Screening and Characterization of Nitroglycerin Degrading Microorganisms
Padmavathy S., Ananthi V., Praveen Raja P. and Asha Devi N. K. 1. Department of Zoology and Microbiology, Thiagarajar College (Autonomous), Madurai - 625009, Tamilnadu, India.2. Pharma Division, Aurolab, Madurai, India.Abstract
Biodegradation process is a novel and economically feasible one for the degradation of many toxic compounds present in the environment in a sustainable manner. Nitroglycerin degrading organisms were isolated from soil samples using enrichment technique. The nitroglycerin biodegradation assay was carried out using all the bacterial species capable of growing in minimal medium containing nitroglycerin. Among the 5 isolates, three isolates were found to be potent in nitroglycerin degradation. It was selected based on their efcacy in substrate utilization and spectrophotometric analysis. The crude enzyme was extracted from the selected isolates by cell lysis method. Nitroglycerin biodegradation assay was also carried out using the enzyme extracts and subjected to UV spectrophotometric analysis. The selected isolates were tentatively identied asArthrobacter sp., Agrobacterium sp.and Pseudomonas sp., as per the standard methods. Based on the results obtained above, one potent isolate N5 (Pseudomonas sp.,) was selected and utilized for chemical characterization studies. Chemical characterization of the degraded samples was done by FTIR analysis, which showed potential degradation of nitroglycerin carried out by Pseudomonas sp. Keywords: Biodegradation; Biotransformation; Recalcitrant; Nitroglycerin, glycerol trinitrate; Flavoprotein nitroester reductase.
hydrolysis yield glycerol, nitrite or nitrate. However, these techniques
suffer from high operational cost, the presence of excess reactants that
Nitroglycerin (NG) or Glycerol trinitrate (GTN) is an aliphatic nitrate
remain dissolved in the efuent, and the necessity for secondary
ester containing compound that is important for manufacturing of
treatment to remove nitrogenous products. As more stringent
explosives and rocket propellants and as a pharmaceutical vasodilator. It
environmental regulations are enacted at the state and federal levels,
is commonly found in the waste streams and soils of munitions and re
these techniques are no longer considered viable. Preference would
cracker manufacturing facilities and pharmaceutical plants (Husserl et.
therefore be given to environmentally friendly biological treatment
al., 2010). Concerns about toxicity and explosion hazards have led to
methods, provided that a robust GTN biotransformation technology that
increased efforts to develop safe and cost effective methods for treating
ensures complete transformation i.e. completed denitration without
GTN laden waste streams. A number of early studies on environmentally
accumulation of glycerol dinitrates (GDN) or glycerol mononitrate
fate of NG revealed toxicity to algae, invertebrates and vertebrates and
(GMN) and economic practicability could be developed. Completed
further suggested that NG was recalcitrant to degradation (Kalderis et.
denitration is preferred since GDNs and GMNs are more soluble than
al., 2011). NG affects the cardiovascular system, blood and nervous
system of experimental animals and was suffered by hypotension,
Few reports on the metabolism of NG have been demonstrated in
tremors, ataxia, lethargy etc. Acute exposure to NG can cause headache,
bacterial species (Binks et. al., 1996; Blehert et. al., 1996; Spain et. al.,
nausea, vomiting, occasionally diarrhoea, sweating and light
2000). Husserl et. al., (2010) reported on the ability of pure bacterial
headedness. High exposure can cause abdominal cramps, vomiting,
cultures to utilize GTN as sole Nitrogen source with the help of the
depression or mania, mental confusion, convulsions, paresthesia or
enzyme called Flavoprotein nitroester reductase puried from two
paralysis, apasia, impaired vision, breathing difculties,
Pseudomonas species. Since the avoprotein nitroester reductase
methaemoglobinemia and blue skin (Cyanosis), bradycardia, circulatory
enzyme production is often associated with the capability of
collapse or death (Mirecki et. al., 2006). Chronic exposure to NG can
microorganism to utilize nitroglycerin as substrate (Fox and Karpalus,
lead to the development of tolerance, and sudden withdrawal from
1994; French et. al., 1994; Zenno et. al., 1994; Binks et. al., 1996;
exposure can result in angina like chest pains which may be
Blehert et. al., 1997; Williams and Bruce, 2002).
accompanied by malaise, weakness, vomiting, dizziness, headache or impaired vision. Sudden death may also result. Chronic exposure may
NG was metabolized to DNG & MNG intermediates by various bacterial
also result in severe headache, hallucinations and skin rashes. Allergic
cultures (Gorontzy et. al., 1994; Husserl et. al., 2010), and in some cases
contact dermatitis can occur secondary to topical exposure to NG
glycerol nitrates could be removed from the culture medium. Recently,
(Yinon, 1990; EPA, 2007; Rittman and McCarty, 2001).
