption phase, then lag phase from 1, 112, 737, or
2,400 h, after which CL-20 and RDX are desorbed from the column (Fig. 36).
Both CL-20 and RDX showed smaller apparent desorption Kd values with
greater aging time (Fig. 37a, b), where plots show the ratio of desorption (aged)/
sorption (no aging), Kd was observed in each experiment. A value of \1 indicates
weaker sorption binding over time, whereas a value [1 indicates stronger sorption
binding over time. In these oxic, aseptic conditions of the experiments, RDX is not
degraded, so the smaller Kd values for RDX reflect less mass desorption (i.e. a
desorption resistant fraction). CL-20 will undergo some abiotic degradation, and a
larger decrease in the desorption Kd was observed.
Both CL-20 and RDX showed greater breakthrough curve tailing with more
aging time (Fig. 36). A model simulating breakthrough with a first-order sorption
and first-order mass loss was used to quantify the desorption rate. The CL-20
desorption rate showed little change with aging (Fig. 37c). In contrast, the RDX
desorption rate showed a large decrease with aging time for the high foc sediment,
and no change for the low foc sediment (Fig. 37d). This indicates that RDX would
(a) (b)
CL-20
/
CL-20
(c) (d)
RDX / RDX
Fig. 36 CL-20 and RDX sorption and desorption in aged columns for the Westmoreland
sediment (2 % organic carbon). CL-20 and RDX sorption shown in (a) and (c). CL-20 and RDX
were desorbed: (b) after 1 h, and (d) after 2,245 h of aging with no flow
desorb more slowly with long sediment/solute contact times for sediments with
organic carbon possibly due to greater binding with the organic matter over time.
CL-20 was abiotically degraded slowly during the aging experiments. The CL-
20 degradation rate was averaged for all sorption experiments (horizontal bar,
Fig. 37e) and was used to calculate the mass of CL-20 that should be degraded
during the aging experiments. The observed rate of CL-20 degradation was cal-
culated from both aqueous effluent data and methanol extractions of CL-20 from
the sediments. Surprisingly, there was greater CL-20 mass remaining than the
predicted. This indicated that the CL-20 degradation rate during no-flow aging
experiments was apparently decreasing with increasing aging time (Fig. 37e).
6 Conclusions and Implications for Field Scale

Remediation of Energetics
Uncontrolled release of energetics to the surface and subsurface environment can

occur through munition manufacture, storage, and deployment (UXO and partial
detonations). RDX and HMX are common groundwater contaminants due to slow
aerobic degradation in soils and vadose zone sediments and minimal sorption. In
contrast, TNT is a common soil/shallow sediment contaminant with limited TNT
migration because of aerobic (Boopathy et al. 1994) and anaerobic (Funk et al.
1993) degradation in soils as well as higher sorption. CL-20 is a relatively new
(a) 1.5
Desorb. Kd/sorb Kd
(b) 1.5
Desorb. Kd/sorb Kd
1.0 1.0
0.5 0.5
0.0 0.0
1 10 100 1000 104 1 10 100 1000 104
Aging time (h) Aging time (h)
(c) 101 (d) 103
Desorption rate (1/h)

Desorption rate (1/h)
102
0
Sassafrass, 0.3% foc
10
101
Westmoreland, 2% foc
10
-1
100
1 10 100 1000 10
4
1 10 100 1000 104
Aging time (h) Aging time (h)
(e) 10
0
Degradation rate (1/h)
-1
10
-2
10
10-3
1 10 100 1000 104
Aging time (h)
Fig. 37 Influence of energetic/sediment contact time (aging) on: a CL-20 sorption mass, b RDX
sorption mass, c CL-20 desorption rate, d RDX desorption rate, and e CL-20 degradation rate.
The Sassafrass sediment (circles) had low (0.3 %) fraction organic carbon (foc), and the
Westmoreland sediment (triangles) had 2 % organic carbon
energetic, exhibiting generally low adsorption, but highly varied degradation rates
in the sediments. CL-20 is likely to be persistent in low-water content sediments
(i.e. vadose zone), and in low-clay or iron content groundwater systems. NDMA is
a known groundwater contaminant, as it shows nearly no adsorption to the sedi-
ment, slow to no degradation in a wide variety of subsurface conditions, and is
toxic even at ppt levels.
For groundwater contamination of RDX and HMX in a limited number of

energetic-contaminated aquifer sediments, coupled abiotic/biotic remediation
shows the most rapid RDX and HMX mineralization rates and extent (Fig. 38a, b)
compared with abiotic treatment only or biostimulation only. The amount of iron
reduction in sediment (by chemical reduction or addition of zero valent iron)
exerts the greatest impact on mineralization. Additional biostimulation (carbon or
trace nutrient addition) increases the mineralization rate marginally (in addition to
iron reducing conditions). For RDX, the mineralization rate in dithionite-reduced
(a) (b)
5 5
10 10
Mineralization half-life (h)

untreated sed.
4 4
10 10
untreated sed.
bioremediation
3 3
10 10
ZVI + sed.
faster, chemically-
more CO2 reduced sed.
chemically-reduced sed.
chemically-reduced sed. + nutrients faster,
2 more CO2
10 102
0 20 40 60 80 100 0 20 40 60 80 100
Mineralization extent (%) Mineralization extent (%)
(c) (d)
105 10
6
untreated sed.
Degradation half-life (h)
chemically-reduced sed.
105
chem.-reduced sed.
104
anaerobic
sediment
sed.
oxic
104
3
10
3
10
toADNTr
DANT
faster, toDANTr
TAT
faster,
more CO2 more TAT
2 2
10 10
0 20 40 60 80 100 0 0.2 0.4 0.6 0.8 1
Mineralization extent (%) Fraction to DANT or TAT

(e) (f)
105 104
rate,red
red. Ft Lewis, Wa oxic sediments
oxic sed rate
rate,ox
oxic Ft Lewis, Wa anoxic sediments
anox rate
rateRocky Flats, Co
oxic reduced
red rate sediments
4 rate
red. Rocky Flats, Co
10 microbial
bug rate isolates
ISOox
oxic isolates (oxic)
oxicA
oxic Aerojet, Ca
redA
red. Aerojet, Ca
anoxicA
anoxic Aerojet, Ca
103
zvi
red. then oxic Aero.
3 puch
10 Puchack nat. red. sed.
faster, faster,
more CO2 more CO2
102 102
0 20 40 60 80 100 0 20 40 60 80 100
Mineralization extent (%) Mineralization extent (%)
Fig. 38 Energetic mineralization rate (half-life) and extent for different abiotic, biotic, and
abiotic/biotic remediation technologies on the same sediment: a RDX, b HMX, c TNT, d TNT
cometabolic degradation rate and extent to diaminonitrotoluene (DANT) or triaminotoluene
(TAT, both irreversibly sorb), e NDMA, and f CL-20. Mineralization rate and extent shown for
multiple sediments in (e)
sediment was 98 times more rapid than untreated sediment (31,000 h half-life), and
in dithionite-reduced sediment with biostimulation it was 277 times more rapid
than untreated sediment (112 h half-life, Table 4). Biostimulation (carbon addition
to anoxic sediment) increased RDX mineralization \10x). For HMX, the miner-
alization rate in dithionite-reduced sediment was 48 times more rapid than
untreated sediment (7,800 h half-life), and in dithionite-reduced sediment with
biostimulation, it was 58 times (162 h half-life) more rapid than untreated sedi-
ment. Addition of 0.4 % zero valent iron was nearly as effective as high miner-
alization rates, but 60 % mineralization extent compared with 78 % mineralization
extent for dithionite-reduced sediments.
In the same energetic-contaminated aquifer sediments, remediation of TNT
would best be effected by glucose addition, to stimulate the co-metabolic TNT/
glucose degradation amino-degradation pathway, producing 2-aminodinitrotolu-
ene (2-ADNT) ? 4-aminodinitrotoluene (4-ADNT) ? 2,4-diaminonitrotoluene
(2,4-DANT) ? 2,6-diaminonitrotoluene (2,6-DANT) ? triaminotoluene (TAT).
While the initial monoamino- products are more toxic than TNT, both diamino-
and triamino-toluene irreversibly sorb and thus are immobilized in the sub-surface
environment. Interestingly, TNT cometabolic degradation is most rapid in
dithionite-reduced sediment (half-life 610 h, producing TAT), compared to just
glucose addition (half-life 8,030 h; untreated sediment 55,000 h, Fig. 38c, d).
Therefore, glucose addition alone increased the TAT production rate by 6.8 times,
whereas glucose addition and dithionite reduction increased the TAT production
rate 90 times. The 610 h half-life for TNT degradation to triaminotoluene is still
slow for a viable groundwater remediation technology. Typically, a reaction half-
life of 100 h or faster is needed to have sufficient residence time in a subsurface
treatment zone to achieve full degradation.
Transformation of CL-20 to intermediates in aquifer sediments appears most
rapid under abiotic iron reducing conditions or biotically-created reducing condi-
tions or by alkaline hydrolysis (pH [ 10). CL-20 degradation to intermediates may
be insufficient to mitigate environmental impact, as the toxicity of many of these
intermediates is still unknown. CL-20 mineralization rate was the highest for oxic
sediments with carbon (and not N) additions. For an arid region sediment, untreated
sediment had a CL-20 mineralization half life of 980 h and an extent of 13 %. With
carbon addition, the mineralization half-life was reduced to 230 h and minerali-
zation extent was 69 % (Fig. 38f). However, the CL-20 mineralization rate in the
presence of carbon additions was investigated in several other aquifer sediments
(not arid sediments) with no sign of increased mineralization. Although the CL-20
mineralization level was significantly less under iron reducing conditions
(40–52 %) compared to oxic conditions of the same sediment, the CL-20 miner-
alization rate was slightly more rapid. The highest CL-20 mineralization extent was
observed in anoxic sediment. Clearly, a better understanding of the multiple steps in
CL-20 mineralization is needed to evaluate optimal in situ remediation.
A comparison of NDMA mineralization rate (as a half-life) to mineralization
extent in a limited number of sediment/water systems shows a general correlation
(Fig. 38e) which indicates that oxic bioreactors were the most efficient. Certainly,
364
Table 4 Sediment primary and secondary treatment and energetic degradation

