Isolation of Three Hexahydro-l,3,5-Trinitro-l,3,5-Triazine-Degrading Species of the Family Enterobacteriaceae from Nitramine Explosive-Contaminated Soil

CHRISTOPHER L. KITTS, DARYL P. CUNNINGHAM, AND PAT J. UNKEFER CST-IB, Los Alamos National Laboratory, Los Alamos, New Mexico

Three species of the family Enterobacteriaceae that biochemically reduced hexahydro-l,3,5-trinitro-l,3,5­triazine (RDX) and octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine (HMX) were isolated from nitramine explosive-contaminated soil. Two isolates, identified as Morganella morganii and Providencio rettgeri, completely transformed both RDX and the nitroso-RDX reduction intermediates. The third isolate, identified as Citrobacter freundii, partially transformed RDX and generated high concentrations of nitroso-RDX interme­diates. All three isolates produced 14COZ from labeled RDX under 0z-depleted culture conditions. While all three isolates transformed HMX, only M. morganii transformed HMX in the presence of RDX.

The nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-tria­zine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazo­cine (HMX) are used by the military in high-yield munitions, often in combination. Manufacture and testing of explosives have resulted in soils contaminated with mixtures of these nitramine explosives. Bioremediation is proposed as a safe and cost-effective means of site cleanup (9, 11, 15). Attempts to biodegrade RDX and HMX aerobically have not been success­ful (8, 12, 15, 19). However, biodegradation of both RDX and HMX has been reported to occur under anaerobic or anoxic conditions (6, 9, 12, 13, 16, 19).

The first step of biological RDX degradation involves reduc­tion of the nitro groups to form nitroso compounds. Stable mono-, die, and trinitroso derivatives of RDX have been isolated (12, 13). Mono-, die, trio, and tetranitroso intermedi­ates of biological HMX reduction have also been observed (13, 19). McCormick et al. used results obtained with a sewage sludge inoculum to propose a degradation pathway for RDX wherein the biochemical reduction of mono-, die, and trini­troso-RDX intermediates to unstable hydroxylamino com­pounds is followed immediately by hydrolytic ring cleavage (12). Hydrolysis of the remaining carbon-nitrogen bonds and reduction of all nitro groups to amines complete the postulated pathway. Strictly anaerobic cultures did not produce I4C02 from labeled RDX; however, I4C02 production was observed in composting experiments (9).

Previous work with RDX degradation used sewage sludge, horse manure, or amended soils; however, no microorganisms were isolated or identified (6, 9, 12, 13). In this report, we describe the isolation and identification of three individual members of the family Enterobacteriaceae, each of which degraded RDX in pure culture.

Isolation and identification of RDX-reducing bacterial strains. The numerically predominant, aerotolerant bacteria from a nitramine explosive-contaminated soil inoculum were the subject of this investigation. An aqueous extract of nitra­mine explosive-contaminated soil from Los Alamos National Laboratory (110 mg of HMX g of soil- I and 16 mg of RDX g of SOil-I) was used to inoculate nutrient broth (Difco) con­taining 0.33 mM RDX and 0.05 mM HMX. The culture was shaken (150 rpm, 30°C) in a stoppered flask for 2 weeks, when 1 ml was transferred to fresh medium and incubated for 7 more days. During this time, both RDX and HMX were completely

transformed. The predominant organisms from this culture were isolated by dilution plating onto nutrient agar (Difco) and subsequently tested for the ability to degrade RDX. Several of the isolated bacterial strains tested did not transform RDX after 2 weeks of growth under the conditions described below. However, three isolated species were able to transform RDX in pure liquid culture. These isolates were identified as Provi­dencia rettgeri B1, Morganella morganii B2, and Citrobacter freundii NS2 with the Biolog system (2) and the API20E system (18) (Table 1). The identifications were confirmed by reference to Bergey's Manual of Systematic Bacteriology (3). Standard bacterial physiology tests (7) were also applied (Table 1). These strains were sent to the American Type Culture Collec­tion for storage and dissemination.