Historically, the destruction of energetic materials and explosive mixtures has been accomplished through open – air burning, detonation
or incineration techniques. Physicochemical methods of GTN
destruction involve adsorption on activated carbon followed by reduction with inorganic chemicals (eg. Na SO ) or by alkaline
19 THE SCITECH JOURNAL VOL. 01 ISSUE 02 FEBRUARY 2014
the biodegradation of Bacillus thuringiensis, Bacillus cereus and
counted and calculated as the number of utilizing bacteria. All tests were
Enterobacter agglomerans has been reported and a hydrolytic reaction
mechanism was suggested based on studies of dialysed cell extracts (Meng et. al., 1995). The denitration of NG by pure cultures of
Selection of Bacterial Isolates Agrobacterium radiobacter has been also reported and in vivo nuclear
The 3 isolates were selected from the UV spectrophotometric analysis
magnetic resonance measurements showed that both isomers of DNG
and colony forming units obtained in substrate utilization.
accumulated with 1, 3 DNG preferred by a roughly 8:1 ratio (corresponding to selectivity for denitration at the C- 2 position (White
Nitroglycerin Biodegradation Assay using Selected Bacterial et. al.,1996; Marshall et. al., 2004). The main objective of the present
study is to reveal the biodegradation of nitroglycerin by the soil microorganisms.
The nitroglycerin biodegradation assay for the selected bacterial isolates was carried as per the procedure referred before. Materials and Methods Enzyme Extraction Isolation of Nitroglycerin degrading Microorganisms
The enzyme avoprotein nitroester reductase responsible for the complete denitration of NG can be extracted by cell lysis method. The
Soil sample collected was used as a source for isolating nitroglycerin
incubated samples obtained from nitroglycerin biodegradation studies
degrading bacteria. An enrichment culture technique was used to isolate
were taken, centrifuged and the resultant supernatant was used for crude
nitroglycerin degrading microorganisms. Nitroglycerin used in the
enzyme extraction. The separation of crude enzyme extract was done by
present study was obtained from re factory in Sivakasi. Erlenmeyer
precipitating the protein by adding 10% TCA solution to the supernatant
ask (250ml), containing 50ml of minimal medium was sterilized. 0.1%
solution obtained from the degradation studies.
(w/v) nitroglycerin was added to the ask. One gram of the soil sample was added to the above contents and incubated on the shaker at 150rpm at
Nitroglycerin Biodegradation Assay using Enzyme extract
30ºC. Repeated subculturing was carried out by adding 2ml of the enriched culture from the above ask to the fresh medium. This was done
The nitroglycerin biodegradation assay for the selected bacterial isolates
in step wise manner after every one week of incubation and the
by using their respective crude enzyme extracts was carried as per the
concentration of the nitroglycerin was increased from 0.1% to
procedure mentioned above. After incubation, the resultant samples
0.5%.After subsequent enrichment the sample was inoculated on the
were subjected to UV spectrophotometric analysis for determining the
minimal agar plate with nitroglycerin. The process was repeated twice to
degradation ability shown by the enzyme extracts.
obtain pure culture. The pure cultures were then transferred to freshly prepared nutrient agar slants and stored at 4ºC for further studies. Five
Nitroglycerin Biodegradation Assay using Selected Bacterial
different isolates were obtained by purication based on colonial
The nitroglycerin biodegradation assay for the selected bacterial isolate
Nitroglycerin Biodegradation Assay
(N5) was carried out again for chemical characterization as per the procedure referred before.
The enrichment media supplemented with 0.1% of nitroglycerin sample were used for the degradation studies. The nitroglycerin sample was
Characterization of Bacterial Isolate
prepared by lter sterilized in 0.2 micron membrane lter, in order to
The selected bacterial isolates were identied based on microscopic and
make them free from contamination. The inoculum used for the
biochemical characteristics and screened as per the guidelines of
biodegradation studies were prepared by inoculating a loopful of isolated
Bergey's Manual of Bacteriology (Holt et. al., 1994; Cappucino and
culture in 50ml of enrichment media, incubated in shaker at 37ºC for 24
hrs. After incubation 10ml of inoculum was centrifuged and supernatant was discarded. The cells were washed in 50mM Phosphate buffer pH
(7.0) and absorbance was read at 660nm to obtain an O.D. value of 0.2. Then 1ml culture suspension of ve different isolates were inoculated in
After incubation, control sample and the degraded sample were analyzed
5 separate conical asks containing 50ml of enrichment medium
by Fourier Transform Infra-Red Spectroscopic (FTIR) analysis. FT-IR -
supplemented with 0.1% nitroglycerin sample. The asks were placed in
8400S, SHIMADZU model was used for the analysis and the spectrum
rotatory shaker at 37ºC and kept for 5 days of incubation.
was taken in the mid IR region of 400 – 4000cm .