Energetic Untreated sed. Primary treatment Treated sed. Secondary treatment Treated sed.
mineralization mineralization mineralization
half-life (h) half-life (h) half-life (h)
RDX 31,000 Dithionite reduction of sediment 315 Carbon addition trace nutrient 140
112
HMX 7,800 Dithionite reduction of sediment 162 Carbon, trace nutrient 135
TNT* 55,000 (to TAT) Glucose addition 8030 Dithionite reductio of sediment 610
(product = TAT)
CL-20 980 Oxic sediment, nutrient addition 230 Sequential reduced then oxic sediment
NDMA 2,960 Oxic sediment 471 Propane addition ? prestimulation 150
*TNT degradation to triaminotoluene (TAT), which irreversibly sorbs
J. E. Szecsody et al.
the highest mineralization (70–80 %) was found in the oxic sediments. However,
in 1-D columns (Fig. 38e), it showed that the NDMA mineralization rate in the
oxic systems was inefficient, but in reduced sediment columns, it was as fast as any
batch bioreactor (and 55 times faster than 1-D columns with oxic, biostimulated
sediment). The mineralization extent in the reduced sediment columns was low to
moderate (2.2, 6.2, and 16.8 %) compared to 20–80 % in oxic bioreactors. The
oxic bioreactors with the most rapid and greatest extent of NDMA mineralization
had propane addition and were pre-stimulated for months before NDMA addition.
Since these ex situ bioreactors were more successful than stimulation of in situ
microbial activity in sediments, a field scale remediation of NDMA should focus
on comparison of in situ abiotic NDMA mineralization (under iron-reducing
conditions) to ex situ biomineralization.
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Phytoremediation of TNT and RDX
Shree Nath Singh and Shweta Mishra
1 Introduction
Besides other organic contaminants, soil contamination by explosives also poses a

serious environmental concern. Explosive compounds are released into the envi-
ronment during manufacturing, handling and disposal operations at military sites
to contaminate surface and ground waters, soils and sediments (Sunahara et al.
2009) In addition, aquatic environments are also contaminated with unexploded
ordnance (UXO) and dumped ammunition wastes (Darrach et al. 1998; Rodacy
et al. 2000; Dave 2003; Ek et al. 2006).
Explosive compounds are heterocyclic nitramines and mostly nitro derivatives
of benzene, toluene and phenol. They can be classified into two groups i.e. primary
and secondary, based on their susceptibility to initiation when exposed to stimuli,
such as heat, shock, friction etc. Primary explosives are highly susceptible to ini-
tiation and hence, often used to ignite secondary explosives, such as TNT (2,4,6-
trinitrotoluene), RDX (1,3,5-trinitro-1,3,5-triazinane), HMX (1,3,5,7-tetranitro-
1,3,5,7-tetrazocane), and tetryl (N-methyl-N,2,4,6-tetranitroaniline).They exhibit
low bioaccumulative potential due to weak hydrophobicity (Lotufo et al. 2009).
Only a few natural nitroaromatic compounds, such as chloramphenicol, nitropyo-
luteorin, oxypyrrolnitrin and phidolopin are known to date. Apart from them, no
other natural nitoaromatic compounds are available which show recalcitrance to
biological degradation. These are carcinogenic and mutagenic in nature and also
cause a disease to human, known as pancytopenia as a result of bone marrow failure
and also have harmful effects on the liver (Amdur et al. 1991).
The most common contaminants, found around active military firing ranges, are
TNT, RDX and HMX. TNT and RDX are also priority pollutants in the list of
United States Environmental Protection Agency (USEPA 2004). The distribution
of these contaminants in sub-surface environments occurs through the dissolution
S. N. Singh (&) S. Mishra

CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India
e-mail: drsn06@gmail.com

372 S. N. Singh and S. Mishra
of mixed solid-phase energetic residues, which are found to be spread around

military ranges after detonation events. The dissolution of the residues occurs
through the direct impact of precipitation events and by flowing surface runoff, or
by percolating soil pore water.
TNT, first time synthesized in 1863, was initially used in the dye industry
before becoming in the 20th century, the main conventional explosive used by
military forces worldwide. TNT is obstinate to oxygenolytic transformation due to
mutual steric and electrophilic effects of multiple nitro substitutions on the aro-
matic nucleus (Esteve-Núñez et al. 2001; Preuss and Rieger 1995). On the other
hand, RDX, which was formerly used as a rat poison, is today considered a
carcinogen by the EPA (Binks et al. 1995; Lachance et al. 1999). RDX has low
aqueous solubility (*40 mg/l) (Talmage et al. 1999). However, once dissolved,
RDX can migrate with groundwater to pollute down gradient aquifers. A lifetime
health advisory of 2 lg/l of TNT in drinking water and a water-quality limit of
105 lg/l of RDX have been recommended (Etnier 1989; Ross and Hartley 1990).
Even though, many reports have suggested that this compound can be readily
biodegraded, but RDX persists in the sub-surface environments for a longer time
(Meyers et al. 2007).
2 Physical and Chemical Properties of RDX and TNT
The uptake and transformation of energetic substances, such as TNT and RDX, by
plants are regulated by both their physical and chemical properties (Table 1). TNT
is a nitroaromatic compound and chemically known as 1-methyl-2,4,6-trinitro-
benzene and commonly known as tolite. TNT is a highly reactive energetic
compound, as three nitro functional groups are attached to an aromatic ring. It can
undergo oxidation and reduction in both aerobic and anaerobic conditions (Hawari
et al. 2000). But due to the presence of aromatic ring, TNT is resistant to elec-
trophilic attack and hence rarely metabolized (Spain 1995).
RDX - a hetrocyclic nitramine also known as hexagen, hexolite, trinitrohexa-
hydrotriazine and cyclotrimethylenetrinitramine, is a major component of military
explosives, such as Composition B (Comp B) and Composition 4 (C4) (Hewitt
et al. 2007). Since RDX is fairly soluble, it does not get easily sorbed to soil
particles, and hence, more transportable in the environment as compared to TNT.
Octanol water partition coefficient is an important factor for the uptake of
compounds by the plants from the soil and also for their movement through the
membrane of the roots (Yoon et al. 2005). Many studies have reported that
hydrophilic compounds, having log KOW less than 1.8, are not able to penetrate the
lipid-rich membrane of roots, while hydrophobic compounds with log KOW greater
than 3.8, will be easily taken up into the roots, but not translocated to the shoots
(Yoon et al. 2005). The major difference between these two explosive compounds
is the logarithm of their soil organic carbon–water coefficient (KOC). Since log
KOC of TNT is over a hundred fold greater than RDX, it is strongly adsorbed to the
Phytoremediation of TNT and RDX 373
Table 1 Physical and chemical properties of RDX and TNT (USEPA 2011a)
Properties RDX TNT
State at room temperature White crystalline solid Yellow, odorless solid
Molecular weight (g/mol) 222 227
Water solubility (mg/l) 42 (at 20 °C) 130 (at 25 °C)
Octanol-water partition 0.87 1.6
coefficient log (Kow)
Soil organic carbon water 1.80 300
coefficient log (Koc)
Vapour pressure at 25 °C 4.0 9 10-9 1.99 9 10-4
(mm Hg)
Henry’s law constant (atm- 1.96 9 10-11 (at 25 °C) 4.57 9 10-7 (at 20 °C)
m3/Mol)
Molecular structure
other organic matter present in the soil and gets immobilized, whereas RDX
mainly moves deeply through the soil to the groundwater (Kalderis et al. 2011).
3 Phytoremediation of Ammunition Wastes
Traditional treatments for the remediation of the toxic ammunition wastes (e.g.,
open burning and open detonation, adsorption onto activated carbon, photooxi-
dation, etc.) are very costly and also deteriorate the environment. In many cases, it
was found practically infeasible. Therefore, an inexpensive and environment-
friendly treatment was developed which is based on either microorganisms or
plants or their combination.
Phytoremediation is an attractive technology which uses green plants for the
partial degradation of explosive compounds present in the soil and water. It was
developed a few decades ago based on our knowledge that plants are capable of
metabolizing toxic pesticides. It utilizes a variety of biological and physical
characteristics of plants to aid in site remediation. Different classes of organisms,
such as bacteria, fungi and plants, have been reported for the biotransformation of
TNT, RDX, and HMX. The transformation occurs through a sequential reduction
of the nitro groups to form toxic aromatic amino derivatives which are further
transformed. Transformation is based on the ‘‘Green Liver’’ model which
describes the fate of organic contaminants within the plant tissues (Sandermann
1994; Burken and Schnoor 1997; Salt et al. 1998; Hannink et al. 2002).
Phytoremediation encompasses several different technologies (1) phytoextrac-
tion involves bioconcentrating contaminants in the harvestable zones of the plant;
(2) phytostabilization allays the bioavailability of contaminants by binding them to
plant tissues; (3) phytodegradation degrades toxic compounds by the enzyme
systems of the plants and plant-associated microorganisms and (4) phytovolatil-
ization volatilizes the contaminants by the plants.
Many plants, such as poplar trees, reed grass and agronomic plants have been
reported to take up RDX and TNT (Sikora et al. 1997; Price et al. 2002; Best et al.
2004; Vila et al. 2007a) and concentrate them mainly in new growth (Seth-Smith
et al. 2002). Harvey et al. (1991) also studied the uptake, translocation and
transformation of RDX by plants, but the results were found quite different from
that of TNT (Adrian et al. 2003). Besides, maize (Zea mays L.) and broad beans
(Vicia faba L.) are also able to remove TNT from the soils (Van Dillewijn et al.
2007).
Plants have developed the ability to take up the chemicals from the vapor,
liquid and solid phases, but the movement of the organics within the plant usually
occurs in solution. The uptake efficiency of the plants generally depends on fol-
lowing factors, such as pH, pKa, soil water, organic content, water partition
coefficients (log Kow) and plant physiology (MacFarlane et al. McFarlane et al.
1990). Only chemicals, having log octanol: water partition coefficients (log Kow)
between 0.5 and 3.0, are taken up by the plants. Among nitroaromatic explosives,
nitrotoluene has log Kow 2:37, while 2,4-DNT possesses log Kow 1:98
(Briggs et al. 1982). Besides, water solubility and uptake of the contaminants can
be enhanced by the use of both synthetic surfactant (Triton X-100) and naturally
produced biosurfactants (rhamnolipids) (Salt et al. 1998). Once the explosive
compounds have entered the plant tissues, they can be metabolized, stored (often
in the root system) or volatilized.
The biological degradation of contaminants can also be enhanced by another
process of phytoremediation, known as rhizodegradation, in which, a symbiotic
relationship between plants and microorganisms exists. Bacteria and fungi increase
their activity in the rhizosphere of plants (Susarla et al. 2002) and result in the
reduced toxicity and reduced nutrient deficiency in both bacteria and plants
(Wenzel 2009). An increased removal of TNT has been also observed from an
active rhizospheric zone of the prairie grass (Wolfe et al. 1994). The plant secretes
some sugars, alcohols and acids which encourage the growth of rhizospheric
bacteria around the root system (Schnoor et al. 1995). The bacteria humidify the
organics and secrete the degradative enzymes, such as peroxidases and thereby,
augment the degradation of contaminants (Dec and Bollag 1994). Besides, a few
enzymes, such as nitroreductases, laccases and peroxidases, have been reported to
be involved in the phytodegradation of nitroaromatic compounds (Schnoor et al.
1995). In addition, another process, known as rhizofiltration, also plays an
important role in the remediation by mediating absorption and adsorption of the

contaminants to the roots of the plant. Thus, plant root system plays an active role
in the remediation of explosive contaminants (Salt et al. 1998).
4 ‘‘Green Liver’’ Model
The uptake and transformation of energetic substances in plants is driven by

simple diffusion and degradative enzymes. The assimilation of organics in plants is
essential for the contact between plant cell enzymes and organic contaminants.
The ‘‘Green Liver’’ model illustrates the process of transformation of explosive
contaminants in plants, once they are taken up from the soil. According to early
studies, plants deal with organic explosives, such as RDX and TNT in three phases
as depicted in Fig. 1 (Van Dillewijn et al. 2008; Rylott and Bruce 2009): Phase I
(transformation)—The contaminant is metabolized into a more soluble and less
toxic intermediate products by several reactions, such as oxidation, reduction, or
hydrolysis. The oxidative metabolism of explosive compounds is generally med-
iated by cytochrome P450 mono-oxygenase in plants. Infact, hydrophobic pollu-
tants are emulsified to make them highly reactive electrophilic compounds for
conjugation. In plants, cytochrome P450 forms a largest group of plant protein
which plays an important role in degradation of explosives (Morant et al. 2003).
Phase II (conjugation)—In conjugation between organic contaminant and endog-
enous hydrophilic molecules, such as D-glucose, glutathione, or amino acids,
soluble or insoluble substances are produced to be subsequently sequestered in
different cellular compartments of the plant for storage (Yoon et al. 2005; Schnoor
et al. 2006). Conjugation also enhances metabolic activity which is further cata-
lyzed by glycosyl-, malonyl-, and glutathione S-transferases. Phase III (compart-
mentation)—The soluble contaminants and breakdown products are sequestered
into vacuoles or cell wall from the cytosol of the plants via ATP-binding, ABC
transporters and multi-drug resistant proteins which play an important role in
sequestration or compartmentation to reduce their toxicity and finally, the insol-
uble compounds are stored into the cell wall (Yoon et al. 2005).
5 Biotransformation
5.1 TNT Transformation by Plants
Periwinkle (Catharanthus roseus) and parrot feather (Myriophyllum aquaticum)