Nitramine explosive degradation in liquid culture. Explo­sive degradation was accomplished by aerobic culturing of the organisms followed by culturing under 02-depleted conditions. YE medium (minimal salts (1] with 0.8% yeast extract [Difco]) was supplemented with either a mixture of RDX (0.33 mM) and HMX (0.05 mM) or HMX (0.07 mM) alone. Thirty­milliliter cultures were initially grown aerobically in 50-ml fluted flasks at 30°C with shaking (150 rpm). Although the aerobic growth rate of each bacterial isolate was unaffected by these concentrations of RDX and HMX, they were unable to transform either compound when grown aerobically. There­fore, once aerobic cultures reached an A S60 of 1.0 (6 to 8 h of growth), the flasks were plugged with rubber stoppers to obtain 02-depleted conditions. Each culture was momentarily ex­posed to air once every 2 to 3 days when samples were taken. Samples (400 fl.l) were centrifuged for 5 min in a microcentri­fuge, and 350 fl.l of the culture supernatant was set aside. The cell pellet was resuspended in acetonitrile (400 fl.\) to recover any hydrophobic compounds that may have been attached to cell wall material. After centrifugation, 350 fl.l of acetonitrile supernatant was combined with the culture supernatant and filtered (0.2-fl.m-pore-size filter). The filtrate was analyzed for RDX, HMX, and the products of biotransformation. After 45 days of growth, cultures were plated on nutrient agar. Inspec­tion of the colonies growing on these plates after 2 days of

1. of and testsTABLETABLE 1. ResultsResults of genusgenus and speciesspecies identificationidentification tests

% ConfidenceConfidenceOxidase C t I Gram M t·l·ty Nitrate Lysine OrnithineOrnithine Citrate Urease H2S. P-Galacto-Strain Gram Nitrate Lysine Citrate H2S I3-Galacto­ ConversionConversion ofof % Motility production tryptophantryptophan toto fofB·BiologItttesta a ase stainstain 0 1 1 reductasereductase decarboxylasedecarboxylase decarboxylasedecarboxylase utilizationutilization productIOn sidasesidase indole 0 10 og es

B2B2 - + - + + - + - + - - + 9292+ + + + + + BlBi - ++ - ++ + - ­ ++ ++ - - + 9595+ + NS2 - + - + + - ­ + - + + - 96NS2 + + + + + + 96

that the the and Allgrowthgrowth showedshowed that eacheach cultureculture containedcontained onlyonly the originallyoriginally generategenerate the aqueousaqueous and ether-solubleether-soluble fractions.fractions. All fractionsfractions by (ininoculatedinoculated bacterialbacterial strain.strain. werewere thenthen analyzedanalyzed by liquidliquid scintillationscintillation countingcounting (in PackardPackard

of and of XR to the finalQuantitationQuantitation of explosivesexplosives and intermediatesintermediates of biologicalbiological UltimaUltima GoldGold XR scintillationscintillation fluid)fluid) to determinedetermine the final and ofreduction.reduction. RDX,RDX, HMX,HMX, and theirtheir biotransformationbiotransformation productsproducts distributiondistribution of radioactivity.radioactivity.

were by liq­ of the 14CO2were separatedseparated by usingusing reverse-phasereverse-phase high-performancehigh-performance liq­ EachEach of the threethree isolatesisolates releasedreleased 14C02 whenwhen incubatedincubated uid a C-18 [14C]RDX 2). of the P. anduid chromatographychromatography (HPLC)(HPLC) withwith a 25-cm25-cm DynamaxDynamax C-18 withwith P4C]RDX (Table(Table 2). MostMost of the labellabel fromfrom P. rettgerirettgeri and

and a of MeOH-H20 M. in the Thecolumncolumn (Rainin)(Rainin) and a mobilemobile phasephase of 28:7228:72 MeOH-H20 M. morganiimorganii culturescultures remainedremained in the aqueousaqueous phase.phase. The at a flow rate of 0.5 ml min-'. were of RDX(Baker)(Baker) at a flow rate of 0.5 ml min-1. TheThe explosivesexplosives were ether-solubleether-soluble fractionsfractions containedcontained residualresidual amountsamounts of RDX