UV Spectrophotometric Analysis
After incubation, the degraded samples using 5 isolates were subjected
The preliminary screening of nitroglycerin degrading bacterial strains
to UV spectrophotometric analysis. The absorption shown by the treated
was isolated by their growth in the presence of nitroglycerin as a
samples were compared with the peaks obtained for control broth
criterion. By using enrichment culture technique, 5 native isolates from
the soil sample were found to possess the ability to grow in nitroglycerin
Enumeration of substrate utilization
containing medium and named as N1 to N5 (Figure.1).The nitroglycerin biodegradation assay was carried out by using the ve different isolates
Serial dilutions of overnight culture of 5 different isolates were made and
obtained in enrichment procedure. After incubation, the resultant
the diluted samples were spread plated on the solidied minimal media
samples were subjected to UV spectrophotometric analysis. After
coated with nitroglycerin as a sole carbon and energy source. The plates
incubation, the samples were used for determining the degradation
were incubated at 37ºC for 48 – 72 hrs. The colony forming units were
ability of the nitroglycerin by 5 isolates using UV spectrophotometer.
20 THE SCITECH JOURNAL VOL. 01 ISSUE 02 FEBRUARY 2014
Figure 1. Nitroglycerin Biodegradation Assay Using Screened Cultures
Figure 2. Nitroglycerin Biodegradation Assay Using Crude Enzyme Extract
Figure 3. Pure Cultures on Nitroglycerin Containing Minimal Plates
Figure 4. FTIR Spectrum of Nitroglycerin sample (Control)
Figure 5. FTIR Spectrum of Degraded Nitroglycerine Sample – Treated sample
The maximum absorption peak for the degraded samples by the bacterial
enzyme extracts from the selected isolates as per the procedure referred
cultures was compared with the peak obtained for control (Figure.4 ;
before. After incubation they were subjected to UV spectrophotometric
Table.1).Enumeration procedure was done to nd out the ability of the
analysis for determining the degradation ability shown by the enzyme
bacterial species capable of utilizing nitroglycerin. In this, no of colony
extracts (Figure.2 ; Table.3).According to Bergey's Manual of
forming units were counted for each bacterial species growing on
Bacteriology, the efcient native isolate was tentatively identied as
minimal medium containing nitroglycerin (Table.2). Selection of strains
Arthrobacter sp., Agrobacterium sp., and Pseudomonas sp., (Figure.3 ;
was mainly based on the results obtained from UV spectrophotometric
Table.4).The nitroglycerin biodegradation assay was carried out using
analysis and enumeration procedure. Three isolates were found to show
the one selected isolate based on earlier results. The samples were used
maximal growth in utilizing nitroglycerin as substrates. The crude
for FTIR analysis. Degradation analysis of Nitroglycerin samples was
enzyme was extracted from the selected isolates by cell lysis method.
done using the one selected bacterial isolate. The analysis was carried out
The nitroglycerin biodegradation assay was carried out using the crude
in a control and test ask (Figure. 4 & 5).
21 THE SCITECH JOURNAL VOL. 01 ISSUE 02 FEBRUARY 2014
Table.1 UV Spectrophotometric Analysis of
Table .2 Substrate Utilization Pattern of the Isolates on
Peak absor Substrate the Isolate ption (nm)
Table. 3 UV Spectrophotometric Analysis for Nitroglycerin Biodegradation Assay
the Isolate absorption Discussion
found to be much more effective in degradation of nitroglycerin. The results are in conformity with early ndings (Blehert et.