are two plants involved in the transformation of TNT. In this process, two main
metabolites i.e. 2-amino-4,6-dinitrotoluene (2-ADNT)) and 4-amino-2,6-dinitro-
toluene (4-ADNT), are formed as primary reduction products during the
Organic Compound in
Environment
Uptake Transport
Transport,
Xylem Flow
Organic Compound in
Root Tissue
Organic Compound Transformation Reactions

in Xylem or Leaf
Metabolite in Plant
Conjugation
Conjugate Bound or Soluble
Sequestration
Compartmentalized
Conjugated Compound
Fig. 1 Green liver model for the metabolism of xenobiotics in plants (Burken et al. 2000)
degradation of TNT by plants (Palazzo and Leggett 1986; Thompson et al. 1998;
Bhadra et al. 1999). The formation of diaminotoluenes (2,4-diamino-6-nitrotolu-
ene and 4,6-diamino-2-nitrotoluene) and azoxy compounds was also observed
under strong reducing conditions and by the condensation of hydroxylamines,
respectively (Pavlostathis et al. 1998; Sens et al. 1999; Thompson et al. 1998).
TNT transformation pathway has been proposed by Rylott and Bruce (2009) as
reflected in Fig. 2. However, Bhadra et al. (1999) studied the oxidation of TNT by
plants and identified six oxidized metabolites, such as 2-amino-4,6-dinitrobenzoic
acid, 2,4- dinitro-6-hydroxy-benzyl alcohol, 2-N-acetoxyamino-4,6 dinitrobenz-
aldehyde, 2,4-dinitro-6-hydroxytoluene, and two binuclear metabolites from
azoxytetranitro toluenes during oxidative transformation of TNT (Fig. 3). The
oxidized metabolites were detected in parrot feather (Myriophyllum aquaticum)
during degradation of TNT (Subramanian 2004). Besides, they also concluded that
oxidation of TNT could occur before the reductive transformation in plants.
The reductive transformation of TNT has also been reported in non-axenic
culture and aquatic plant systems where nitro groups of TNT undergo reduction
with the formation of 2-hydroxylamino-4,6-dinitrotoluene (2HADNT) and
4- hydroxylamino-2,6-dinitrotoluene (4HADNT) (Pavlostathis et al. 1998; Wang
Vacuole
Fig. 2 Proposed TNT degradation mechanism in plants (Rylott and Bruce 2009)
RDX DNX
MNX
Light-mediated Breakdown
CH2O CH3OH
CO2
Fig. 3 RDX degradation mechanism in plants (Rylott and Bruce 2009)

et al. 2003). These hydroxylamines were also observed in the axenic hairy roots of
Catharanthus and axenic Arabidopsis seedlings (Subramanian 2004; Subramanian
et al. 2005). According to Subramanian and Shanks (2003) and Wang et al. (2003),
hydroxylamines are the first transformation products which form other metabolites
by undergoing reduction, oxidation, conjugation, and polymerization process.
After transformation, the products of the TNT move to another phase called
conjugation and are sequestered in the plant cells. Bhadra et al. (1999) reported
that monoamines were the precursors to the conjugates. They have characterized
four conjugates of TNT metabolites having a 6-carbon moiety in Catharanthus
roseus and Myriophyllum aquaticum and found that out of four conjugates, two
were similar to 2-ADNT and others were similar to 4-ADNT in molecular struc-
tures. Similarly, Vila et al. (2005) have also reported conjugates of TNT metab-
olites formed by conjugation of glucose on the hydroxylamine group of either
2HADNT or 4HADNT and also various other diglycoside conjugates with gen-
tiobioside or sophoroside including monoglycosides by tobacco cell cultures. The
conjugation of plant sugars with monoamines and hydroxylamines was also
observed by Subramanian (2004) and Subramanian et al. (2005).
5.2 RDX Transformation by Plants
The degradation of RDX was studied by Van Aken et al. (2004) in poplar tissue
cultures and crude extracts of leaves (Fig. 4). In plant cells, during transformation
process, RDX undergoes reduction and the metabolites produced were identified as
hexahydro-1-nitroso-1, 3-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitr-
oso-5-nitro-1,3,5-triazine (DNX) (Fig. 5). Subsequently, MNX and DNX were
transformed to formaldehyde and methanol, both in crude extracts and in intact
cultures in the presence of light. In the final step, light-independent mineralization
of one-carbon metabolites by intact plant cultures was observed, but not reported
in crude extracts. Some of transformed products may be assimilated by the plants.
In plants, enzymes mediate conjugation of formaldehyde to form S-formyl-gluta-
thione (Just and Schnoor 2004). After complete mineralization of RDX by plants,
small quantities of CO2 are produced to be re-assimilated in the photosynthesis
process (Van Aken et al. 2004).
6 Phytoremediation
6.1 Phytoremediation of TNT
The uptake and fate of energetic substances in plant system were found different
for nitroaromatic and nitramine explosives. The degradation of TNT is largely
found in the plant roots, where TNT remains due to its high biochemical activity of
TNT 2HA46DNT 4HA26DNT
2A46DNT 4A26DNT
Fig. 4 TNT and its major metabolites
RDX MNX DNX TNX
MDNA BHNA NDAB
Fig. 5 RDX and its major metabolites
aromatic nitro group of TNT. It forms oxidative couplings on roots and hence,
little or no translocation to leaves and stems occurs as examined by phosphor
imager autoradiography (Schneider et al. 1996; Brentner et al. 2010). The most
observed transformation process in the case of TNT is the aerobic reduction by the
plants (Burken et al. 2000) and the most commonly observed reduction products
formed in plants were monoaminated TNT metabolites (2-amino-4,6-dinitrotolu-
ene and 4-amino-2,6-dinitrotoluene). However, only a few plants have the
potential to translocate TNT to leaves (Schneider et al. 1996).
Hughes et al. (1997) also reported partial degradation and formation of
metabolites in the aqueous medium by hairy root cultures of axenically grown
Catharanthus roseus and Myriophyllum aquaticum plants. This observation was
also endorsed by Bhadra et al. (1999) who reported 25 ppm of TNT degradation
within a few weeks by the hairy root cultures of Catharanthus roseus. Similarly,
Pavlostathis et al. (1998) observed 100 % removal of TNT with the concentration
up to 49 lM by Myriophyllum spicatum (Eurasian watermilfoil). However,
smooth bromegrass (Bromus inermis L.), grown in a sterilized environment, was
found to remove and/or break down TNT into less toxic by-products.
Nelson (2001) reported that hydroponic cultures of sago pondweed (Pota-
mogeton pectinatus L.) were able to dissipate TNT from water at a faster rate
(below HPLC detection limits within 48–96 h) as compared to non-cultured plants,
where only 37–56 % of the added TNT was lost. It was also observed during the
experiment that when TNT was applied in successive doses (once every 4 days),
sago pondweed was able to tolerate up to 0.5 mg/l TNT. However, a concentration
of TNT 60 mg/l did not influence tuber germination of sago pondweed.
Bae et al. (2004) observed that Indian mallow (Abuliton avicennae) removed
76.8 % of TNT from the soil, while 31.6 % was recovered in the soil as ADNTs
and 0.2 % as TNT and ADNTs in the shoots and roots, respectively. However, in
unplanted column, 51.9 % of the TNT was mineralised in the soil and 37.3 % was
recovered as ADNTs.
Working on degradation of TNT through plants, such as, Phragmites australis,
Juncus glaucus, Carex gracillis and Typha latifolia, Vanek et al. (2006) observed a
maximum of 90 % of TNT transformation within 10 days of cultivation. Among
four plants, the most potential degrader was found to be Phragmites australis
which transformed about 90 % of TNT within 10 days and 4-amino-2,6-dinitro-
toluene (4-ADNT) and 2-amino-4,6-dinitrotoluene (2-ADNT) were the first stable
products formed during the degradation process. Similarly, Lee et al. (2007)
worked on four plant species i.e. barnyard grass (Echinochloa crusgalli), sun-
flower (Helianthus annuus), Indian mallow (Abutilon avicennae) and Indian
jointvetch (Aeschynomene indica), for the remediation of TNT contaminated soil
and observed that all the four species had a high potential to remove TNT and its
metabolites, regardless of whether the culture was grown single or mixed. The
concentrations of TNT and its metabolites, 2-amino-4,6-dinitrotoluene (2-ADNT))
and 4-amino-2,6 dinitrotoluene (4-ADNT) were found very high in H. annuus,
A. indica and A. avicennae except Echinochloa crusgalli.
Ouyang et al. (2007) observed 25 % of the TNT removal from the soil by the
poplar tree in 90 days by the use of UTCSP model (dynamic model for Uptake and
Translocation of Contaminants from a Soil–Plant ecosystem). They also monitored
a diurnal variation pattern in the uptake of TNT by roots and observed that TNT
uptake was enhanced during the day and decreased during the night, most likely
due to changes in leaf water transpiration as result of diurnal variations in xylem
water potentials. Earlier also, Ouyang et al. (2005) made similar observation using
CTSPAC model (mathematical model for coupled transport of water, solutes, and
heat in the soil–plant-atmosphere continuum) on TNT removal from contaminated
sandy soil by a single poplar (Populus fastigiata) tree. According to CTSPAC
model, no TNT was found in the stem and leaves and only about 1 % of total TNT
was observed in the roots due to rapid biodegradation and transformation of TNT
into its intermediate products. About 13 % of the soil TNT was removed by root
uptake of the poplar tree. Brentner et al. (2010) also investigated the localization of
RDX and TNT in the plant tissues of Populus deltoids x nigra DN34 (poplar) and
Panicum vigratum, Alamo (switchgrass) by the use of phosphor imager autora-
diography. They observed that in both plants, TNT and/or TNT-metabolites
remained predominantly in the root tissues, while RDX and/or RDX metabolites
were readily translocated to leaf tissues.
Makris et al. (2007) studied the uptake of 40 mg TNT/l for 8 days by vetiver
grass in a hydroponic system and found that in aqueous medium, the concentration
of TNT reached to method detection limit (1 mg/l) within 8 days, indicating
vetiver high affinity for TNT with no visible toxicity. Das et al. (2010) also
reported that vetiver grass potentially removed TNT when treated together with
different TNT (0–100 mg/kg) and urea (0, 125, 350 and 1,000 mg/kg) concen-
trations. In the presence of urea, the removal rate of TNT was found as high as
91 %, indicating fast translocation of TNT from root to shoot. Major TNT
metabolites, such as 2-ADNT, 4-ADNT and 1,3,5-TNB were detected in the plant
tissues. In addition, Chekol et al. (2002) reported reed canary grass (Phalaris
arundinacea L.) and switch grass (Panicum virgatum L.) as the most effective
plant species which enhanced TNT transformation. About 77 and 73 % transfor-
mations of TNT (100 mg/kg) were observed by switch grass and reed canary grass,
respectively.
Jiamjitrpanich et al. (2012) discovered a new technology known as nano-
phytoremediation which is a combination between phytoremediation and
nanoscale zero valent iron (nZVI) for removal of trinitrotoluene (TNT) from
contaminated soil. In this study, a hyperaccumulator plant purple guinea grass
(Panicum maximum) was used for nano-phytoremediation in soil with the TNT/
nZVI ratio of 1/10 (100 mg/kg initial TNT concentration) and observed a complete
TNT remediation within 60 days.
6.2 Phytoremediation of RDX
RDX is fairly soluble and mobile in the environment, as it does not bind well to
organic or soil fractions. Therefore, it is readily translocated to shoots and leaves
of the plants after its uptake as compared to TNT (Thompson et al. 1999). About
70 % of RDX was accumulated in the aerial parts of the plant. Photolytic trans-
formation, that occurs in the aerial parts of the plant, is the primary mechanism of
transformation during the degradation of RDX (Just and Schnoor 2004). Photolysis
occurs mainly when water containing organic contaminants is taken up by plants
and is released into the air through their leaves. Phytophotolysis phenomenon for
the mineralization of RDX in poplar plant tissues was also observed by Van Aken
et al. (2004). According to them, the mineralization of RDX occurs in three steps
(1) a light independent reduction of RDX to MNX (hexahydro-1-nitroso-3,5-
dinitro-1,3,5-triazine) and DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine)
by plant cells, (2) a plant/light mediated breakdown of RDX, MNX, or DNX into
metabolites (formaldehyde and methanol), (3) a light-independent mineralization