on the of A230. By this the and its nitroso as by The C.quantifiedquantified on the basisbasis of theirtheir A 230• By this method,method, the and its nitroso derivatives,derivatives, as shownshown by HPLCHPLC analysis.analysis. The C. of and had the of labelminimumminimum detectabledetectable concentrationconcentration of RDXRDX and nitroso-RDXnitroso-RDX freundiifreundii cultureculture had the highesthighest percentagepercentage of radioactiveradioactive label

was 1.3 ,uM. The at 230 nm of in the of the isolatesintermediatesintermediates was 1.3 f..LM. The molarmolar extinctionsextinctions at 230 nm of remainingremaining in the ether-solubleether-soluble fraction.fraction. NoneNone of the isolates the of and are to of andthe nitrosonitroso derivativesderivatives of RDXRDX and HMXHMX are identicalidentical to producedproduced significantsignificant amountsamounts of volatilevolatile compounds,compounds, and

of the The of RDX did notthosethose of the parentparent compoundscompounds (10).(10). The nitrosonitroso derivativesderivatives of intermediatesintermediates producedproduced duringduring RDX degradationdegradation did not RDX and were by of their to to cell material.RDX and HMXHMX were identifiedidentified by comparisoncomparison of their appearappear to adhereadhere to cell material.

times and their to In contrast to results with anaer­retentionretention times duringduring HPLCHPLC and their molecularmolecular weightsweights to In contrast to results obtainedobtained previouslypreviously with strictlystrictly anaer­those of and obic CO2 was evolved in this by usingthose of authenticauthentic compoundscompounds synthesizedsynthesized and generouslygenerously obic liquidliquid cultureculture (12),(12), CO2was evolved in this studystudy by using

with us by J. of the New Mexico Technical an O2-depleted culture. Each of the three isolates testedsharedshared with us by J. OxelyOxely of the New Mexico Technical an 02-depleted liquidliquid culture. Each of the three isolates tested (Hewlett- 14CO2 when with ['4C]RDX 2).Institute.Institute. LiquidLiquid chromatography-masschromatography-mass spectrometryspectrometry (Hewlett­ releasedreleased 14C02 when incubatedincubated with [14C]RDX (Table(Table 2).

5989A mass was with a our isolates were all able to break the RDX ring becausePackardPackard 5989A mass spectrometer)spectrometer) was performedperformed with a Thus,Thus, our isolates were all able to break the RDX ring because and the HPLC above 14C02 could be released only if the ring carbons of RDX werethermospraythermospray interfaceinterface and the HPLC methodmethod describeddescribed above 14C02 could be released only if the ring carbons of RDX were

with of 0.1 M acetate to the mobile phase. This is consistent with awith additionaddition of 0.1 M ammoniumammonium acetate to the mobile phase. completelycompletely metabolized.metabolized. This findingfinding is consistent with a For all and their this of 14C02 released from RDXFor all nitraminenitramine explosivesexplosives and their reductionreduction products,products, this previousprevious reportreport of 14C02 beingbeing released from RDX degradeddegraded

gave a molecular ion mass of +59 (an acetate in a where a mixture of and anoxicprocedureprocedure gave a molecular ion mass of +59 (an acetate in a compostcompost where a mixture of aerobic,aerobic, anaerobic,anaerobic, and anoxic as the peak in the detection mode. conditions could have existed (9). The distribution of label inadduct)adduct) as the majormajor peak in the negative-ionnegative-ion detection mode. conditions could have existed (9). The distribution of label in

