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SAMANTHI
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 efcacy 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 identied 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 Introduction
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 efuent, 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 puried from two paralysis, apasia, impaired vision, breathing difculties, 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 Isolates
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 purication based on colonial Isolates
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 identied 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 Chemical Characterization
(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 solidied 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 efcient native isolate was tentatively identied 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 conrmed by dened 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 efcient native isolate Conclusion
was tentatively identied 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 identied to genus level and thus comparison in an environmentally responsible manner. Assessing the potential for between degradative ability of specic species is difcult.
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 rene efcacy 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. Identication of the Selected Isolates Biochemical Tests
Arthrobacter sp. (N1)
Agrobacterium sp. (N3)
Pseudomonas sp. (N5)
Acknowledgements
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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.
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