of metabolites to CO2. The uptake of RDX by plants generally occurs, because log
Kow of RDX is 0.87 (Burken and Schnoor 1997; Talmage et al. 1999). Brentner
et al. (2010) monitored the translocation and transformation of RDX in the plant
leaves by phosphor imager autoradiography.
Thompson et al. (1999) studied plant uptake of RDX from both soil and
hydroponic system and reported that due to decreased bioavailability of RDX in
soil, the RDX uptake was slower than in the hydroponic system. About 71 % of
RDX was taken up by hybrid poplar from the hydroponic system in 7 days. Similar
observation was also made earlier by Harvey et al. (1991). They reported that less
than 16 % of RDX was taken up by bush beans (Phaseolus vulgaris) from soil
after 60 days, while 60 % was removed from the hydroponic system by the same
plant after 7 days. It was also observed that the transformation and translocation of
RDX were different from its co-contaminant TNT (Adrian et al. 2003).
Thus, various studies have confirmed the uptake of energetic substances, such
as RDX and TNT, by many plant species i.e. terrestrial, agronomic and wetland
(Price et al. 2002; Vila et al. 2007a, b). Many agricultural crops such as lettuce
(Lacutca sativa), tomato (Lycopersicon esculentum), corn (Zea mays), and cyperus
(Cyperus esculentus) also play an important role in the removal of RDX through
accumulation (Larson 1997).
Bhadra et al. (2001) studied the uptake and transformation of 8 mg/L RDX by
two plants i.e. Catharanthus roseus hairy root cultures and whole Myriophyllum
aquaticum (parrot feather) and found that both plants have a high potential to
remove RDX from the hydroponic system. They also pointed out that C. roseus
had an intrinsic capability for removal of RDX. Similar observation for RDX
removal by C. roseus and production of bound metabolites, was also made by
Hughes et al. (1997). The formation of polar metabolites and bound residues of
RDX metabolites was also observed during transformation of RDX (Hannink et al.
2002; Just and Schnoor 2004). Besides, mineralization of RDX was also reported
by Van Aken et al. (2004). The production of mononitroso and dinitroso trans-
formation products in the plant tissues during RDX degradation by plants has also
been reported by other workers (Vila et al. 2007a; Reynolds et al. 2006; Larson
et al. 1999).
Thus, phytoaccumulation is the main process involved in the phytoremediation
of RDX, as more than 90 % of soluble residues of RDX were detected as the
parent compound (Price et al. 2002; Hannink et al. 2002; Vila et al. 2007a, b). In
contrast to this hypothesis, some workers have suggested that during phytoreme-
diation of RDX, phytodegradation may also act as a possible technology for its
removal. Panicum maximum was reported as an effective species for the removal
of RDX in Hawaii by Paquin et al. (2004). Lamichhane et al. (2012) observed that
in the presence of molasses, the phytoremediation of RDX by guinea grass
(Panicum maximum) was enhanced which resulted in RDX disappearance mainly
in the root zone.
6.3 Enzymes
Phytodegradation (phytotransformation) is a mechanism by which plants tissue

degrades contaminant by plant enzymes or enzyme co-factors (Susarla et al. 2002).
For example, nitroreductase and laccase are a few enzymes reportedly involved in
the breakdown of TNT and the metabolites are incorporated into new plant
materials (Schnoor et al. 1995). The reduction of nitro groups of nitroaromatic
compounds generally occurs through a group of enzymes known as nitroreductases
(Bryant and DeLuca 1991). These enzymes use flavin mononucleotide (FMN) or
flavin adenine dinucleotide (FAD) as prosthetic groups and nicotinamide adenine
dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH)
as reducing agents to catalyse the reduction of nitro-substituted compounds
(Bryant et al. 1981; Bryant and DeLuca 1991).
According to Schnoor et al. (1995), Myriophyllum aquaticum, an aquatic plant,
produces a nitroreductase enzyme which mediates the partial degradation of TNT
(128 ppm) within one week in the flooded soil. Transgenic plants are found to be
more efficient degrader of explosive compounds than the wild type. These plants
minimize the phytotoxic effects by expressing the bacterial genes which are
reported to be involved in the degradation of TNT and RDX (Rylott and Bruce
2009; Van Aken 2009). For example, when pentaerythritol tetranitrate (PETN)
reductase gene from Enterobacter cloacae strain PB2 was expressed in transgenic
tobacco, an improved tolerance of transgenic tobacco to TNT was reported.
Similarly, when bacterial nitroreductase gene (nfsI) was expressed in transgenic
tobacco, the degradation of TNT was found much faster than the control plants
(Hannink et al. 2001) as shown in Fig. 6. Van Dillewijn et al. (2008) had also
reported that when Pseudomonas strain containing nitroreductase gene (pnrA) was
expressed in poplar plant, it resulted in increased uptake of TNT from both water
and soil.
Gandia-Herrero et al. (2008) also observed overexpression of many important
enzymes in plants in response to nitroaromatic compounds through microarray and
other gene expression assay. According to them, phytoremediation of TNT can be
improved not only by upregulating genes involved in the nitroreductase step, but
also in the conjugation step. During microarray analysis, an over-expression of two
uridine glycosyl transferases from Arabidopsis has resulted in both conjugate
production and TNT detoxification. Similarly, oxophytodienoate reductases
(OPRs) and glutathione-S-transferases (GSTs) were also upregulated in A. thali-
ana and Populus trichocarpa, respectively, in response to TNT exposure (Mezzari
et al. 2005). Gene expression analysis in poplar trees exposed to TNT has clearly
shown that genes for glutathione S-transferases may be mainly responsible for
detoxification of TNT in the plants (Brentner et al. 2008).
Rylott et al. (2006) reported that Rhodococcus rhodochrous 11Y had xplA gene
(CYP177) encoding an enzyme, known as flavodoxin-cytochrome P450 which
played a central role in the biodegradation of RDX. The tolerance and removal of
RDX from soil was found ten times higher when xplA gene was expressed in the
Nitroreductase 2
nitroso dinitrotoluene hydroxylamino dinitrotoluene aminodinitrotoluene

(NODNT) (HADNT) (ADNT)
Pentaerythritol
tetranitrate reductase
hydride-Meissenheimer dihydride-Meissenheimer
complex (H- - TNT) complex (2H- - TNT)
Fig. 6 Proposed transformation pathway of TNT by pentaerythritol tetranitrate reductases and

nitroreductase (Williams et al. 2001)
transgenic Arabidopsis plant as compared to non-transgenic plant. Jackson et al.

(2007) also reported that the transformation of RDX by plants could be increased
by 30 folds through the co-expression xplA and xplB (a flavodoxin reductase) in
transgenic plants as compared to xplA alone. Similarly, the co-expression of nfsI
and xplA genes in Poplar plants increased the removal of both TNT and RDX.
6.4 Rhizodegradation
In phytoremediation, the degradation of pollutants is mainly mediated by rhizo-

spheric microbes, a process called rhizodegradation. Rhizosphere is a zone
between roots and soil which can be characterized by low redox potentials,
abundant energy and nutrients, low pH, and high microbial activities due to root
activities. The rhizoremediation is generally mediated by three step processes i.e.
(a) sequestration or immobilization or retention of toxicants within a confined area,
(b) removal of contaminants from the soil/waste water, and (c) destruction/deg-
radation of organic pollutants by plant-microbial association. The contaminated
soil is mainly treated by these three strategies either individually or in combination
with each other. The microbes found in the plant roots are also involved in
xenobiotic metabolism. Both bacteria and fungi present in the rhizosphere show
catabolic activity mediated by the enzymes involved in the degradation process.
Organic chemicals, released from both living and dead roots, used to modulate
enzyme activity. It was observed that neither a single plant or microbe worked
extremely well in immobilization, removal and destruction properties, nor a single
species had shown faster degradation of organic contaminants. Hence, the con-
taminated soil may be successfully treated by a combination of plant species with
appropriate remediation properties, aided by rhizospheric communities (bacteria
and fungi) which are active against the specific contaminants present in the soil.
The microbes in the rhizosphere have been also observed to possess plant growth
stimulating properties (Campbell and Greaves 1990). They may fix nitrogen,
synthesize siderophores and phytohormones, like auxins and cytokinins, and sol-
ubilize soil minerals for the plant growth (Glick 2003). The beneficial microor-
ganisms in the rhizosphere are closely attached to the plant roots and are also
known as plant growth promoting bacteria (PGPR). These microbes play an
important role in recycling of plant nutrients, maintenance of soil structure,
detoxification of noxious chemicals, and control of plant pests (Mackova et al.
2006; Rajkumar et al. 2009, 2010). Among the rhizospheric microorganisms, the
Plant Growth Promoting Rhizobacteria (PGPR) and Arbuscular Mycorrhizal Fungi
(AMF) have gained prominence all over the world to treat the contaminated soil
(Ma et al. 2011). The microbes in the rhizosphere utilize root exudates containing
sugars, organic acids and amino acids as source of nutrient and energy (Vancura
and Hovadik 1965). The growth and activity of microbes in the rhizosphere can be
increased by specific plant species which target the biodegradation of explosive
contaminants. The rhizospheric microbes also reduce the plant toxicity of explo-
sives through increased biodegradation process of explosives.
Several studies have reported that grass species harbour a large population of
bacteria in their vigorous root system which are found suitable for rhizodegra-
dation. This process can be enhanced by increasing aeration of the soil by the plant
roots, which penetrate the soil with highly developed fine roots and also by
enhancing the contact of colonized bacteria with the organic pollutants (Kuiper
et al. 2004). Yang (2010), during his experiment on rhizodegradation of TNT and
RDX by two selected Missouri native grasses i.e. eastern gamma grass
(EG, Tripsacum dactyloides) and switchgrass (SW, Panicum virgatum L.),
observed that when 14C-spiked RDX and TNT rhizosphere soils were incubated
for 8 weeks, both grasses were able to stimulate the rhizodegradation of RDX,
TNT and its metabolites. More than 13 % of applied RDX was converted into CO2
as compared to 5 % as observed in the control. Eastern gamma grass was found
more effective in augmenting RDX rhizodegradation than switch grass. But in case
of TNT, more than 95 % of applied TNT was degraded in the first 7 days, but less
than 2 % TNT was transformed into CO2 and six major degradation metabolites
were identified. In contrast to RDX degradation, switchgrass appeared to be more
effective for degrading TNT than eastern gamma grass.
The degradation of explosive compound can also be enhanced by inoculating
bacteria in the rhizosphere soil. The bacteria can be acclimatized in the rhizo-
sphere by inoculating bacteria with coating seeds in the rhizospheric zone.
Pseudomonas spp., are predominant plant growth promoting bacteria found in the
rhizosphere. Other than bacteria, fungi are also reported to colonize plant roots,
and increase the plant uptake of nutrients. The synergistic interaction between
microbes and plants enhances the feasibility of the application of phytoremediation
technology on a large scale at relatively high explosive concentrations. Yang