with ['4C]RDX. The fate of RDX carbons was the also well to the amount of RDXExperimentsExperiments with [14C]RDX. The fate of RDX carbons was the organicorganic phasephase also correspondscorresponds well to the amount of RDX monitored by using the described above and nitroso-RDX intermediates after 45 days in themonitored by using the culturingculturing proceduresprocedures described above and nitroso-RDX intermediates remainingremaining after 45 days in the with a few modifications. YE medium (120 ml in a 500-ml unlabeled described below.with a few modifications. YE medium (120 ml in a 500-ml unlabeled experimentsexperiments described below. flask) was with a mixture of unlabeled RDX and Biochemical reduction of RDX by three individual strains.flask) was supplementedsupplemented with a mixture of unlabeled RDX and Biochemical reduction of RDX by three individual strains. [U-14C]RDX (1.2 radiochemical >93%; Chem­ All three isolates transformed RDX in pure culture during the[U_14C]RDX (1.2 Ci/mol;Ci/mol; radiochemical purity,purity, >93%; Chem­ All three isolates transformed RDX in pure culture during the syn Science Labs) to make a final RDX concentration of 0.15 phase of growth. P. rettgeri and M. morganii trans­syn Science Labs) to make a final RDX concentration of 0.15 stationarystationary phase of growth. P. rettgeri and M morganii trans­mM (72 mCi mol-1) and autoclaved to ensure sterility. Cul­ formed all of the RDX, whereas C. freundii did not (Fig. 1).mM (72 mCi mol-I) and autoclaved to ensure sterility. Cul­ formed all of the RDX, whereas C. freundii did not (Fig. 1).tures were inoculated with the strain aerobically Mono- and dinitroso derivatives of RDX accumulated totures were inoculated with the appropriateappropriate strain aerobically Mono- and dinitroso derivatives of RDX accumulated to grown to an A560 of 1.0, at which point the flasks were plugged maximum concentrations within 10 days. M. morganii accumu­grown to an A 560 of 1.0, at which point the flasks were plugged maximum concentrations within 10 days. M. morganii accumu­and incubated for 45 days. At this time, the labeled volatile lated the lowest concentrations of these nitroso compounds. P.and incubated for 45 days. At this time, the labeled volatile lated the lowest concentrations of these nitroso compounds. P.organics and CO2 were recovered and trapped by first sparging rettgeri and M. morganii removed each of these nitroso-RDXorganics and CO2were recovered and trapped by first sparging rettgeri and M. morganii removed each of these nitroso-RDXthe of each flask with air for 6 min and passing the intermediates to levels below 5% of the initial RDX concen­the headspaceheadspace of each flask with air for 6 min and passing the intermediates to levels below 5% of the initial RDX concen­exit gasses through a volatile organic trap (Supelco) and a trations (Fig. 1A and B). As corroborated by the radiolabelexit gasses through a volatile organic trap (Supelco) and a trations (Fig. 1A and B). As corroborated by the radiolabelsolution of 5 N NaOH. The culture medium had become basic experiments described above, disappearance of the nitrososolution of 5 N NaOH. The culture medium had become basic experiments described above, disappearance of the nitroso (pH 8 to 9), requiring that it be acidified (1 ml of H2SO4) to intermediates also implies ring breakage. Assuming that the(pH 8 to 9), requiring that it be acidified (1 ml of H2S04) to intermediates also implies ring breakage. Assuming that the ensure complete recovery and trapping of 14C02. Cellular pathway suggested by McCormick et al. (12) applies, these ensure complete recovery and trapping of 14C02. Cellular pathway suggested by McCormick et al. (12) applies, thesematerial was removed by centrifugation. The culture superna­ bacteria may reduce the nitroso groups to unstable hydrox­material was removed by centrifugation. The culture superna­ bacteria may reduce the nitroso groups to unstable hydrox­tant was extracted with an equal volume of ethyl ether to ylamino groups, which decompose, thus hydrolyzing the ring.tant was extracted with an equal volume of ethyl ether to ylamino groups, which decompose, thus hydrolyzing the ring.

TABLE 2. Distribution of radioactivity' from metabolism of ['4C]RDXTABLE 2. Distribution of radioactivirya from metabolism of P4C]RDX

Avg % radioactivityAvg % radioactivity

Cell- Ether-Organism Total at Total at Cell- Volatile Ether- AqueousOrganism AqueousTotal at Total at associated Volatile CO2 soluble fractionstart end associated organics CO2 soluble start end fraction organics fraction fraction

fraction fraction

M. morganii B2 100 75 (5) 1 (0.2) <1 5 (1) 7 (1) 62 (7)M. morganii B2 100 75 (5) 1 (0.2) <1 5 (1) 7 (1) 62 (7)P. rettgeni B1 100 75 (7) <1 1 8 (2) 14(2) 52(5)P. rettgeri Bl 100 75 (7) <1 ~1 8 (2) 14 (2) 52 (5)C. freundii NS2 100 65 (9) <1 1 (0.2) 9 (2) 33 (4) 22 (3)C. freundii NS2 100 65 (9) <1 1 (0.2) 9 (2) 33 (4) 22 (3)

a Distribution by percentage of initial [14C]RDX added (total, 2.8 x 107 cpm). The values shown are averages of duplicate samples with one standard deviation in a Distribution by percentage of initial [14C]RDX added (total, 2.8 X 107 cpm). The values shown are averages of duplicate samples with one standard deviation inparentheses.

parentheses.

