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Indian Journal of Experimental Biology Vol. 41, September 2003, pp. 991-1001

Biodegradation of nitro-explosives

Pradnya Kanekar*, Premlata Dautpure & Seema Sarnaik

Microbial Sc iences Di vision, Agharkar Research Institute, G. G. Agarkar Road, Pune 4 I I 004, Indi a

Environmental contamination by nitro compounds is associated principally with the explosives industry. However, global production and use of explosives is unavoidable. The presently widely used nitro-explosives are TNT (Trinitrotoluene), RDX (Royal Demolition Explosive) and HMX (High Melting Explosive). Nevertheless, the problems of these nitro-explosives are almost parallel due to their simil arities of production processes, abundance of nitro-explosives and resembling chemical structures . The nitro-explosives per se as well as their environmental transformation products are toxic, showing symptoms as methaemoglobinaemia, kidney trouble, j aundice etc. Hence their removal/degradation from soil/water is essential. Aerobic and anaerobic degradation of TNT and RDX have been reported, while for HMX anaerobic or anoxic degradation have been described in many studies. A multisystem involvement using plants in remediation is gaining importance. Thus the information about degradation of nit ro-explosives is avai lable in jigsaw pieces which needs to be arranged and lacunae fill ed to get concrete degradative schemes so that environmental pollution from nitro-explosives can be dealt with more successfully at a macroscale. An overview of the reports on nitro-explosives degradation, future outlook and studies done by us are presented in this review.

Keywords : Biodegradation, Bioremediation, Nitro-explosives, Waste waters

Explosives are chemical compounds that detonate at rates of krnls and thus rapidly produce large vo lumes of hot gases when properly initiated. Exothermic oxidation-reduction reactions provide the energy released during detonation . Majority of explosives and propellants are organic nitro-compounds. The presently widely used explosives are TNT (Trinitrotoluene) [nitroaromatic compound] , RDX (Royal Demolition Explosive, I ,3,5-trinitro-l ,3,5-triazacyclohexane) [nitramine compound] and HMX (High Melting Explosive,l ,3,5,7-tetranitro-l ,3,5,7-tetrazacyclooctane) [nitramine compound] (Fig . 1).

The nitroaromatic and nitramine explosives are used in military applications such as burster charges for artillery shells, component of solid-fuel rocket propellants, and to implode fiss ionable materi al in nuclear devices I. The problems of these nitro explo-

Cli] 0 , NO, I I

'~y.'O' / N", N -CH, / " ,I CH, CH, CH, N- NO,

L ~ I I N CH,

0 , 0 , rf '" /" /" /

CH, O, N O, N CH,- N I

NO, (a) (b) (e)

Fig. I - (a) 2,4,6-Trin itrotoluene, (b) RDX , and (c) HM X

*For correspondence: E-mail : kancka rp @rediffmai l. colll Fax: 020-565 1542

sives are almost parallel due to their similarities of production processes, abundance of nitro- groups, resembling chemical structures.

Production TNT is produced by nitration of toluene with the

mixture of sulphuric acid and nitric acid. Both RDX and HMX are produced by the Bachmann process, that is nitration of hexamine by nitric acid and ammonium nitrate in presence of acetic anhydride and acetic acidt.2.

Toxicity Toxici ty of nitrobodies such as explosives is

widely known. Some of the common sy mptoms are irritation of the digestive tract, methaemoglobinaemia, disturbed heart function, kidney trouble, dysfunction of the whole vascular system, and severe jaundice3

.

Not just the nitroaromatic explosives but their environmental transformation products, including arylamines, ary lhydroxy lamines, and condensed products such as azoxy- and azo- compounds, are equall y or more toxic as the parent nitroaromatic. Aromatic amines and hydroxy lamines are implicated as carcinogenic intermediates as a resu lt of nitrenium ions formed by enzymatic ox idation4

.

The toxicological properties of these compou nds are expected to result from metabolic activation of the nitro-group undergoing anaerobic reductive biotrans­fo rmation in microorganisms and mammalian tissues.

992 INDI AN J EXP BIOL, SEPTEMB ER 2003

The vasodilating, cardio-vascular, and tox ic effects o f such compounds can be linked to the formation o f nitri c oxide, a biologically active free radical species. It was proposed that formatio n of nitrite is an important initi a l step in fo rmatio n o f the bi o logically acti ve nitric ox ides.

T NT is known to cause toxic hepatiti s, apl asti c anaemi a, cyanosis, pallor, and di sturbances of ex ternal secretory and incretory fun ctio n o f pancreas3

.

TNT showed mutagenic acti vity with Salmonella/ mammalian' microsome test6

.

In a toxicity test using spiked sediment with two freshwater invertebrates: Chironomus lentans (midge) and Hyalella azteca (amphipod), tox ic effec ts were seen after exposure to TNT and its degradation prod ucts7

.

TNT is on the li st of US EPA priority po llutants. It is a known mutagen and can cause pancytopenia as a result of bone marrow failure . Mutatox and green alga bioassays have confirmed it to be the most toxi c o f the nitroaromatic explos ives4.