(2010) reported that when 14C-TNT spiked rhizospheric soils of both eastern
gamma grass and switch grass were inoculated with Pseudomonas putida KT2440
and incubated for 8 weeks, more than 90 % TNT disappeared in the first 7 days
and less than 1.2 % TNT was mineralized into harmless CO2. It was also observed
that TNT degradation followed a second order kinetics of degradation in soils.
Although P. putida KT2440 suppressed mineralization, more non-extractable
residues were detected in the soil. Overall, switchgrass with P. putida KT2440
acted to possess the best capacity to degrade TNT in soil.
7 Conclusions
Recently, phytoremediation has gained importance as an eco-friendly and self-

sustaining technology against the conventional technologies available. It has got
many advantages which include in situ applicability, low costs, no need for spe-
cific equipment and no introduction of new chemical substances into the envi-
ronment to deal with various environmental toxicants. Over the years, there has
been a substantial increase in our knowledge of the mechanisms involved in the
uptake, transport, and detoxification of pollutants by plants and their associated
microbes. However, phytoremediation efficiency has been not fully explored due
to lack of our knowledge about basic plant processes and plant microbe interac-
tions. Besides, there is a need for more phytoremediation field studies to dem-
onstrate its effectiveness for enhanced acceptance by the public.
Several researches are still ongoing for the development of plant-based reme-
diation technology to make it a commercially viable industry. However, some key
technical hurdles have to be overcome to make it a commercially viable tech-
nology. These include identifying more species with high remediation potentials,
optimizing phytoremediation processes, such as appropriate plant selection and
agronomic practices, understanding more about how plants uptake, translocate,
and metabolize contaminants, identifying genes responsible for uptake and/or
degradation for transfer to appropriate high-biomass plants, decreasing the length
of time needed for phytoremediation to work, devising appropriate methods for
contaminated biomass disposal, particularly for heavy metals and radionuclides
that do not degrade to harmless substances, and protecting wildlife from feeding
on plants used for remediation.
Although, plants have the ability to remove explosives from the environment,
but the processes for removal and transformation of TNT, RDX and HMX are
different from the metals and regulated by enzymes and other several factors. Their
products are conjugated and stored largely in vacuoles and cell walls of plants.
Transgenic plants are specifically designed with bacterial genes to have a higher
phytoremediation capacity than wild-type plants. Transgenic lines are more
resistant to the toxic effects of RDX and TNT, take up higher quantities of
explosives and more effectively degrade these substances. But, research on the use
of transgenic plant lines in phytoremediation is still in the laboratory phase. The

next phase would include experiments in the greenhouse and field trials to test
their efficacy in explosive transformation and mineralization.
No doubt, transgenic studies are very encouraging for the future of phyto-
remediation technology. The enhanced metabolism of GTN and TNT, as dem-
onstrated in transgenic tobacco, indicates that the introduction of PETN reductase
and the bacterial nitroreductase into grasses or fast-growing deep-rooted trees,
such as poplars, more suitable for phytoremediation purposes, could significantly
increase explosive removal in the field application. Besides, use of specific plant
promoters to direct transgene expression to specific plant tissues is of great
potential interest in enhancing the phytoremediation properties of plants. Appli-
cation of new genomic technologies will provide an invaluable help in the iden-
tification of genes which enable explosives tolerance and their regulatory systems.
The identification of enzymes mediating the detoxification systems will provide
new targets for future rounds of genetic engineering which may provide robust
plants for degradation of explosive compounds. Thus, a multi-approach is needed
targeting to develop efficient plants for phytoremediation of explosive compounds.
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About the Editor
I have been presently working as Chief Scientist and Area Co-coordinator, Plant
Ecology and Environmental Science Division at CSIR—National Botanical
Research Institute (NBRI) Lucknow—a premier R & D laboratory of plant
sciences in the network of Council of Scientific and Industrial Research, New
Delhi. During my 31 years of research career, I have worked in the areas of air
pollution, monitoring and mitigation, climate change as well as bioremediation
and biodegradation of organic wastes.
My interest in microbial degradation was generated when we started working on
a research project on biodegradation of oily sludge sponsored by Government of
India. In this project, we studied biodegradation and biotransformation of several
recalcitrant alkanes and polyaromatic compounds usually found in oily sludge in
laboratory conditions by potential individual bacterial strains and their several
combinations. Apart from degradation study, we also elucidated the degradation
pathways with involvement of several degradative enzymes through proteomics.
Subsequently, we also carried out microcosmic study by a combination of high
degrading fungal and bacterial strains in optimum conditions with biostimulation
and bioaugmentation. Now this study has to be scaled up to pilot scale to develop a
microbial technology for degradation oily sludge or oil spills for field application.
Working on this aspect, we have published more than dozen papers in high impact
journals and a few more are in the pipeline. Enthused with this success, we have
now targeted microbial degradation of polychlorinated biphenyls (PCBs) largely
used in transformer oil, adhesives, paints etc.
In 1992, I visited UK for six months to work on plant responses to elevated
levels of CO2 and temperature at Institute of Ecology, Bangor. Again in 2006, I
attended a workshop in USA on Agricultural Air Quality: State of Science and
delivered a lecture on invitation on GHGs emission from crop fields.
Working on these aspects, I published 75 research papers in the international
journals of repute with good impact factor and four edited volumes, such as Trace
Gas Emission and Plants published by Kluwer Academic Publishers in 2000,
Environmental Bioremediation Technologies in 2007, Climate Change and Crops
2009 and the latest one Microbial Degradation of Xenobiotics in 2012 by Springer,
Germany.

Environmental Science and Engineering, DOI: 10.1007/978-3-319-01083-0,
Subject Index
0–9 2-amino-4,6-dinitrotoluene (2-ADNT), 213,

a-TNT, 71 375, 380
ß-galactosidase activity, 1 2-amino-4-formamide-6-nitrotoluene, 213
p-electrons, 90 2-hydroxyamino-4,6-dinitrotoluene
1,2-GDN, 47–49, 51–53 (2HA46DNT), 243
1,3,5-TNB, 381 2-N-acetoxyamino-4,6 dinitrobenzaldehyde,
1,3-GDN, 47, 49–51, 153 376
13
C enrichment, 272, 274, 277–279 2-nitrotoluene 2,3-dioxygenase (2NTDO), 183
14
C-GTN, 55 3,30 -dimethylbutan-2-yl)-methylphosphonoflu-
15
N enrichment, 271, 272, 274, 275, 278, 279 oridate, GD, 179
3
16S rDNA gene sequences, 6 H-3He method, 274
16S rRNA gene-targeted oligonucleotide 3-methyl-4,6-dinitrocatechol, 211
microarray (RHC-PhyloChip), 184 4,6-diamino-2-nitrotoluene, 376
1-methyl-2,4,6-trinitrobenzene, 372 4,6-dinitrohexanoate, 7, 8
2,20 ,6,60 -tetranitro-4,40 -azoxytoluene and 4-ADNT, 20, 28, 30, 122, 215, 340, 378
4,40 ,6,60 -tetranitro-2,20 -azoxytoluene, 243 4-ADNT degradation, 340
2,4-dinitro-6-hydroxy-benzyl alcohol, 223 4-amino-2,6-dinitrotoluene (4A26DNT), 243
2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexa- 4-amino-2,6-dinitrotoluene (4-ADNT), 213,
azaisowurtzitane (HNIW), 89 375, 380
2,4,6-triaminotoluene (TAT), 16, 18, 203 4-aminodinitrotoluene sorption, 326
2,4,6-trichlorophenol monooxygenase, 4
2,4,6-trinitrophenol (TNP), 1, 3, 7, 89
2,4,6-trinitrotoluene, 7, 9, 15, 71, 73, 74, 179, A
201, 202, 205, 235, 239, 241 ABC transporters, 375
2,4,6-trinitrotoluene (TNT) Abiotic alkaline hydrolysis of RDX, 273, 278
2,4-DANT, 28, 122, 327, 341, 363 Abiotic degradation of energetics, 315
2,4-DANT degradation, 341 Abiotic mechanism, 18, 55
2,4-diamino-6-nitrotoluene, 30, 376 Abiotic reduction of RDX, 316
2,4-dinitro-6-hydroxytoluene, 223, 376 Abiotic transformations, 288, 303
2,4-dinitroanisole (DNAN), 117, 179 Abuliton avicennae, 380
2,4-dinitrophenol, 2, 6, 8 Acetobacterium paludosum, 74, 76, 118
2,4-dinitrotoluene (2,4-DNTA), 77 Achromobacter, 26, 119
2,4-DNP, 6, 8 Acinetobacter sp, 20
2,4-DNP-degrading strains, 6 Actinomycetes, 16, 71–73
2,6-diaminodinitrotoluene sorption, 327 Activated carbon, 24, 46, 78, 113, 128, 151,
2-amino-4,6-dinitrobenzoic acid, 376 165, 373
2-amino-4,6-dinitrotoluene, 25, 30, 126, 223, Activated sludge, 49, 54, 59, 78, 113, 128,
243, 379 151, 165, 373

Environmental Science and Engineering, DOI: 10.1007/978-3-319-01083-0,
396 Subject Index
Advanced oxidation processes, 127, 237 Barnyard grass (Echinochloa crusgalli), 241,
Aerobic biodegradation, 71, 208, 275, 288 380
Aerobic degradation of nitroexplosives, 71 Behavior of GTN, 45
Aerobic denitration, 272, 278 Benzo[a]pyrene contamination, 115
Aerobic nitrobenzene-degradation pathways, Bioassays, 130
277 Bioaugmentation, 25, 56, 116, 124, 129, 155,
Agrobacterium radiobacter, 51, 53, 59, 153 180, 218
Aldehyde oxidase, 117, 207, 221 Bioavailability restrictions, 273
Alfalfa (Medicago sativa), 239, 240, 241 Biochemical transformation, 223
Alkaline hydrolysis of RDX, 265 Biodegradation, 49, 69, 73, 74, 96, 115, 119,
Alkaline-hydrolysis degradation of HNIW, 131, 151, 168, 181, 187, 237, 289, 292, 316
289 Biodegradation of GTN, 52, 153
Amino metabolites, 117 Biodegradation of HMX, 77
Amino-4,6-dinitrotoluene(2-ADNT), 213, 373, Biodegradation of nitroaromatic compounds, 2
380 Biodegradation of TNT, 28, 114, 315
Amino-dimethyl-tetranitrobiphenyls, 302, 308 Biodegradation of TNT by fungi, 213
Aminodinitrotoluene (ADNT), 73 Biodegradation pathways, 73, 293
Ammonium perchlorate (NH4ClO4), 166 Biological degradation, 70, 71, 204, 371, 374
Anaerobic bacteria, 17, 70, 75, 118, 286 Biological methods, 24, 26, 70, 71, 301
Anaerobic bacterial consortia, 50 Biological perchlorate treatment, 175
Anaerobic biodegradation, 96, 123, 207 Biological treatment method, 170
Anaerobic degradation of nitroexplosives, 76 Biological treatments of TNT, 219
Anaerobic denitrifier, 286 Biologically-mediated degradation, 56
Anaerobic digester, 51, 77, 155 Biomagnification, 129, 248
Anaerobic incubation, 76, 115 Bioreactor processes, 25
Anaerobic microcosms, 156 Bioreactors, 49, 123, 124, 170, 224
Anaerobic nitro group reduction, 272 Bioreduction of perchlorate, 170
Anaerobic reduction of RDX, 278 Bioremediation approaches, 122, 135, 136
Anaerobic sludge, 77, 158, 319 Bioremediation of explosive chemicals, 201
Anaerobic treatment, 25, 77, 124 Bioremediation of FOX-7 wastewater, 78
Anion exchange, 167 Bioremediation of GTN, 47, 54
APCI-MS mass spectrometry, 304 Bioremediation of TNT, 16, 218, 223, 302
Application of CSIA, 262, 274 Bioremediation strategy, 120
Arabidopsis thaliana, 215, 249 Bioremediation technologies, 24
Arbuscular mycorrhizal fungi (AMF), 385 Bioreporters, 190
Aromatic amino derivatives, 373 Biosensors, 80, 121
Aromatic ring reduction of TNT, 302 Bioslurry, 47, 123, 219, 224
Arthrobacter, 3, 52, 114, 118, 155 Bioslurry treatment, 123, 220
Arthrobacter protophormiae RKJ100, 74 Biostimulants, 155
Atmospheric pressure chemical ionization Biostimulated zone, 314
mass spectrometric (APCI-MS) detection, Biostimulation, 25, 114, 180, 218, 221
303 Biotic degradation of CL-20, 102, 286
Autoradiography analysis, 243, 245 Biotransformation, 17, 31, 47, 51, 76, 102,
Azoxy-product formation, 243 114, 150, 218, 240, 295, 373
Azoxytetranitrotoluene, 18, 208, 213 Biotransformation of RDX, 75
Bjerkandera adusta, 20
Brevibacterium linens, 74
B Bulking agents, 56
Bacillus thuringiensis, 154 Burkholderia cepacia SH-1, 221
Bacillus thuringiensis/cereus, 51, 53
Bacterial genes encoding enzymes, 249
Bacterial metabolism, 49, 50 C
Bacterial nitroreductase gene (nfsI), 383 Cabbage (Brassica rapa), 239
Bahiagrass (Paspalum notatum), 243, 252 Cabbage leaf extract, 115
Subject Index 397
Caenorhabditis elegans, 184 Corynebacterium simplex, 91