1.8100 1.8100

9595 C 90 1.690 1.6 8585 8080 1.41.47575 7070 1.21.2650 0 65

c '"0'0 CD

a = 6060 1.0 Q55 1.0 !55

~-o 500 0. 50 0.8..Ec.. cL _ 45 0.8 CD0

C45 0.8 000 Qow _ 0 4040 "ii

35 u35 0.6 ;:0.0.'­ Q,

-0 03030 0

Xe 25wJ . 25 0.40.420

20 1515 0.210 0.2

10 55 0 0.0

OM~~~=;:::;=-a-:;:::;::::B=sp...e~-r-r-""T""J"-.,......,~)---r(:r-........,......,r-Ot).(J¥.!IQ':;=,-......-r-~......-,--..--r--t- 0.0 0 15 30 45 15 30 45 15 30 45o 15 30 45 15 30 45 15 30 45

Time (d)Time (d) FIG. 1. Degradation of an RDX-HMX mixture in YE medium by M. morganii B2 (A), P. rettgeri Bl (B), and C. freundii NS2 (C). Each nitrosoFIG. 1. Degradation of an RDX-HMX mixture in YE medium by M morganii B2 (A), P. rettgeri B1 (B), and C. freundii NS2 (C). Each nitrosointermediate concentration is expressed as a percentage of the parent compound's initial concentration. Symbols: C, RDX; 0, mononitroso-RDX;intermediate concentration is expressed as a percentage of the parent compound's initial concentration. Symbols: 0, RDX; 0, mononitroso-RDX;A, dinitroso-RDX; O, trinitroso-RDX; *, HMX; 0, mononitroso-HMX; A, dinitroso-HMX; X, cell growth (A560). Nota bene: because the initial

~,dinitroso-RDX; 0, trinitroso-RDX; _, HMX; e, mononitroso-HMX; A, dinitroso-HMX; x, cell growth (AS60). Nota bene: because the initial concentration of HMX was an order of magnitude less than that of RDX, the absolute rate of HMX transformation by M. morganii was an orderconcentration of HMX was an order of magnitude less than that of RDX, the absolute rate of HMX transformation by M morganii was an order of magnitude less than that of RDX transformation.of magnitude less than that of RDX transformation.

C. freundii transformed the least RDX and accumulated thec. freundii transformed the least RDX and accumulated the most nitroso-RDX compounds (Fig. 1C). Only this isolatemost nitroso-RDX compounds (Fig. lC). Only this isolate produced detectable amounts of trinitroso-RDX, which mayproduced detectable amounts of trinitroso-RDX, which may indicate a reduced ability to transform these nitroso com­indicate a reduced ability to transform these nitroso com­pounds.pounds.Biochemical reduction of HMX by three individual strains.

Biochemical reduction of HMX by three individual strains. Each isolate catalyzed the disappearance of another nitramineEach isolate catalyzed the disappearance of another nitramine explosive, HMX, although the process was slower and lessexplosive, HMX, although the process was slower and less complete than that for RDX. After 45 days, M. morganiicomplete than that for RDX. After 45 days, M. morganiitransformed approximately 60% of the initial HMX, P. rettgeritransformed approximately 60% of the initial HMX, P. rettgeritransformed -60%, and C. freundii transformed -50%.transformed ........60%, and C. freundii transformed ........50%.Mono- and dinitroso intermediates of HMX also accumulatedMono- and dinitroso intermediates of HMX also accumulated in the media of all three cultures.in the media of all three cultures. The extent of HMX transformation by M. morganii was not