In another such testing, the biotransformation products of TNT and metabo lites in mammalian cells in culture and their cy totoxicity were studied. The most prevalent biotransformati on product is NG 1 08 neurobl astoma cells, monoamino-dinitroto luene (2Am-DNT) caused tox ic effects based on try pan blue exclusion and LDH-release colo rimetric assays8. The EPA has ambient crite ri a of 0 .06 mg/I and drinking water criteria of 0 .049 mg/I for TNT9.

RDX is class C poss ible human carcinogen and has adverse effects on centra l nervous system (CNS) in mammals. In Italy, workers in munition manu­facturing factory showed RDX intoxication symptoms as VOlTIltll1g, insomnia, loss o f consciousness, irrit ability, anxiety, verti go, con vulsio ns2

. Sunderman repon ed that fumes emanating d uring the RDX production by Bachmann process produced skin lesions (transient erythema and ede ma about the eye)2.

HM X also has some C NS effects but furth er chroni c carcinogenicity and tox ic ity studies are needed I. HMX was found to be tox ic to fresh water a lgae and fi sh species (bluegill , channel catfi sh, fathead minnow, rainbow trout) tested2

.9

.IO

•

In a study , sublethal and chronic effects of HMX in arti ficia l so il were assessed using earthworm (Eisenia alldrei). HMX sho wed some reproducti ve consequences (number of juvenile and their bi omass)

d . I I on cocoon pro uctIOn . The EPA has ambient crite ri a of 0 .3mg/l and

drinking water crite ri a of O.03Smg/1 fo r RDX/HMX9.

Environmentalfate and recalcitrance Nitroaromatics fo rm an important g roup of

recalcitrant xenobioti cs . Onl y few aromati c compo unds, bearing o ne nitro gro up as a substituent o f the aromatic ring, are produced as secondary metabo lites by microorgani sms. Majority of these compounds therefore are sy nthetic and are recalcitrant to bio logi cal treatment and remain in the bi osphere where they constitute a source of po llution due to both toxic and mutagenic effects on humans, fish, a lgae and microorganisms l2

. Consequently, they are environmentall y persistent, and remediatio n o f waste streams and contaminated ground water is difficult. Aromatic nitro compo unds are res istant to chemical or bio logical ox idatio n and to hydrol ys is because of the e lectron withdraw ing nitro groups4 However, relatively few microorgani sms have been described as being able to use nitroaromatic compounds as nitrogen and/or carbon and energy source l 2

.

Structure property and structure actl vlty re lati onships studies are important tools in current pharmacology, toxicology, environmenta l research and medicinal chemi stry for predicting effec ts o f chemi cals on bio log ical environment. Because such effects pertain to additi onal processes such as metabo li sm and distribution , these greatly depend on the molecul ar structures and phys ico-chemical property of the chemicals. It is worth mentioning that the physico-chemical parameters of RDX and re lated co mpounds that have been corre lated with W (Wi ener index) and Sz (Szeged index) are a lso valuable data for determining environmental behav iour and biological acti vity i.e. toxicity of organic ex plos ives. It should be noted that the phys ico-chemical properties o f che micals dete rmine their biological action . ego onl y those chemicals that in te rfe re with a parti cular transport system can be introduced into the bio log ical worldS.

TNT has very low mobility once adsorbed to so il4. A small amount o f HMX can be re leased to the a ir as dust or ash fro m fac ilities that burn waste contaminated with HMX. Some HM X may be re leased to the soi I as a result o f acc idental spi li s, the settling o f HMX containing dust parti cles from the a ir, or the disposal of waste containi ng HMX in land fill s. In surface water, HMX does not evaporate or bind to sediments to any large ex tent l3

.

Pollution Activities associated with manu fac turing, training,

waste disposal and closure o f bases have resul ted in

, -t-

i

KANEKAR el a/: BIODEGRADATION OF NITRO-EXPLOSIVES 993

severe soil and groundwater contamination with ex plosives 14. Contaminated wastewaters are generated from the productio n, handling, loading Qf explos ive materi als in Army Ammunitio n Plants, etc. These wastewaters are contaminated with li ve explosives as well raw materials used for productio n of

I . 15 exp oSlves .

Tn addition to contaminated gro undwater, co ntami­nated wastewaters may also be generated by treatment of contamin ated so il , such as so il leaching and so il

h· h ' 16 was 1I1g tec nlques .

Bioremediatioll The tox ic effects o f explos ives, the ir persistence in

nature and their po lluting nature necess itate remediation of the ex plos i ves and generated waste waters.

Treatment processes can be of three types, viz. phys ical, chemical and bio logica l.

Physical methods such as settling, incinerati on, filtration have been used fo r remedi ating waste water. Many of the commonl y used remediation techno logies in this fie ld are separations rather than destructi on processes. They inc lude res in adsorption, surfactant complexing, liquid-liquid ex tracti on, ultrafiltratio n, reverse osmosis, all o f which concentrate the nitroaromati c ex pl os ives from so il or water into another medium fo r further treatment or landfill di sposal, but do not modify them to nonhazardous materi a ls4

.

Chemical methods such as ad vanced ox idatio n processes (AOPs) empl oy ing ox idi zing agents as H20 2, 0 3, which generate hydroxy l radicals [a strong

ox idant ; Eo = -2.8V], addition of which to aromatic rings InItI ates nng opening and ul timate minera li zation to CO2 and H20 . Bu t limitati on is that UV+03 despite remov ing TNT from soluti on, it does not necessarily ameliorate tox icity . The capita l cost also increases. Other AOPs are hydrothermal technologies which e mpl oy wet a ir oxidati on and superficial water ox idatio n, these cause minera lization of organi cs by oxygen or H20 2 at high temperatures and pressures4

.