Capillary electrophoresis, 163 Coupled degradation, 331
Carbon isotope signature, 274 Cress (Lepidium sativum), 239
Carbon monoxide dehydrogenase, 207, 221 Cryoprotectants, 31
Carbon skeletons, 221 CTSPAC model, 380
Carbon–carbon bonds, 286 c-type cytochromes, 101
Cardiac microcirculation, 149 Culture-independent molecular tools, 180
Cardiovascular diseases, 149 Cunninghamella echinulata var elegans, 119
Carex gracillis, 380 Cyclic nitramine explosives, 74, 77
Cartharanthus roseus, 379 Cyclic nitramines, 71, 75, 76, 88, 97, 106, 287
Catabolic diversity, 180 Cyclodextrin, 115
Catabolic pathways, 9, 105, 118 Cyctochrome, 69
Catabolic protein, 187 Cyperus esculentus, 382
Cell-based biosensors, 190 Cytochrome P450, 74, 75, 97, 126, 248
Central nervous system (CNS), 95 Cytochrome P450 mono-oxygenase, 375
CF-IRMS, 265 Cytochrome P450 system, 76, 97
Chemical warfare agents (CWAs), 179 Cytochrome P450s, 248
Chemotaxis-mediated biodegradation, 77
Chlorinated aliphatic compounds, 165
Chlorinated aliphatics, 262 D
CL-20, 74, 77, 89, 253, 322, 329, 344 DCB-extractable, 325, 330
CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12- Dearomatization of the benzene ring, 277
hexaazaisowurtzitane), 67 Dechlorosoma (Azospira), 273
CL-20 alkaline hydrolysis, 289 Degradation half-life, 320, 326, 350, 355
CL-20 contamination, 288 Degradation of CL-20, 105, 292, 344
CL-20 degradation, 104, 115, 286, 363 Degradation of HMX, 71, 72, 74, 77
CL-20 degradation pathway, 322, 345 Degradation of TNP, 90, 92–95, 124
CL-20 mineralization, 292, 347, 363 Degradation products, 24, 26, 30, 158, 238,
CL-20 nitroso-functional groups, 322 243, 246, 247, 288, 236, 246, 248
CL-20 sorption, 329 Dehydrogenase enzyme, 75, 295
CL-20 toxicity, 101 Dehydrogenases, 117
Cladosporium resinae, 119 Denaturing gradient gel electrophoresis
CLE, 115 (DGGE), 6, 30, 182, 206
Clostridium acetobutylicum, 23, 75, 118, 183 Denitration, 25, 47, 49, 53, 117
Clostridium bifermentans KMR-1, 31 Denitration of GMN, 52
Clostridium pasteurianum, 4 Denitration of GTN, 48, 50, 51, 153
Clostridium sp., 17, 75, 77, 102, 287 Denitration pathways, 51
Co-expression of nfsI and xplA, 384 Denitrifying bacteria, 77
Combustion reactor, 263 Denitrohydrogenated intermediate
Co-metabolic degradation of TNT, 338 (C6H7N11O10), 104
Common reed (Phragmites australis), 242 Designer microorganisms, 189
Complete degradation of TNT, 217 Desorption-resistant, 359
Complete mineralization, 4, 49, 50, 114, 131, Desulfovibrio, 77, 118, 220
156, 246 Desulfovibrio gigas, 4
Complete NG mineralization, 156 Detonation of ordnances, 204
Composting, 23, 24, 47, 56, 121, 218 Detoxifying enzymes, 74, 76
Compound-specific isotope analysis (CSIA), Diamino derivatives, 208
259 Diaminodinitroethylene (FOX-7), 67, 72
Conjugation, 120, 186, 215, 243, 383 Diaminonitrotoluene, 73, 207
Conjugation of formaldehyde, 378 Diaminonitrotoluenes (DANT), 16, 23, 204
Conjugation of p-bonds, 295 Diarylamines, 19, 21, 302, 308
Constructed wetland treatment, 124 Dihydride meisenheimer complexes, 21, 22
Conventional bioreactors, 170 Dihydrophilic amide dehydrogenase, 221
Corn steep liquor, 115 Dinitroaminotoluenes, 203, 340
398 Subject Index
Dinitroazoxytoluenes, 122, 203 Explosives, 1, 3, 15, 24, 30, 39, 46, 68, 116,
Dinitroglycerine (DNG) isomers, 248 117, 202, 248
Dinitroglycerine isomers, 248, 253 Explosive compounds, 67, 252, 371, 373
Diode-array (DAD), 303 Explosive organic compounds, 262
Dinitrotoluenes, 71, 307, 309 Extracellular fluid, 292
Dioxygenase, 2, 7, 70, 117
Dissimilatory sulfite reductase b-subunit, 182
Dithionite chemical treatment, 138 F
Dithionite reduction, 350 F420-dependent reductase, 8
Dithionite-reduced sediments, 335–338, 363 Facultative anaerobic organisms, 220
DNA-microarray, 184 Fate of explosives, 241
DNG isomers, 53 Fermentation inhibitors, 158
DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5- FGA-based detection, 184
triazine), 381 Field scale remediation, 360
Dynamites, 41 Film packed-bed bioreactor, 171
Fingerprint of bioreporter signals
Fingerprinting techniques, 181
E Flavin adenine dinucleotide (FAD), 105, 217,
Earthworms, 68, 122, 129, 237, 340 383
Echinochloa crusgalli, 241, 280 Flavin mononucleotide (FMN), 22, 217, 383
Ecotoxicological considerations, 129 Flavin-moiety (FMN), 105
Electrochemical reduction, 167, 169 Flavobacterium, 3
Electrodialysis, 167, 169 Flavodoxin-cytochrome P450, 251, 383
Electrolysis of brine, 169 Flavodoxin reductase, 251, 384
Electron acceptors, 75, 118, 175 Flavoproteins, 22
Electron deficient molecule, 203 Flax (Linumus itatissimum), 248
Electron donors, 77, 114, 123, 172 Flax seed, 57
Electron shuttle-mediated RDX reduction, 101 Fluidized-bed bioreactors, 171
Electrophilic characteristics, 206 Fluorescence in situ hybridization (FISH), 181
Electrospray mass, 9 Fluorescent conjugated polymers, 191
Enchytraeus albidus, 101, 122 Fluorescent single strand conformation poly-
Endophyte-assisted phytoremediation, 126 morphism (F-SSCP), 182
Energetic compounds, 39, 113, 135, 314, 332 Fluxomics, 188
Energetic residues, 45, 372 Functional gene arrays (FGAs), 184
Energetics, 285, 313, 358, 360 Fungal culture, 293
Enhanced biodegradation of energetics, 314 Fungal degradation of nitrate esters, 156
Enhanced biodegradation of RDX, 315 Fungal metabolism, 52, 288
Enrichment factor, 261, 273 Fusariumsolani, 156
Enterobacter agglomerans, 51, 53, 155
Enterobacter cloacae, 22, 59, 73, 249, 306
Enterobacter cloacae PB2, 118, 154, 217 G
Environmental health hazard assessment, 164, Gasoline additives, 262
167 GC-IRMS method, 264, 265
Environmental pollutants, 237, 259, 261 GDN, 47–53, 55, 57, 59, 153–158
Environmental protection agency, 201, 237, Gene cloning and sequencing, 187
285, 371 Gene expression, 183–185, 243, 248
Enzymatic hydrolytic ring cleavage, 96 Gene expression analysis, 383
Enzymatic mechanisms, 248 Genes-encoding nitroreductases, 118
Enzymatic pathways, 152 Genetic variability, 126
Enzymatic production of 1-H--TNT, 306 Genetically engineered organisms, 194
Enzyme engineering, 185 Genetically modified plants, 237, 249, 252
Escherichia coli, 17, 22, 75, 104, 118, 183 Geno-toxicological assays, 129
Ethylene glycol dinitrate (EGDN), 158 Geobacillus thermoglucosidasius, 4
Ex situ bioremediation, 121 Geochip’ microarray, 184
Subject Index 399
Geotrichium candidum, 156 Hydride transferase II (HTII), 94

Glutamic-oxaloacetic transaminase, 206 Hydride-meisenheimer complex, 3
Glutathione S transferases (GST), 244 Hydrodynamic conditions, 358
Glutaraldehyde-binding processes, 244 Hydrogenases, 75, 117, 221
Gluteraldehyde, 332, 350 Hydrogenation of H-TNP, 91
Glycerol dinitrates (GDNs), 47, 154 Hydrogen-peroxide-generating oxidases, 289
Glycerol kinase, 53 Hydrolytic pathway, 54
Glycerol trinitrate (GTN), 67, 149 Hydroquinone pathway, 3
Glycosyltransferase, 215 Hydroxyamino-2,6-dinitrotoluene
GMN, 47, 49, 52, 154, 157 (4H26DNT), 243
GMN isomers, 50, 51, 155, 156 Hydroxylamines, 71, 378
Gradient oxic column, 354 Hydroxylamino dinitroluene, 21
Green liver model, 375, 376 Hydroxylamino metabolites, 117, 208
Green technology, 252s Hydroxylamino-2,6-dinitrotoluene, 213, 217,
GTN, 39–41, 45, 54, 151 223, 376
GTN biotransformation, 49, 59, 154 Hydroxylamino-dinitrotoluenes (HANTs), 17,
GTN degradation, 47, 59, 158 215, 302
GTN denitration, 49, 52, 59, 155 Hydroxylaminolyase, 2, 3
GTN metabolism, 47, 51 Hydroxypropyl-b-cyclodextrins, 116
GTN mineralization, 155 Hydroxyquinol 1,2-dioxygenase, 4
GTN treatment, 46, 53, 59 Hydroxyquinol pathway, 3
GTN-acclimated cultures, 50 Hyperthyroidism, 166
GTN-contaminated soil, 46, 50
I
H Immobilized cell bioreactors, 72
Helophytes, 241 Immobilized fungi, 124
Hepatotoxicity, 205 Immobilized microorganisms, 29, 32
Herbicide transformation, 317 Immune system dysfunction, 205
Heterocyclic nitramines, 235, 247, 371 Immunoassay, 212
Heterocyclic ring cleavage, 246 In situ bioremediation technologies, 237
Heterotrophic biological treatment, 172 Indian mallow (Abutilon avicennae), 242, 380
Heterotrophic conditions, 76, 118 Initrotoluenes, 17, 71, 134, 201, 302, 307, 309
Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine Intrinsic degradation mechanisms, 278
(MNX), 75, 246 Ion chromatography, 163, 280
HMX, 56, 67, 80, 89, 113, 115, 119, 122, 179, Ion exchange membrane bioreactor, 172
244, 253, 264, 313, 318, 336, 355 Isomers of 3,5-2H--TNT.H+, 302, 304, 308,
HMX mineralization, 320, 336, 362 309
HMX phytoremediation, 246 Isotope fractionation, 183, 261, 265, 273
HMX transformation products, 247 Isotope ratio mass spectrometer (IRMS), 263
HNitClO4 (complex of nitron and perchlorate), Isotopic composition, 260
168 Isotopic enrichment, 261, 273
HNIW degrader, 287 Isotopic shifts, 260, 261
Horizontal packed bed bioreactor Isowurtzitane cage, 286, 293, 295
(HPBBR), 77
Horizontal transfer of genes, 186
HPLC analysis, 49, 245, 247 J
Hydride complexes, 302, 304 Juncus glaucous, 242, 248, 380
Hydride ion transfer, 104, 287
Hydride meisenheimer complex, 8, 20, 70, 90,
117, 218, 303 K
Hydride meisenheimer r-complex, 94 Kinetic modeling, 289
Hydride pathway, 302 Kinetics of aerobic denitration, 156
Hydride transferase enzyme, 218 Klebsiella oxytoca, 72, 155
400 Subject Index
L Microbial degradation of CL-20, 20, 102