The extent of HMX transformation by M. morganii was not inhibited by RDX (Fig. 1A). In contrast, RDX stronglyinhibited by RDX (Fig. lA). In contrast, RDX stronglyinhibited transformation of HMX by either C. freundii or P.inhibited transformation of HMX by either C. freundii or P.rettgeri (Fig. 1B and C). M. morganii was probably able torettgeri (Fig. IB and C). M morganii was probably able to further metabolize nitroso-HMX intermediates in the pres­further metabolize nitroso-HMX intermediates in the pres­ence of RDX, as supported by accounting for the initial HMX. ence of RDX, as supported by accounting for the initial HMX. At the end of the incubation, 15% remained as HMX, 25% wasAt the end of the incubation, 15% remained as HMX, 25% wasmononitroso-HMX, and 25% was dinitroso-HMX, leavingmononitroso-HMX, and 25% was dinitroso-HMX, leaving about one-third further metabolized, presumably in a mannerabout one-third further metabolized, presumably in a manner similar to the fate of the nitroso-RDX intermediates.similar to the fate of the nitroso-RDX intermediates. Summary. We have isolated three RDX-degrading members

Summary. We have isolated three RDX-degrading members of the family Enterobacteriaceae from explosive-contaminatedof the family Enterobacteriaceae from explosive-contaminated soil. All three isolates released 14C02 from [14C]RDX under soil. All three isolates released 14C02 from P4C]RDX under02-depleted conditions and were therefore able to break the 02-depleted conditions and were therefore able to break the RDX ring. All three isolates also transformed HMX; however,RDX ring. All three isolates also transformed HMX; however, P. rettgeri and C. freundii were unable to do so when RDX was P. rettgeri and C. freundii were unable to do so when RDX waspresent. The best demonstrations of RDX degradation have present. The best demonstrations of RDX degradation have been obtained with inocula derived from either sewage sludgebeen obtained with inocula derived from either sewage sludge (11, 12) or horse manure (9). Each of these inocula are good(11, 12) or horse manure (9). Each of these inocula are good

sources of members of the family Enterobacteriaceae, and sources of members of the family Enterobacteriaceae, andtherefore members of this family may be responsible for mosttherefore members of this family may be responsible for most of the nitramine explosive degradation witnessed under theseof the nitramine explosive degradation witnessed under thesecircumstances. Coincidentally, many enteric bacteria are rec­circumstances. Coincidentally, many enteric bacteria are rec­ognized for their aromatic nitroreductase activities (4, 5, 14, 17,ognized for their aromatic nitroreductase activities (4, 5, 14, 17, 20). We are currently examining an aromatic nitroreductase20). We are currently examining an aromatic nitroreductase activity in M. morganii to determine its involvement in nitra­activity in M. morganii to determine its involvement in nitra­mine reduction.mine reduction.

REFERENCESREFERENCES

1. Beardsmore, A. J., P. N. G. Aperghis, and J. R. Quayle. 1982.1. Beardsmore, A. J., P. N. G. Aperghis, and J. R. Quayle. 1982.Characterization of the assimilatory and dissimilatory pathways of

Characterization of the assimilatory and dissimilatory pathways of carbon metabolism during growth of Methylophilus methylotrophuscarbon metabolism during growth of Methylophilus methylotrophus on methanol. J. Gen. Microbiol. 128:1423-1439.on methanol. J. Gen. Microbiol. 128:1423-1439.2. Bochner, B. R., and M. A. Savageau. 1977. Generalized indicator

2. Bochner, B. R., and M. A. Savageau. 1977. Generalized indicator plate for genetic, metabolic and taxonomic studies with microor­plate for genetic, metabolic and taxonomic studies with microor­ganisms. Appl. Environ. Microbiol. 33:434.ganisms. AppI. Environ. MicrobioI. 33:434. 3. Brenner, D. J. 1984. Family I. Enterobacteriaceae, p. 409-516.