Thus the biofriendly treatment is therefore bioremediation. Microorgani sms are known for their versatile metaboli c acti vity and have evolved di verse pathways all owing the m to minera li ze specifi c nitro­componds. Despi te this, re lati vely fe w micro­organisms have been described as be ing able to use nitroaromati c compounds as N and/or C and e nergy source l2

.

The high nitrogen contents o f nitramines suggest that they are potentially good nitrogen sources fo r microorgani sms. The res ident microfl ora may be incapable o f explo iting such resources. Under these c ircumstances, inoculati on with no nindigenous organisms able to use the pollutant can lead to the c reati on of stable po llution-degrading communities. If the surviva l of the inoculant is linked to the avail ability o f the po llutant, the no n ind igenous organi sms will eventually die back afte r the bioremedi ation has been successful. The success of an inoculant depends on its environmental competence and degradati ve capac ity. As a result, a considerable effort is being made to isolate microorgani sms that are abl e to degrade nitroaromatic and nitram ine po llutants l 7

. Although one can isolate bacteria, including thermophilic species, that degrade nitroaromatic explos ives, most authors employ natura ll y occurring co nsorti a to degrade contami nants, rather than selecting or engineering-speci fic strains. The latter usually lack survivability in the fie ld whatever their promise under laboratory conditions4

Microorgani sms have been postul ated to degrade nitroaro matic compounds by aerobic as well as anaerobic means.

Aerobic mechanism - Remova l or product i ve metaboli sm of nitro groups can be accompli shed by fo ur di ffe rent strategies : (a) Some bacteria can reduce the aromatic ring of

dinitro and trinitro compo unds by the additio n of a hydride io n to fo rm a hydride- Meisenheimer complex, whi ch subsequently rearomati zes with the e liminatio n of nitrite.

(b) Monooxygenase enzy mes can add a sing le oxygen ato m and e liminate the nitro group from nitropheno ls.

(c) Dioxygenase enzy mes can insert two hydroxy l groups into the aromatic ring and precipitate the spontaneous e limination of the nitro group fro m a variety of nitroaro matic compounds.

(d) Reduction o f the nitro group to the correspo nding hydroxy lamine is the initi a l reac tion in the producti ve metabo li sm of nitro­benzene, 4-nitrotoluene, and 4-nitrobenzoate. The hydroxylamines undergo enzyme-catalyzed

. rearrangements to hydroxy lated compounds that are substrates for ring- fi ss ion reacti ons.

Anaerobic mechanism - Anaerobic microorgan isms can reduce the nitro group via nitroso and hydroxy lamino inte rmedi ates to the correspo nding amines l8

.

994 [NDIAN J EXP BIOL, SEPTEMBER 2003

Bioremediation usually employs aerobic co nditions, both to promote mineralizati on of organic compounds to CO2 and H20 , and because aerobic bioreactors usually achi eve higher throughput than anaerobic sys tems, with less sludge formation and by­product off-odou rs.

TNT The nitroaro mati c exp los ive 2,4,6-trinitrotoluene

(TNT) is a reactive mo lecule that undergoes biotransformat ion read ily under both aerobic and anaerobic conditi o ns to give aminoclinitrotoluenes . The resulting am ines biotransform to g ive several o ther products, including azo, azox y, acety l and phenolic derivatives , leav ing the aromatic ring intact. Although some Meisenheimer complexes, initi ated by hydride ion attack on the ring, can be formed during TNT biodegradatio n, little or no minera li zation IS

d d . b . I 19 encountere un ng actena treatment . A number of biotechnological app l ica tions of

bacteria and fungi , including slurry reac tors, composting, and land farming, to remove TNT from po lluted so ils have been worked o ut. These treatments have been des igned to achieve minerali zation or reduction of TNT and immobili zation of its amino derivatives on humic material. These approaches are highly effici ent in remov ing TNT, and increas ing amounts of research into the potential usefulness of phytoremediation , rhizophytoremedi ati on, and transgenic plants with bacterial genes for TNT removal are be ing done2o

.

Most if not a ll aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxy lamine inte r­medi ates. However condensation of the latter compounds y ie lds highly recalcitrant azoxy tetra­nitrotoluenes2o

.

Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons20

. Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external mjlieu, where they are substrates for ligninolytic enzymes20

.

The ability of Phanerochaete chrysosporium to biore mediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-l ,3,5-trinitro-l ,3,5- triazine; 3,000

ppm) and HMX (octahydro-I ,3,5,7-tetranitro-1 ,3 ,5,7-tetrazocine ; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives ex tracted from the so il were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Myce lium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt ex tract broth , spore- inocu lated cultures minera li zed 10% of added [14C]TNT (5 ppm) in 27 days at 37°C. No minera li zation occurred during [ '4C]TNT biotrans­formation by mycelium-inoc ulated cultures, although the TNT was transformed21

.