Laccases, 20, 23, 75, 374 Microbial degradation of nitroglycerol, 151
Lacutca sativa, 382 Microbial degradation of RDX, 95, 97, 104
Lag phase, 289, 291, 292, 359 Microbial degradation of TNP, 90, 94
LC-IRMS, 264, 280 Microbial entrapment, 26
Lepidium sativum, 222 Microbial fuel cell, 172, 190
Lignified tissues, 246 Microbial remediation of TNT, 220
Lignin peroxidase, 20, 75, 119, 213, 289 Microbial strategies, 32
Ligninolytic enzyme, 20, 213, 215, 291 Microbial toxicology, 206
Ligninolytic systems, 213, 215 Military energetic compounds, 259, 265, 273,
Linimumus itatissimum, 57 276, 280
Logistic kinetic model, 290 Mineralization of RDX, 115, 381
Low maintenance bioreactors, 172 Mineralization of [14C]-CL-20, 292
Low water saturation, 358 Mineralization of GTN, 50
Lycopersicon esculentum, 382 Mineralization of TNT, 20, 213, 338, 343
Lysobacter taiwanensis, 118 Minimal salt medium, 27, 97
Mitosporic fungi, 156
Mn-dependent peroxidase, 20
M MNX, 72, 96, 101, 331, 378, 381
M. aquaticum, 79, 246 Molasses, 26, 115, 315
Macromolecules, 249 Molecule fluorophores, 191
Madagascar periwinkle (Catharanthus roseus), Monoamino derivatives, 207
245 Monoaminodinitrotoluenes (ADNT), 16, 19
Maltophilia, 71, 101, 110 Monohydride-Meisenheimer complex, 302
Manganese peroxidases, 75 Mononitrate isomers, 49
Manganese-dependent peroxidases, 289 Mononitroglycerin (MNGs),, 150
Marine macroalgae, 126 Mononitroso derivative of CL-20 (IV), 295
MDNA degradation, 320, 335 Monooxygenase, 2, 7, 74, 117
Meisenheimer complexes, 16, 18, 304, 309 Munitions wastewater, 50
Membrane bioreactors, 175 Mutagenic properties, 206
Membrane-based techniques, 167 Mutase, 2
Metabolic by-products, 171 Mutation frequency, 186
Metabolic engineering, 185 Mycobacterium sp, 302
Metabolic enzymes, 89, 116 Myriophyllum aquaticum, 215, 241, 378, 383
Metabolic inhibitors, 23
Metabolic pathway engineering, 185, 188
Metabolic pathways, 73, 105, 286, 315, 338 N
Metabolic products, 80, 207, 221 N15-TNT, 25
Metabolism of explosives, 115 NAC biodegradation, 183
Metabolism of TNT, 75, 118, 207, 221 NACs, 179, 183, 185, 191
Metabolomics, 188 NACs-based explosives, 184, 185, 191
Metagenomic array (MGA), 184 NAD(P)H, 22, 54, 75, 213, 218
Metagenomic libraries, 185, 186 NADPH-dependent F420 reductase (NDFR), 94
Metagenomics, 183, 185 Nano-phytoremediation, 223, 381
Metaproteomics, 187 Nanoscale zero valent iron (nZVI), 223, 281
Metatranscriptome sequencing, 184 Nanotechnology, 223
Methaemoglobin, 3 Natural attenuation, 25, 45, 106, 116, 260
Methaemoglobinaemia, 68 Natural biodegradation, 314–316
Methanogenic conditions, 55, 76 NDAB, 72, 73
Methemoglobinemia, 3, 40, 150 NDMA, 317, 322, 350, 363
Methylene dinitramine (MDNA), 318 NDMA degradation, 322, 350, 353, 354
Methylobacterium, 73, 79, 118 NDMA mineralization, 351, 352, 354, 365
Microarrays, 183, 184 Negative inductive effect, 90, 94
Microbe-catalyzed reactions, 47 Nematoloma frowardii, 20, 213
Subject Index 401
Nitramine, 68, 70, 71, 77, 89, 118, 241 Organic contaminants, 55, 158, 371, 374, 381
Nitramine explosives, 68, 76, 264, 286, 378 Oxidation of haemoglobin, 206
Nitramines, 68, 70–74, 76–78, 96, 101, 118, Oxidative metabolism, 375
121, 179, 235, 264, 272, 280, 285–290, Oxidative phosphorylation pathway, 6
319, 372, 378 Oxidative stress, 129, 205
Nitramines detoxification, 248 Oxidoreductase, 20
Nitrate esters, 39, 48, 51 Oxidoreductase flavoproteins, 53
Nitrate reductase enzyme, 97 Oxido-reductases, 20
Nitric oxide, 52, 68, 303, 308 Oxophytodienoate reductases (OPRs), 244,
Nitrite elimination, 94 383
Nitro compounds, 16, 69, 70, 71, 291 Oxygen deficiency, 17
Nitro group reduction, 119, 271, 272, 301, 302, Oxygenolytic transformation, 372
308
Nitroaromatic compounds, 16, 22, 67, 75, 90,
117, 201, 271, 316 P
Nitroaromatics (NACs), 15, 22, 24, 89, 90, P. chrysosporium, 51, 52, 119, 156, 289, 291,
181, 201, 265, 271 292
Nitroaromatics (NACs)-based explosives, 179, P. fluorescens, 50, 53, 59
184, 185, 191, 192 P-450, 20, 69
Nitroaromatics degrading microorganisms, Packed bed bioreactor, 123
189 Packed bed reactors, 51, 54
Nitrobenzene, 2, 23, 71, 73, 183, 201, 271, 277 Pancytopenia, 371
Nitrobenzene reduction, 128 Panicum maximum, 381, 382
Nitrocatechol (MNC) monooxygenase, 188 Panicum virgatum, 381, 385
Nitrocellulose, 42, 52, 114, 149 Parrot feather (Myriophyllum aquaticum), 79,
Nitroexplosive waste waters, 68, 69, 71, 77 375, 376
Nitroexplosives, 67–69, 75, 76, 80 Pathways of TNT degradation, 303
Nitroglycerin, 149, 150, 158, 235, 240 PCR-denaturing gradient gel electrophoresis, 6
Nitroglycerin degrading ability, 152 Penicillium corylophilum, 52, 157
Nitroglycerine phytoremediation, 251 Pentaerythritol tetranitrate (PETN), 67, 118,
Nitronate monooxygenases, 76 179
Nitrophenol compounds, 1, 2, 3 Pentaerythritol tetranitrate (PETN) reductase
Nitrophenol degradation, 9 gene, 383
Nitrophenols, 1, 70, 71, 117, 201 Pentaerythritol tetranitrate reductase, 217, 306,
Nitroreductase gene (pnrA), 383 308
Nitroreductase NfsA, 249 Perchlorate, 59, 124, 163, 164, 166, 273, 276
Nitroreductases, 16, 21, 75, 205, 221 Perchlorate contaminated water, 172, 175
Nitroso-dinitrotoluenes (NSDNTs), 302 Perchlorate contamination, 164, 166, 170
Nitro-substituted explosives, 236 Perchlorate degradation, 172, 276
Nitrotoluene-responsive regulator NtdR, 186 Perchlorate kinetic barrier, 168
Nocardioides sp., 8, 94 Perchlorate reduction pathway, 170
Nonligninolytic conditions, 213 Perchlorate respiring bacteria (PRB), 164, 170
Novel partial reductive pathway, 186 Perchlorate treatment technologies, 167
npc genes, 5 Periplasmic proteins, 101
Nuclear magnetic resonance spectral Permeable redox barrier, 318
analyses, 9 PETN reductase, 57, 218, 387
Nutsedge (Cyperus esculentus), 246 PETNr enzyme, 217
Peudo-first-order reaction, 358
Phalaris arundinacea L, 381
O Phanerochaete chrysosporium, 51, 72, 102,
Octanol–water coefficients, 237 119, 156, 289
Octanol–water partition coefficient, 329 Phase I (transformation), 215, 375
Old yellow enzyme (OYE), 21, 53, 76, 217 Phase II (conjugation), 215, 375
Ordinance related compounds (ORCs), 179 Phlebia radiata, 214
402 Subject Index
Phosphonium moieties, 168 Pseudomonas putida, 19, 22, 28, 31, 50, 97,
Phosphor imager autoradiography, 246, 379, 115, 152, 155, 183, 220, 221, 250, 251, 386
381 Pulsed-field gel electrophoresis (PFGE)
Photoactivation of perchlorate, 168 analysis, 5
Photocatalysis, 127 Pyrazolo-pyrazine aromatic molecule, 295
Photochemical smog, 39
Photodegradation of CL-20, 294
Phragmitesaustralis, 158, 380 Q
Phylogenetic analysis, 6 Quantitative fingerprinting method, 183
Phylogenetic oligonucleotide arrays (POAs),
184
Phytoaccumulation, 238, 382 R
Phytodegradation, 57, 238, 374, 382 Radio chromatographic methods, 247
Phytoextraction, 125, 238, 374 Radio labeled tracers, 211
Phytophotolysis, 246, 381 Radiochromatogram, 28
Phytoplankton, 204, 206 Rayleigh equation, 262, 274
Phytoremediation, 25, 47, 57, 58, 78, 125, 243, RDX, 56, 59, 67, 76, 78, 89, 95, 119, 125, 179,
248, 373, 378 244, 325, 331, 354
Phytoremediation mechanisms, 158 RDX biodegradation, 97, 118, 275
Phytoremediation of explosives, 131, 222, 241 RDX mineralization, 315, 332, 336
Phytoremediation of nitroexplosives, 79 RDX phytoremediation, 244
Phytoremediation of RDX, 381 RDX sorption, 325, 326
Phytoremediation of TNT, 222, 378 RDX teratogenicity, 239
Phytoremediation process, 222, 224, 248, 386 RDX transformation, 246, 275, 335, 378
Phytoremediation technologies, 238, 248 RDX-degradation products, 246
Phytoslurry, 128 Reactive transport, 354, 358
Phytostabilization, 125, 238 Real-time monitoring biosensor
Phytotechnologies, 78 system, 191
Phytotoxicity of explosives, 239 Real-time PCR, 181, 182
Phytotoxicity of HMX, 240 Recalcitrant intermediates, 313
Phytovolatilization, 125, 238, 374 Recombinant penta erythritol tetra nitrate
Ping-pong bi–bi mechanism, 217 (PETN), 154
Plant growth promoting rhizobacteria (PGPR), Reductive degradation of TNT, 221
385 Reductive denitration, 51, 155
p-Nitrophenol (PNP), 3 Reductive transformation, 30, 74, 90, 376
PNP bioremediation, 115 Regioselectivity, 50, 51, 52, 188
PNP-degrading strains, 9 Regulatory challenges, 194
Polycyclic nitramine CL-20, 101 Remediation strategies, 113
Polymerase chain reaction (PCR), 27, 30 Remediation technologies for GTN, 46
Polystyrene immobilized bacteria, 29 Removal of nitroaromatics, 15
Populus deltoides x nigra, DN34, 243 Rhizodegradation, 57, 238, 374, 384
Populus fastigiata, 125, 380 Rhizofiltration, 57, 238, 374
Potamogeton pectinatus, 380 Rhizoremediation, 106
Poultry feathers, 115 Rhizosphere-enhanced phytoremediation, 58
Preventive approaches, 31 Rhizospheric communities, 385
Programmed bioremediation, 189 Rhizospheric degradation of nitroglycerin, 57
Properties and uses of nitroglycerin, 40 Rhodococcus opacus, 4, 118
Proteomics, 184, 187, 190 Rhodococcus sp. strain DN22, 75, 97
Providencia rettgeri, 71, 72, 74 Ribosomal intergenic spacer analysis
Pseudomonas aeruginosa, 28, 71, 218 (RISA), 182
Pseudomonas fluorescens, 22, 97, 186 Rieske-type dioxygenases, 188
Pseudomonas pseudoalcaligenes strain JS45, rRNA-targeted POA, 184
277 Ryegrass (L. perenne), 247
Subject Index 403
S TNB (1,3,5-trinitrobenzene), 77
Secondary metabolites, 291, 294 TNP degradation, 9, 90, 91, 95
Sediment-energetic aging, 358 TNP toxicity, 89
Selective bifunctional anion-exchange resin, TNP-degrading strains, 8
265 TNT, 15–31, 56, 67, 68, 71, 74, 79, 89, 113,
Self polymerization, 204 114, 116–119, 123, 125, 126, 179, 180,
Sequencing batch reactor, 50, 114 183, 202–204, 207, 213, 215, 218,
Sequential anaerobic–aerobic treatment, 314 220–224, 235, 237, 239, 242, 249, 252,
Sequential denitration, 49, 51, 153, 158 253, 274, 275, 302, 303, 304, 307, 309,
Sequential denitration pathway, 49, 153, 158 313, 326, 340, 357, 371, 372, 374, 375,
Sequential reduced-oxic sediment systems, 378, 380, 381, 383, 385, 386
354 TNT bioavailability, 130
Sex pheromones, 39 TNT biodegradation, 20, 30, 31, 115, 123, 187,
S-formyl-glutathione, 378 308, 315
Signal transmission, 186 TNT biotransformation, 26, 117, 124, 302
Single strand conformation polymorphism TNT chips, 220
(SSCP), 182 TNT conjugates, 215
Slurry bioreactor, 123 TNT contamination, 23, 31, 32, 79, 113, 205,
Soil columns, 54 206, 224
Soil organic carbon–water coefficient, 372 TNT degradation, 19, 23, 26, 27, 30, 115, 116,
Soil quality criteria, 128 122, 125, 128, 129, 220, 221, 224, 275,
Solidification point, 203 302, 309, 315, 316, 320, 326, 338, 356,
Solid-phase micro-extraction (SPME), 264 363, 379, 386
Sorption of energetics, 323 TNT degradation by bacteria, 17
Southern hybridization analysis, 5 TNT degradation by fungi, 19
Soybean (Glycine max), 240, 242, 245 TNT detoxification, 125, 215, 243, 383
Stable isotope probing technique (SIP), 183 TNT metabolites, 23, 27, 28, 30, 31, 125, 223,
Stenotrophomonas maltophilia, 118, 188 378, 379, 381
Subsequent oxidation, 318, 343 TNT mineralization, 116, 185, 338, 342
Sulfate-reducing bacteria, 77, 118 TNT reductase, 213
Superchannels, 189 TNT reduction, 114, 203, 205, 220, 265, 302,
Superoxide anion radical, 129, 205 303
Surfactants, 54, 116, 123, 186, 374 TNT reductive derivatives, 223
Switch grass (Panicum vigratum), 243 TNT transformation pathways, 302
Synthetic biology, 189, 190 TNT-dihydride complex, 21, 304, 309
TNT-hydride complexes, 302, 304, 306–309
TNX degradation, 335
T Total reductive capacity, 318
TAT, 118, 204, 207, 220, 320, 328, 338, 342, Toxic ammunition wastes, 373
363 Toxicity of explosives, 68, 129, 385
TAT degradation, 320 Toxicity of TNT, 25
TCE dechlorination, 317 Toxicogenomic techniques, 131
Terminal electron acceptors (TEA), 101 Transcriptional control, 186
Terminal restriction fragment length poly- Transcriptional fusion, 187
morphism, 181, 206 Transcriptional regulator DntR, 185
Testicular atrophy, 206 Transduction, 120
Tethered triphenylarsonium, 168 Transformation, 17, 21, 23, 28, 30, 31, 50, 52,
Tetranitroazoxytoluenes, 17, 28 53, 71, 74, 79, 90, 97, 113, 115, 119, 120,
Thermodynamic evaluation, 155 127, 154, 158, 206, 207, 213, 215, 218,
Thermophilic composting, 56 221, 223, 224, 247, 251, 264, 265, 272,
Thin layer chromatography, 49 286, 302, 303, 306, 308, 309, 315, 331,
Thyroid hormone, 166 335, 337, 363, 372, 374, 375, 376, 378,
TiO2-photocatalysis, 127 380–382, 384, 386
TLC purification methods, 246 Transformation of nitroglycerin, 153, 158
404 Subject Index
Transformation products, 19, 26–28, 215, 239, V