3. Brenner, D. J. 1984. Family I. Enterobacteriaceae, p. 409-516. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of In N. R. Krieg and J. G. Holt (ed.), Bergey's manual ofsystematic bacteriology, vol. 1. The Williams & Wilkins Co.,systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. Baltimore.4. Bryant, C., and M. DeLuca. 1991. Purification and characteriza­

4. Bryant, C., and M. DeLuca. 1991. Purification and characteriza­tion of an oxygen-insensitive NAD(P)H nitroreductase from En­tion of an oxygen-insensitive NAD(P)H nitroreductase from En­terobacter cloacae. J. Biol. Chem. 266:4119-4125. terobacter cloacae. J. BioI. Chern. 266:4119-4125. 5. Einisto, P., M. Watanabe, M. Ishidate, Jr., and T. Nohmi. 1991.

5. Einisto, P., M. Watanabe, M. Ishidate, Jr., and T. Nohmi. 1991.Mutagenicity of 30 chemicals in Salmonella typhimurium strains Mutagenicity of 30 chemicals in Salmonella typhimurium strainspossessing different nitroreductase or o-acetyltransferase activi­ties. Mutat. Res. 259:95-102.possessing different nitroreductase or o-acetyltransferase activi­

ties. Mutat. Res. 259:95-102. 6. Funk, S. B., D. J. Roberts, D. L. Crawford, and R. L. Crawford. 1993. Initial-phase optimization for bioremediation of munition6. Funk, S. B., D. J. Roberts, D. L. Crawford, and R. L. Crawford.

1993. Initial-phase optimization for bioremediation of munition compound-contaminated soils. Appl. Environ. Microbiol. compound-contaminated soils. Appl. Environ. MicrobioI. 59:2171-2177. 59:2171-2177.

7. Gerhardt, P., R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.). 1981. Manual of7. Gerhardt, P., R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A.

Wood, N. R. Krieg, and G. B. Phillips (ed.). 1981. Manual of

for formethodsmethods for generalgeneral bacteriology.bacteriology. AmericanAmerican SocietySociety for Microbi­Microbi­D.C.ology,ology, Washington,Washington, D.C.

8.8.Hotfsommer,Hoffsommer,J.J.C.,C.,etetaleal. 1978.1978. BiodegradabilityBiodegradability ofof TNT:TNT: a a pilot TR 77-136.three-yearthree-year pilot plantplant study.study. ReportReport NSWC/WOLNSWC/WOL TR 77-136.

Va.DefenseDefense TechnicalTechnical InformationInformation Center,Center, Alexandria,Alexandria, Va. 9. J. D., R. C. and J. F. 1982.9. Isbister,Isbister, J. D., R. C. Doyle,Doyle, and J. F. Kitchens.Kitchens. 1982. EngineeringEngineering

and of for the U.S.and developmentdevelopment supportsupport of generalgeneral decondecon technologytechnology for the U.S. Task 11. Composting ofArmy'sArmy's installationinstallation restorationrestoration program.program. Task 11. Composting of

DAAK11-80-C-0027. U.S. andexplosives.explosives. ReportReport DAAKll-80-C-0027. U.S. ArmyArmy ToxicToxic and Md.HazardousHazardous MaterialsMaterials Agency,Agency, Aberdeen,Aberdeen, Md.

1950. The spectraspectra ofof aliphaticaliphatic nitramines,nitramines, nitrosaminesnitrosamines and nitrates.nitrates. Can. J.and Can. J.

10.10. Jones,Jones, R.R.N.,N.,andandG.G.D.D. Thorn.Thorn. 1950. The ultravioletultraviolet absorptionabsorption

Res. 27:828-860.Res. 27:828--860. 11. D. L. 1990. of hazardous11. Kaplan,Kaplan, D. L. 1990. BiotransformationBiotransformation pathwayspathways of hazardous

p. In D. A.energeticenergetic organo-nitroorgano-nitro compounds,compounds, p. 155-181.155-181. In D. Kamely,Kamely, A. and G. S. andChakrabarty,Chakrabarty, and G. S. OmennOmenn (ed.),(ed.), BiotechnologyBiotechnology and biodeg­biodeg­

radation. Advances in series, vol. 4. Portfolio Pub­radation. Advances in biotechnologybiotechnology series, vol. 4. Portfolio Pub­Co., Tex.lishinglishing Co., Woodlands,Woodlands, Tex.