Study on biodegradation of TNT by using natural mixed flora with supple mentation by organic nutrients was repo rted by Osmon and Klausmeier22 . They studied the degradatio n of TNT using sewage effl uent , so il suspension, pond water and aquarium water as the inoculum and g lucose, yeast ex tract as the ex traneous organic nutrients. They also used a pure culture of Pseudomonas aeruginosa for degradation of TNT supplemented with glucose as the extraneou s nutrient. However they could not achieve complete disappearance of TNT in the absence of other organic nutrient as a supple ment. Thus according to them degradation was rapid and quantitative ly greater, when other organic nutrients were added to the medium; and secondly , with pure cultures the degree of di ssimilation was less and metabolites accumulated transiently in the medium.

A lot of work has been done on biore mediation of TNT and its waste water using Pseudomonas sp. under aerobic conditions. Pseudomonas sp . could metabolically oxidize a-TNT in an attempt to study the metabolic disposition of 2,4,6-trinitrotoluene. With supplementation of yeast extract the degradation of TNT was complete within 24 hr. They demonstrated the metabolites of TNT as 2,2',6,6'­tetran i tro-4-azox ytol ue ne, 2,2',4,4' -tetran i tro-6-azox y­toluene, 2,6-dinitro-4-hydroxylaminotoluene, nitro­diaminotoluene by chromatography. However, they could not achieve cleavage of the benzene ri ng23.

Labeling experiments to study bacterial degradation of a-TNT by Pseudomonas sp. have demonstrated ring cleavage. In this study Traxler et a/.24 used (ring­UL-[ 14C]) TNT. At the end of the experiment residual [1 4C] activity was measured. They could demonstrate release of carbon from TNT as CO2. Since only TNT was labelled, cleavage of benzene ring was confirmed. They could detect removal of nitro group from the

KANEKAR el al : BIODEGRADATION OF NITRO-EXPLOSIVES 995

ring of TNT. They have further stated that since TNT was the only source of carbon and nitrogen, metabolized TNT was used for synthesis of cells. Kanekar and Godbole25 have described Pseudomonas lrinilrololuenophila sp. nov. isolated fro m so il exposed to TNT waste wate r. The culture could degrade 63 % TNT incorporated in sy nthetic nutrient medium.

Biodegradation of TNT by Pseudomonas sp., Bacillus sp. and Cilrobacler sp. under diffe rent environmental conditions, fro m synthetic nutrient medium and TNT waste water was extensively studied by Kanekar and Godbole26

. Another pathway has been described in Pseudomonas sp. Strain JLR II and involves N02- re lease and further reduction to

NH : ' with almost 85% of the N-TNT incorporated as

organic N in the ce lls. It was recently reported that in this strain TNT can serve as a fin al e lectron acceptor in respiratory chains and that the reduction of TNT is coupled to A TP synthes is2o.

Anaerobic studies on reduction of 2,4,6-tri­ni trotoluene (a-TNT ) by using hydrogen in presence o f enzy me preparations from Veillonella alkalescens have resulted only in reductio n products o f TNT namely 4-amino-2,6-dinitroto luene, 2,4-di amino-6-nitrotoluene, 2,2' ,6,6'-tetranitro-4,4'-azox ytoluene. With the strains of Pseudomonas al so the authors co uld get the transformation products namely 4-hydroxyl-amino-2,6-dinitrotoluene, 2,2',6,6' -tetranitro-4,4'-azox ytoluene. The strict anaerobe Veillonella alkalescens also gave the amino compounds. According to these author , there is no evidence o f bio logical c leavage and subsequent degradatio n o f the aromatic nucleus o f TNT27 .

Use of GAC (Granular Acti vated Carbon) as the supporting medium for a fluidi zed-bed bioreactor (FBB) has been used by some workers. In this, a microbial bio film on the GAC created an anaerobic environment for reduc ing the nitroaromatic ex pl os ives to amines. Mineralization was completed in an external (aerobic) acti vated s ludge reacto r or rotating biological contacto r. Fluidi zed beds permit e ffi c ient mass transfer and prevent the formati on of thi ck biofilms (flow causes shearing), while G AC minimi zes shock loads and o ffers a good growth medium. But disadvantages are cost, retentio n o f untreated nitroaromati c compo unds on GAC, incomplete degradatio n of TNT and requirement o f added nutrients4

• Anaerobic microorgani sms can also degrade TNT through di ffe rent pathways. O ne pathway, fou nd in Desulfov ibrio and CLostridium,

involves reduction of TNT to tri aminotoluene; subsequent steps are still not known. Some Closlridium species may reduce TNT to hydroxylaminodi ­nitrotoluenes, which are then furth er metaboli zed20

.

A number o f fung i have a lso been screened fo r TNT degradation. About 98 genera o f the 190

screened showed ability to transform a -T NT. However fung i were found not to degrade TNT and dinitrotoluene. The transformati on products were found to be 4-amino-2,6-dinitrotoluene, 4-hydroxyl­amino-2,6-dinitroto luene and 4,4'-azox y-2 ,2' ,6,6'­tetranitroto luene which accordir.g to them could be tox ic28 .

However res istance to compl ete minerali zatio n has been a major problem in deve loping an effec ti ve method fo r the bioremedi ati on of T NT, even though di sappearance of TNT may occur rap idl y 4. Therefore, despite more than two decades of intensive research to bi odegrade TNT, no bi ominera li zati on-based technologies have been successful to date ' 9

.