241, 243, 248, 249, 252, 303, 304, 382 Vasodilation, 150
Transgenic approach, 223 Vermicomposting, 122
Transgenic plants, 126, 218, 223, 248, 251, Vetiver grass (Vetiveria zizanioides), 125, 242
383, 384, 386
Transgenic tobacco, 126, 249, 383, 387
Treatment of contaminated soils, 24 W
Treatment of contaminated waters, 26 Water partition coefficients, 374
Triacetone triperoxide (TATP), 179 White rot fungi, 19, 20, 102, 105, 224, 291,
Triaminotoluene (TAT), 203, 207, 220, 320, 293, 295
338, 363 Whole-cell bioreporters, 190
Triaminotoluene sorption, 328 Wood rotting Basidiomycetes, 156
Triaminotrinitrobenzene (TATB), 67, 73
Trichloroethylene (TCE), 165
Triethylammonium-trihexylammonium, 168 X
Trinitroglycerin (TNG), 150 Xenobiotic challenge, 150
Trinitroglycerine, 149 Xenobiotic compounds, 87, 246
Trinitrotoluene, 16, 31, 67, 74, 204, 235, 237, Xenobiotic detoxification, 244
241, 242, 249, 301, 371, 381 Xenobiotic reductases, 97
Trinitrotoluene degradation, 338 Xenobiotics, 15, 25, 125, 126, 131, 158, 181,
Tripsacum dactyloides, 385 184, 188, 191, 252
Type I nitroreductase, 22, 220 x-omics’ data, 190
Type II (oxygen-sensitive) nitroreductase, 129 XplA-NR plants, 251
Type II hydride transferases, 22, 117 XplA-XplB, 252
XplA-XplB-NR genes, 251
U
UDP-glycosyltransferase (UGT), 244 Y
Ultra-high mass accuracy, 188 Y. lipolytica AN-L15, 308, 309
Uncoupler compound, 6 Yarrowia lipolytica AN-L15, 119, 303
Unexploded ordnance (UXO), 180, 371 Yellow foxtail (Setaria glauca), 248
Uptake and fate of nitramines, 244
Uptake and fate of TNT, 241
US Environmental Protection Agency Z
(USEPA), 1 Zea mays, 245, 374, 382
UTCSP model, 380 Zero-valent barriers, 317
UV–visible absorbance spectra, 304 Zero-valent iron nanoparticles, 157, 294, 316

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Shree Nath Singh Editor

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Biodegradation of Nitrophenol Compounds . . . . . . . . . . . . . . . . . . . . 1

Microbial Degradation of 2,4,6-Trinitrotoluene In Vitro and

Bioremediation of Nitroglycerin: State of the Science . . . . . . . . . . . . . 39

Bioremediation of Nitroexplosive Waste Waters . . . . . . . . . . . . . . . . . 67

Degradation of TNP, RDX, and CL-20 Explosives by Microbes. . . . . . 87

Assessment of Bioremediation Strategies

Bacterial and Fungal Degradation of Nitroglycrine. . . . . . . . . . . . . . . 149

Bioremediation of Perchlorate Contaminated Environment. . . . . . . . . 163

Bioremediation of Nitroaromatics (NACs)-Based Explosives:

Bioremediation of 2,4,6-Trinitrotoluene Explosive Residues. . . . . . . . . 201

Phytoremediation of Soil Contaminated with Explosive

Stable Isotope Tools for Tracking In Situ Degradation Processes

Biodegradation of Hexanitrohexaazaisowurtzitane (CL-20) . . . . . . . . . 285

Pathways of 2,4,6-Trinitrotoluene Transformation

In Situ Degradation and Remediation of Energetics TNT, RDX,

Phytoremediation of TNT and RDX. . . . . . . . . . . . . . . . . . . . . . . . . . 371

About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

Amy P. Gamerdinger Pacific Northwest National Laboratory, Richland, WA

Jim P. McKinley Pacific Northwest National Laboratory, Richland, WA 99352,

Patrick Shea School of Natural Resources, University of Nebraska, Lincoln, NE

Nobutada Kimura, Wataru Kitagawa and Yoichi Kamagata

Nitrophenol compounds have been used in a number of ways in medicines,

N. Kimura (&) W. Kitagawa Y. Kamagata

S. N. Singh (ed.), Biological Remediation of Explosive Residues, 1

2 Biodegradation of Nitroaromatic Compounds

2,4,6-trinitrophenol through formation of a hydride-Meisenheimer complex before

Fig. 2 Proposed pathway for PNP degradation by bacteria

reported. PNP was reductively converted into p-Aminophenol by Desulfovibrio

Fig. 3 Metabolic pathway for degradation of p-Nitrophenol by Rhodococcus opacus strain

This combination of degradative genes mixture converted 4-NP to hydroxyquinol

The authors also isolated PNP-degrading bacteria by aerobic batch enrichment

2,4-Dinitrophenol (2,4-DNP) is well known as an ‘‘uncoupler’’ compound

koreensis, while an other type was a relative of Nocardioides simplex FJ21-A

2.1.3 2,4,6-Trinitrophenol (Picric Acid)

2,4,6-Trinitrophenol (TNP) is used to be a main source of the military explosives.

Fig. 5 Proposed degradation pathway for Trinitrotoluene (TNT) by Rhodococcus erythropolis

In this chapter, current knowledge on the microbial degradation of nitrophenols

Lenke H, Pieper DH, Bruhn C, Knackmuss HJ (1992) Degradation of 2,4-dinitrophenol by 2

Shimizu S, Kobayashi H, Masai E, Fukuda M (2001) Characterization of the 450-kb linear

2,4,6-Trinitrotoluene (TNT) is a nitroaromatic explosive that was released into soil

S. N. Singh (ed.), Biological Remediation of Explosive Residues, 15

reduction of one or two nitro-groups of TNT to monoaminodinitrotoluenes

2.2 Microbial Degradation of TNT

2.2.1 TNT Degradation by Bacteria

Table 1 Degradation of TNT by microorganisms

CH3 CH3 CH3 CH3

NO2 NO NHOH NH2

2,4,6-TNT 4-Nitroso- 4-Hydroxylamino- 4-Aminodinitrotoluene

Fig. 1 Microbial transformation of TNT by reduction of nitro-groups

to 2,4,6-triaminotoluene (Boopathy and Kulpa 1994; Regan and Crawford 1994;

ring-reduction) may co-exist in the same cell, condensation reactions between

2.2.2 TNT Degradation by Fungi

Responsible for this degradation is the ligninolytic enzyme system of white-rot

2.2.3 TNT as a Source of Nitrogen or Electron Acceptor

Table 2 Microbial utilization of TNT as N-source or electron acceptor