12. N. G., J. H. and A. M. 1981.12. McCormick,McCormick, N. G., J. H. Cornell,Cornell, and A. M. Kaplan.Kaplan. 1981. of Appl.BiodegradationBiodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine.hexahydro-1,3,5-trinitro-1,3,5-triazine. Appl.

Environ. 42:817-823.Environ. Microbiol.Microbiol. 42:817-823. 13. N. G., J. H. and A. M. 1984. The13. McCormick,McCormick, N. G., J. H. Cornell,Cornell, and A. M. Kaplan.Kaplan. 1984. The

anaerobic of RDX, HMX and theiranaerobic biotransformationbiotransformation of RDX, HMX and their acetylatedacetylatedderivatives. AD report no. A149464. U.S. Army Natick Researchderivatives. AD report no. A149464. U.S. Army Natick Research

and Mass.and DevelopmentDevelopment Center,Center, Natick,Natick, Mass. 14. M., T. Y. Y. and H.14. Mori,Mori, M., T. Miyahara,Miyahara, Y. Hasegawa,Hasegawa, Y. Kudo,Kudo, and H. Kozuka.Kozuka.

1984. of by coli1984. MetabolismMetabolism of dinitrotoluenedinitrotoluene isomersisomers by EscherichiaEscherichia coli from Chem. Bull.isolatedisolated from humanhuman intestine.intestine. Chern. Pharm.Pharm. Bull. 32:4070-4075.32:4070-4075.

15. C. A., and W. Sysl. 1991. of15. Myler,Myler, C. A., and W. Sysk. 1991. BioremediationBioremediation of explosivesexplosivessoils, p. In G. S.contaminatedcontaminated soils, p. 137-146.137-146. In G. S. SaylerSayler (ed.),(ed.), Environmen­Environmen­

tal for waste treatment. New York.tal biotechnologybiotechnology for waste treatment. PlenumPlenum Press,Press, New York. 16. J. L., and R. E. Klausmeyer. 1973.16. Osmon,Osmon, J. L., and R. E. K1ausmeyer. 1973. MicrobialMicrobial degradationdegradation

of Dev. Ind.of explosives.explosives. Dev. Ind. Microbiol.Microbiol. 14:247-252.14:247-252. 17. F. J., R. P. and J. L. Holtzman.17. Peterson,Peterson, F. J., R. P. Mason,Mason, J.J. Hovsepian,Hovsepian, and J. L. Holtzman.

1979. and by Esche­1979. Oxygen-sensitiveOxygen-sensitive and -insensitive-insensitive nitroreductionnitroreduction by Esche­richia coli and rat J. Biol. Chem.richia coli and rat hepatichepatic microsomes.microsomes. J. BioI. Chern. 254:4009­254:4009­4014.4014.

18. P. B., K. M. D. L. and A. Balows.18. Smith,Smith, P. B., K. M. Tomfohrde,Tomfohrde, D. L. Rhoden,Rhoden, and A. Balows. 1972. API a multitube method for of Entero­1972. API system:system: a multitube method for identificationidentification of Entero­bacteriaceae. Microbiol. 24:449-452.bacteriaceae. Appl.Appl. Microbiol. 24:449--452.

19. R. J., W. R. T.-W. Chou, S. Lee, D. S. Tse, and19. Spanggord,Spanggord, R. J., W. R. Mabey,Mabey, T.-W. Chou, S. Lee, D. S. Tse, and T. Mill. 1983. Environmental fate studies of HMX. Phase II-T. Mill. 1983. Environmental fate studies of HMX. Phase 11­detailed studies. Final SRI Menlo Park,detailed studies. Final report.report. SRI International,International, Menlo Park,Calif.Calif.

20. L., Y. Ping, and Y. Uanxi. 1987. of TNT-20. Wenzhong,Wenzhong, L., Y. Ping, and Y. Uanxi. 1987. PropertiesProperties of TNT­enzymes in intact cells of Citrobacter freundii. Actadegradingdegrading enzymes in intact cells of Citrobacter freundii. Acta

Microbiol. Sin. 27:257-263.Microbiol. Sin. 27:257-263.