RDXandHMX The manu fac turing processes for RDX and HM X

being a lmost same, each is present as an impurity in the other one's producti on. Because o f the co­presence of RDX and HMX in contaminated waters or at contaminated sites, degradation o f both in each other's presence becomes important. Therefore, bi oremediation o f HMX was te water invo lves degradation of HMXIRDX mi xture. Thus, most biodegradatio n studies are carried out using both RDX and HMX and not indi vidual ones.

The non-aromatic cyclic nitramine explos ives hexahydro-l ,3,5-trinitro-l ,3,5-tri az ine (RDX) and octahydro-l ,3,5,7 -tetranitro- l ,3,5,7 -tetrazocine (HMX) lack the e lectroni c stability enjoyed by TNT o r its transformed products . Predictably, a success ful enzymati c change on one o f the N-N0 2 or C- H bonds of the cyc lic nitramine would lead to a ring cleavage because the inner C-N bonds in RDX become very weak « 2 kcallmol). Recentl y thi s hypothes is was tested and proved feas ible, when RDX produced high amounts of carbo n diox ide and nitro us ox ide fo llowing its treatment with ei ther municipal anaerobic sludge or the fun gus Phanerochaete chrysosporium. Research aimed at the di scovery of new microorgani sms and enzy mes capable o f minera li z ing energeti c chemi cals and/o r enhancing irreversible bind ing (immob ili zation) of their prod ucts to soil is presently receiving considerable attention fro m the scienti fic community' 9.

996 iNDIAN J EXP BIOL, SEPTEMBER 2003

Because nitro-explosives are recalcitrant towards ox idation , severa l researchers have used an organic co-substrate to stimulate their metabolism4

.

In a study of RDX degradation by Rhodococcus sp. nitrite formation was observed with RDX di sappearance. Thi s indicated early enzy mati c denitration step in the degradati on. Thi s is supported by the observation of absence of any of the familiar nitroso products M X(hexahydro- l-nitroso-3 ,S-dinitro- l ,3,S-triazine), DNX (hexahydro- I ,3-dinitroso-S-nitro- I ,3,S-triazine) and TNX (hexahydro- l ,3,S-trinitroso- l ,3,S-tri az ine). Nitrite did not accumul ate in the system and its disappearance was accompanied by mi crobial growth and the formation of N20. This suggested that nitrite was ass imi lated by the bacteria l3

. Even after complete removal of RDX and IlItnte, N20 formation continued, indicating that in addition to nitrite ass imilation, RDX intermediate(s) might be responsible for its formati on. Formation of 2mol of N02° per molecule of RDX is consistent with the stoichiometry. A plausible hypothesis would be that the first loss of N02- produced the cyclohexenyl product whereas the second denitration produced the

N20 + biomass

cyclohexadienyl intermediate. There was transient accumulation of HCHO fo llowed by CO2 formation indicating the cleavage of the RDX ring fo llowing its denitration. Concurrent formation of carbondi ox ide with the di sappearance of HCHO mi ght indicate the direct involvement of the aldehyde in the formati on of CO2. Total yield of CO2 indicated that roughly one carbon atom in each RDX molecul e was minerali zed.

Most of the products (N02-, NH 3, N20 , HCHO, and CO2) detected during RDX biodegradation by Rhodococcus sp. have also been detected during the alkaline hydrolysis of RDX , suggesting a resemblance in the degradation mechani sms of both reac tions. RDX is hydrolyzed in an alkaline so lution (p H 12) via bimolecular elimination of HN02 to initi all y produce a 1,3,S-triaza-3,S-dinitrocyclohex-l-ene intermedi ate. During incubation with Rhodococcus sp. also a product with an MW of 119 with the same retention time was detected. Nitrogen and carbon stoichiometry is consistent with the scheme wherein assumpti on is that both RDX denitration steps occurred prior to ring cleavage l4 (Fig. 2).

02N N02 i 02N~ i 02N ~ "-N~ N / N02·

"-N N N02 N N

~ RDX I t ~ \ ) t ~ \ ~ N/ N N

I I N02 N02

C)H6N60 6 C)HsNs04 C)H4N40 2

II

~ H2O ~ 2H20

t OH

02N,,-~ H2~ ~H NH autodecomposition

~H CO, __ HCHo.-f[NH'CH~ + C2HsN)O) ~

NH NH) IV

(30%in C) (10 %) MW 119Da C)HsN40 4

(64 % in C) III

Fig. 2 - Schematic representation of potenti al steps involved during biodegradation of RDX with Rhodococcus sp.The sche me shows two steps of dcnitra tion to I and II pri or to ring cleavage via the hypothetical hydroxy lated product III. The presence of formamide as an RDX metabolitc rcquires furth er confirmation.