Many biodegradation systems are often set up with little knowledge
about the microbial communities, which are being used. In order to better
Regarding the FTIR analysis the degradation of nitroglycerin by
understand what is happening in an operational biodegradation system
Pseudomonas sp. has been studied by using peaks obtained for
an understanding of the microbial community is needed. A
functional groups in control and treated sample. FTIR spectrum of
comprehensive study using liquid system was chosen, since liquid
control sample showed peaks at 3900 cm , 3854 cm , 3797 cm , 3349
systems are relatively inexpensive, easy to set up and control parameter,
cm indicating aromatic groups; 2962 cm , 2926 cm , 2871 cm ,
which can be monitored and manipulated, suited for large
indicating C-H aliphatic peaks; 2285 cm , 2182 cm , 2032 cm , 1901
cm indicating triple bonded groups; 1464 cm , 1378 cm indicating
methyl groups;723 cm representing mono substituted benzene and 676
Soil samples contain a considerable number of bacteria which are
cm meta substituted benzene which is the characteristics functional
capable of using nitroglycerin with complex structure even as sole
groups of nitroglycerin sample(Fig.4). The spectrum analysis of treated
carbon source. It was conrmed by dened growth on mineralizing
sample showed peaks at 2962 cm , 2926 cm , 2871 cm , indicating C-H
bacteria (Accashian et. al., 1998; 2000). In the present study the isolation
aliphatic peaks (Fig.2). Comparing the degraded samples with the
of nitroglycerin degrading native bacterial isolates from soil was done on
control, the aromatic peaks were not observed. In addition to that, the
minimal medium enriched with nitroglycerin from soil. Similar studies
aliphatic stretch peaks at 6, 7 and 8 positions in the IR spectrum were
were carried out on the isolation of nitroglycerin degrading bacteria from
noticed at 3000nm in the control and the same aliphatic stretch in the
the soil sample (Blehert et. al., 1996; White and Snape, 1993).
treated sample was noticed between 2500 and 1500nm (Acasshian et. al., 1998; 2000).
Of the different isolates, 3 isolates were selected based on substrate utilization and spectrophotometric analysis. The efcient native isolate
was tentatively identied as Arthrobacter sp., Agrobacterium sp., and Pseudomonas sp. Husserl et. al., (2010) have also been reported that
Future research in this area will range from clean-up directives for
Pseudomonas sp., Agrobacterium sp., &Arthrobacter sp., are involved
explosives manufacturing and munitions development to sustaining
in the utilization of nitroglycerin degrading system respectively.
military readiness by appropriately managing training and testing ranges
However, they were only identied to genus level and thus comparison
in an environmentally responsible manner. Assessing the potential for
between degradative ability of specic species is difcult.
explosives contamination and the potential for exposure of environmental and human receptors resulting from various military
The crude enzyme extracts were isolated by cell lysis method and the
activities will be necessary. Research will be needed to rene
efcacy of curde enzyme extract for the biodegradation of nitroglycerin
environmental and human health risk assessment methods and develop
by the isolate has been done by UV-Spectrophotometric analysis. The
tools for effective management of necessary military training operations
results showed that crude enzyme extracts for selected isolates are also
to minimize adverse environmental and human health effects.
22 THE SCITECH JOURNAL VOL. 01 ISSUE 02 FEBRUARY 2014
Table 4. Identication of the Selected Isolates
Biochemical Tests Arthrobacter sp. (N1) Agrobacterium sp. (N3) Pseudomonas sp. (N5) Acknowledgements
Blehert, D.S., Becker, K. and Chambiiss, G.H., 1996. Isolation and Characterization of bacteria that degrade nitroglycerin. Presented at the
Authors thank TNSCST for funding under student project scheme and
Tri – service. Environmental technology workshop.
Department of Zoology and Microbiology, Thiagarajar College
Blehert, D.S., Knoke, K.L., Fox, B.G. and Chambiiss, G.H., 1997.
(Autonomous), Madurai-9 for providing all the necessary facilities.
Regioselectivity of Nitroglycerin denitration by f lavoproteinnitro esterreductases puried from two Pseudomonasspecies. J. Bacteriol. References
Accashian, J. V., Smets, B.F. and Kim, B.J., 2000. Aerobic
Cappuccino, J.G. and Sherman, N., 1999. Microbiology, A Laboratory
Biodegradation of Nitroglycerin in Sequencing Batch Reactor Water
manual. (4 edition) pp. 179 – 182. Environ. Res. 72 (4) : 499-506.
EPA, 2007. National priorities list. Environmental Protection Agency,
Accashian, J.V., Vinopal, R.T., Joon Kim, B. and Smets, B.F., 1998.
Aerobic growth on nitroglycerin as the sole carbon, nitrogen and energy
Fox, K.M. and Karplus, P.A., 1994. Old yellow enzyme at 2 Angstrom
source by a mixed bacterial culture. Appl. Environ. Microbiol. 64 (9):
resolution: Overall structure, ligand binding and comparision with
related avoproteins. Struct. 2: 1089 – 1105.
Binks, P.R., French, C.E., Nicklin, S. and Bruce, N.C., 1996.
French, C.E., Nicklin, S. and Bruce, N.C., 1994. Sequence and
Degradation of Pentaerythritoltetranitrate by Enterobactercloacae PB2.
p r o p e r t i e s o f p e n t a e r y t h r i t o l t e t r a n i t r a t e r e d u c t a s e f r o m
Appl. Environ.Microbiol. 62: 1214 – 1219. Enterobactercloacae PB2. J. Bacteriol. 178: 6623 – 6627.