KAN EKAR el at: BIODEGRADATION OF NITRO-EX PLOSIVES 997

Coleman et af. 29 observed nitrite production from

RDX. Cultures were grown aerobically at 25°C in minimal medium, except that succinate (20mmol/l ) was used as the carbon source and RDX (0. 16-0.19 mmol/I) as the nitrogen sources. Nitrite production is an important step in the ae robic biodegradation of RDX, as it provides a growth substrate for the degradative bacteria. One poss ibility is that two of the nitro-groups fro m RDX are released as nitrite and one of the ring nitrogens is released as ammonium. This hypothesis is consistent with the formation of the dead-end metabolite C2HSN]0 ] reported by Fournier et a/. 14, which contains the remaining three nitrogen atoms from the RDX -molecule

Another mechani sm for RDX biodegradation has been proposed by Sheremata et apo. With RDX as the mai n source of N, white rot fungus Phanerochaele chrysosporium was able to remove it , at the initi al concentration of 62 mg/I in liquid medium studied after 60 days Particularly , MNX and DNX were

formed by the stepwise reduction of -N02 in RDX. Further enzymatic hydrolytic ring cleavage of RDX yielded Methylenedinitramine (02NNHCH2NHN02) and bi s(hydroxymethyl )nitramine ((OHCH2)2NN02) . These four metabolites di sappeared to produce N­

containing products (N20 , NH ; , and traces of N2) as

well as C-containing products (HCHO, CH30H, HCOOH and CO2). In this study , 50-60% mineralization of RDX to CO2 occurred.

Under nitrogen limiting conditions, Stenotropho­monas maltophilia was capable of utilizing RDX . S. maltophilia utilized three N from RDX, yielding a metabolite tentatively ider.tified as methylene-N­(hydroxymethyl )-hydroxlamine-N ' - (hydroxymethyl ) nitroamine. These findings are. in contrast to previous studies on the aerobic biodegradation of RDX and HMX in the environment which show that these compounds are resistant to microbial attack 17.

Most of the studies on biodegradation of both RDX and HMX have been reported to occur under anaerobic or anoxic conditions31 . In anaerobic condition, the biodegradation of RDX in liquid cultures with municipal anaerobic sludge can occur by at least two degradation routes32.

In one route, RDX was reduced to give the famili ar nitroso derivatives MNX (hexahydro-l-nitroso-3,5-dinitro-l,3,5-triazine) and DNX (hexahydro-I,3-dinitroso-5-nitro-I ,3,5-triazine.

In second route, two novel metabolites, methylenedinitrarnine [(02NNH )2CH2] and bis(hydroxy-

methyl)nitramine [(HOCH2)NN02] formed were presumed to be ring cleavage products produced by enzymatic hydrolys is of the inner C-N bonds of RDX . None of the above metabo lites accumulated in the system, and they disappeared to produce nitrous oxide (N20) as a nitrogen-containing end product and HCHO, methanol and formic acid that in turn disappeared to produce CH4 and CO2 as carbon­containing end products (Fig. 3).

The tentati ve conclusion is thus made that, once the RDX ring is cleaved, the fate of the resulting inte rmediates will be determined by microbial and chemical processes. For instance, most of the products di scussed are thermally unstable and undergo fast hydrolytic cleavage in water32

.

Three species of the family Enterobacteriaceae isolated from nitramine explosive contaminated soi l biochemically reduced RDX and HMX. Two isolates, identified as Morganella morganii and Providencia rettgeri , completely transformed both RDX and the nitroso-RDX reduction intermediates . The third isolate, identified as Citrobacter freundii, part iall y transformed RDX and generated high concentrations of nitroso-RDX intermediates. All three isolates produced 14C02 from labeled RDX under O2 depl eted culture conditions. All three isolates transformed HMX, only M. morganii transformed HMX in the presence of RDX31.

The biodegradation of HMX under various electron-acceptor conditions was investigated. HMX was found to biodegrade with various rates under sulfate reducing, nitrate reducing, fermenting , methanogenic, and mixed electron accept ing conditions . However, the rates of degradation varied among the various conditions studied. Fastest removal of HMX was observed under mixed electron-acceptor conditions, followed in order by sui fate reducing, fermenting, methanogenic, and nitrate reducing conditions. Under aerobic conditions, HMX was not biodegraded, which indicated that HMX degradation takes place under anaerobic conditions via reduction . HMX was converted to methanol and chloroform under mixed electron-acceptor conditions. These results are promising since thi s degradation of HMX in anaerobic conditions under mjxed electron acceptor conditions mimics conditions at contaminated field sites, where a heterogeneous or mixed microbial population exists34.

Using strict anaerobes for degradation of hexahydro-I ,3,5- trinitro-l ,3,5-triazine (RDX), and octahydro-l ,3,5,7-tetranitro- l ,3,5 ,7-tetraazocine (HMX)

998 INDIAN J EXP BIOL, SEPTEMBER 2003

revealed that a sulfate-reducing bacterial consortium DesuLfovibrio spp. could use these explosives as sole source of nitrogen for growth. Production of ammonia from the nitro groups of the explosive was observed. This ammonia served as a nitrogen source for the bacterial growth. The sulfate-reducing bacteria thus may be useful in the anaerobic treatment of explosives-contaminated soil35

.

In situ remediation of TNT, RDX and HMX compounds contaminated soil under anaerobic conditions with carbon supplementation in the form of starch showed that within 4 days parent TNT molecules were removed and within 24 days reduced intermediates of TNT and RDX were removed. In this attempt the soil was flooded with phosphate buffer and incubated under static conditions. Aerobic heterotrophs (indigenous or as added inoculum) removed oxygen creating anaerobic conditions which promote biodegradation of these explosives36

.

Based on reports on RDX degradation indicating the process to be in oxygen-limiting conditions, another approach employed use of FeD. This attempt

ON N02 NO MNX

anaerobic sludge t Path b N02

ring cleavage

anaerobic Path a sludge

ChN N02

I N

( ']

NCh

I + N

was based on the assumption that FeU corrosion could rapidly induce anoxic conditions that favor RDX degradation. Production of cathodic (water-derived) H2 by FeD corrosion increases the availability of an electron donor to support microbial reduction of RDX. Homoacetogenic bacteria may participate by cometabolizing RDX (using H2 as primary substrate) and comensalistically supporting heterotrophic activity and Fe (Ill) reduction (by coupling Fe (0) corrosion with acetogenesis). Experiments with other redox-sensitive pollutants such as N03· showed the benefits of enhancing such biogeochemical interactions through bioaugmentation . This mechani sm is doubly beneficial as it can be used to clean not just the parent explosive but also the N03· rich waste waters. Due to chemical similarity HMX biodegradation on similar lines is promising37

.

Biochemical basis of degradation Very little information is available about enzymic

basis of biodegradation of nitro- explosives. Under aerobic conditions, oxidation (most probably

DNX

HCHO +

TNX

MeOH

NO hydroxylamino­

RDX

(CH3 hNNH2 + NH2N~

McCormick et aI (1981)31

"NAN / HOH2C ---- ---- CH20H H H

methylenedinitramine bis(hydroxymethyl)nitramine

HCOOH CO2 + Cl·t.

Fig. 3 - A constructed pathway for the biodegradation of RDX with municipal anaerobic sludge (pH 7.0). Path a, postulated pathway via direct ring cleavage of RDX; Path b, degradation via reduction to the nitroso derivatives followed by ring cleavage (modified pathway Mc Cormick el aJ 33 ) .

KANEKAR el al : BIODEGRADATION OF NITRO-EXPLOSIVES 999

cytochrome P-450 catalyzed) which leads to loss of the nitro group as N02-(nitrite) or catechol formation , depending on the site of the initial oxidation, is suspected to be responsible for biodegradation . Since P-450 enzymes are known to accept polynitro compounds as electron acceptors in place of oxygen, reductive pathways by enzymes (possibly also P-450 catalyzed) lead to reduction of the nitro group to hydroxylamines, azo- and azoxy- compounds, even under aerobic condition, or to replacement of the nitro group by hydrogen . Degradation of nitro explosives by the reductive pathway could be due to non-specific nitroreductase enzymes contained within both aerobic and anaerobic organisms4.

Since nitroaromatic compounds such as a-TNT contain only nitro group substituents, they are not direct substrates for lignin-degrading enzymes (Lignin peroxidase and Manganese peroxidase). Therefore P. chrysosporium reduces the aromatic nitro group to an

.amine in the first step in the degradation of nitroaromatic compounds. It is the amine products that are substrates for peroxidase-catalysed oxidation, and the reduction reaction is the key step in the degradation of nitroaromatic compounds3o

.

The initial reductions in RDX degradation could be catalysed by type I nitroreductase enzymes In Enterobacteriaceae strai ns29

.

Enzymic bas is of degradation suggests a manganese peroxidase enzyme found in the white rot fungus Phelbia radiata that degraded TNT and 2-amino-4,6-dinitrotoluene4.

Although bioremediation overcomes most of the demerits of physical and chemical treatment methods, yet due to various repairable reasons bioremediation efficiency falls on scale-up.

In an attempt to apply bioremediation technology from the flask (bench scale) to the field (full-scale design) , researchers do not understand the phenomena influencing bioremediation. Some of these phenomena are additional mass transport mechanisms, presence of multiple phases, spatial heterogeneities, and unfavorable factors for bacterial growth. Successful bioremediation therefore requires keen examination of such phenomena by using conceptual scales of observation: microscale, mesoscale, and macroscale. Microscale is the level at which chemical/microbial species and reactions can be characterized indepen­dently of any transport phenomena. Since these activities are at the microbial cell level this information comes from bench level work. Mesoscale is the level at which transport phenomena and system

geometry are first apparent, with the exclusion of advective or mixing processes. These are the activities that occur at the pore channel, soil particle or microbial aggregate level. Macroscale is the level at which one can discern advective or rmxIng phenomena. These activities are associated on a site level and are the focus of the design engineer. The critical path, as bioremediation technology is developed from flask to field is to observe and understand the phenomena that exert influence at each scale of observation so that its effects can be incorporated into the final remediation design38

.

Overcoming some of the disadvantages of bioremediation is phytoremediation, which can also be used in combination with bioremediation.

Phytoremediation involves the use of green plants to remediate contaminated soil or water. It is an inexpensive, low maintenance biotechnology that usually tolerates high concentrations of contaminants better than microorganisms. The plants should have a high growth rate and be able to out-compete undesirable invasive species. Phytoremediation comprises several phenomena: phytostabilization (reduces the bioavail­ability of the contaminants by binding them in plant ti ssues), phytoextraction (bioconcentrating contami­nants in the harvestable zones of the plant), phylodegradation (enzyme system of the plants and plant-associated microorganisms degrade the toxic compounds), phytovolatization (plants to volatilize pollutants). Chemicals most likely to be taken up are those with log octanol:water partition coefficients (log Kow) between 0.5 and 3.0. Nitroaromatic explosives are in this range as well nitroaromatic compounds principally undergo phytodegradation rather than phytoextraction, phytoremediation holds promising technology status for nitroaromatics. Nitroreductases and laccases are the enzymes implicated in the phytodegradation of nitromatics4.