23 THE SCITECH JOURNAL VOL. 01 ISSUE 02 FEBRUARY 2014
Gorontzy, T., Drzyga, O., Kahl, M.W., Bruns – Nagel, D., Breitung, E.,
and fate process descriptors for propellant compounds. U.S. Army Corps
Loew, V. and Blotevogel, K.H., 1994. Microbial degradation of
explosives and related compounds. Crit. Rev. Microbiol. 20: 265 – 284.
Oh, S., Chan, D.K., Kim, B.J. and Chiu, P. C., 2004. Reduction of
Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T.,
nitroglycerin with elemental iron: pathway, kinetics, and mechanisms.
1994. Bergey's Manual of Determinative Bacteriology, (eds. Williams,
Environ.Sci. Technol. 38:3723–3730.
Rittman, B. E. and McCarty, P. L.2001. Environmental biotechnology.
Husserl, J., Spain, J.C. and Hughes, J.B., 2010. Growth of
Arthrobactersp. Strain JBH1 on Nitroglycerin as the Sole Source of
Spain, J. C., Hughes, J. B. and Knackmuss, H.-J., 2000. Biodegradation
Carbon and Nitrogen. Appl. Environ. Microbiol. 76 (5): 1689–1691.
of nitroaromatic compounds and explosives. CRC Press LLC, Boca
Kalderis, D., Juhasz, A.L., Boopathy, R. and Comfort, S., 2011.Soils
contaminated with explosives: Environmental fate and evaluation of
White, G.F. and Snape, J.R., 1993. Microbial cleavage of Nitrate esters
state of the art remediation processes (IUPAC Technical Report).P.
defusing the environment. J. Gen Microbial. 139: 1947 – 1957. Appl. Chem. 83 (7): 1407–1484.
White, G.F., Snape, J.R. and Nicklin, S., 1996. Biodegradation of
Marshall, S. J. and White, G. F., 2001. Complete denitration of
glycerol trinitrate and pentaerythryritoltetranitrate by Agrobacterium
nitroglycerin by bacteria isolated from a washwatersoakaway. Appl.
radiobacter. Appl. Environ. Microbiol. 62: 637 – 642. Environ. Microbiol. 67: 2622–2626.
Williams, R. E. and Bruce, N. C., 2002. “New uses of an old enzyme”-
Marshall, S. J., Krause, D., Blencowe, D. K. and White, G. F., 2004.
the old yellow enzyme family of avoenzymes. Microbiology
Characterizationof glycerol trinitratereductase (NerA) and the catalytic
role of active-site residues. J. Bacteriol.186:1802–1810.
Yinon, J.,1990. Toxicity and Metabolism of explosives. CRC Press,
Meng, M., Sun, W.Q., Geelhaar, L.A., Kumar, G., Patel, A.R., Payne,
G.F., Speedie, M.K. and Stacy, J.R., 1995. Denitration of glycerol trinitrate by resting cells and cell extracts of Bacillusthuringiensis /
Zenno, S., Saigo, K., Kanoh, H. and Inouye, S., 1994. Identication of
cereusaand Enterobacteragglomerans. Appl. Environ. Microbiol. 61:
the gene encoding the major NADPH – avinreductase of the
bioluminescent bacterium Vibrioscheri ATCC 7744. J.Bacteriol. 176: 3536 -3543.
Mirecki, J. E., Porter, B. and Weiss, C. A., 2006. Environmental transport
Citation: Padmavathy, S., Ananthi,V., Praveen Raja, P., and Asha Devi, N. K., 2014. Screening and Characterization of NitroglycerinDegrading Microorganisms. The Scitech Journal : Vol 01(2): 19-24.
24 THE SCITECH JOURNAL VOL. 01 ISSUE 02 FEBRUARY 2014
OPINION N° 2001-5 (February 2001) of the French Sustainable Development Commission (CFDD) On the "Charpin - Dessus - Pellat" report entitled AN ECONOMIC SURVEY OF THE FUTURE OF THE NUCLEAR ELECTRIC INDUSTRY An economic survey of the future of the nuclear industry was carried out at the request of the prime minister and published on July 28th 2000. Referring
Effect of Oral Sucralfate on Side Effects of External Radiation Therapy in Patients with Head and Neck Cancer Background: Early side effects of irradiation for head and neck cancers are very common and disturbing. These events may cause a transient disruption in the treatment course. The aim of this study was to evaluate the role of sucralfate on radiation side effects. Patients