Three aquatic species: pondweed, arrowroot, and coontail and one non-aqueous species, poplar were employed for remediating TNT contamination in a constructed wetland. This system could degrade 0.019 mg/l TNT per day4. Sikora et al. 39 used sago pond weed (Potamogeton pectinatus), water stargrass (Heteranthera dubia), elodea (Elodea canadensis) and parrot feather (Myriophyllum aquaticum) for remediating groundwater contamination. The gravel­based wetland reduced TNT concentration from range of 1.2-4.6 ppm to 0.002 ppm. Parrotfeather was studied for treatment of water contaminated with TNT in batch reactors. TNT removal rates increased with

1000 INDI AN J EX P BIOl, SEPTEMB ER 2003

increased plant density and removal kinetics inc reased

with increasing temperature up to 34°C I 6.

Several agricultural and indigenous te rrestri al p lants were examined for their capacity to accumulate and degrade HMX . Traces of mononitroso- HMX were detected in contaminated soil ex tracts and leaf ex tracts. Mechanism for HMX trans location and accumul ation in foliar ti ssue was concluded to be aqueous transpirational flux and evaporation4o.

These reports brighten the potential of applying phytoremediati on for ex pl os ives. Phytoremediation is more rugged than microbi a l bioreactors with respect to physical conditions and changes in contaminant load ing.

However phytore med iation , like bioremediati on suffers from unpredictable climate vanatlOn. Although plants are effec tive remedi ators due to the ir large amount of biomass, they are less efficient per unit of biomass than bacte ria. One majo r drawback is many o f the metabolic products of phytoremediation remain unidentified, making it diffi cult to assess the ir long- te rm fate and tox ic ity. Mo reover accumulation of ex pl os ives in plants to levels s ignificantl y above so il concentration is re levant to the assess ment of both phytoremediation potential and environmenta l ri sks~().

Bioremediation of effluent generated in production ofHMX

We have attempted bioremedi atio n or wastewater generated in production of explosive HMX. Three types o f effluents obtained from HMX manufacturing unit were studied . These were Effluent I (E l )­mother liquor containing acetic acid , nitri c acid, ammo nium nitra te, RDX/HMX ; Eftluent 2 (E2)­mother liquor partially neutrali zed with ammo nia ; Efflu ent 3 (E3) - final effl uent obtained after recovery of acetic acid. pH values of these effluents were in the range o f 1-2.5. Nitration be ing the manufacturing process, the waste waters have hi gh concentration of nitrate. The effluents were characterized in the laboratory for chemical oxygen demand (COD), ace tate and nitrate content by Standard Methods

41.

Soil samples co ll ec ted from factory premises were used for enrichment and isolation of microorganisms for degradation of HMX efflu ent. With so il enrichments, around 35-37% reduc ti on in chemi cal oxygen demand (COD) of the e fflu ents was obtained . Mi croorgani sms were isolated from these so il enrichments . The cultures were tes ted for the ir tolerance to different concentrations of ammonium nitrate and acetic acid incorporated in minimal

medium . The cultures could grow at 5% aceti c acid and 3% ammonium nitrate . Flask culture degradation of E2 by some of the cultures ranged between 20-36% in terms of COD under aeration within 5 days. Based on these results, a two stage process (aerati on followed by fix ed film reactor) was developed for treatment of E2 which res ulted in 48 % reducti o n in COD within one month . Since the concentrat ions of nitrate (- 1,00,000 mg/l ) and acetic ac id (-2,50,000 mg/l) in the effluents are very high , diluted efflu ent ( I: 10 and 1:50) E3 were used for further study. Yeas t culture isolated from this so il could reduce the COD and nitrate of the diluted waste waters. At initi a l pH 6.0 the isolate gave 75% reduction in COD and 65 % reduction in nitrate, while at initi a l pH 7 .0 the culture gave 25 % reductio n in COD and 50% in nitrate after 15 days. These results indicate the feas ibility of appli cati on of bio remediation for treatment of waste waters . To solve the probl em more compl ete ly the natural processes controlling N levels need to be better understood.

Future work There is meagre data o n aerobic deg radatio n of

HMX. Aerobic pathway of degradati on of HMX has not so far been illustrated . These areas are still open for researchers. Future work should focus on the enzymes invol ved in the initial attack on RDX/ HMX and the ro le of enzy mes versus abio ti c mechani sms in the subsequent complex reac tions that take place following ring c leavage. Molec ul ar aspects of degradati on of RDX/HMX also are fields of inte rest.

Acknowledgement The authors are thankful to the au thorities of High

Energy Materi als Research Laboratory (HEMRL), DRDO, Govt. of Indi a, Sutarwadi , Pune 411021 for sponsoring one year proj ect, suppl y ing effluents generated in production of HMX and re levant information for o ur s tudies o n HMX.

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