Biotechnol Lett (2007) 29:411–419 DOI 10.1007/s10529-006-9244-y
ORIGINAL RESEARCH PAPER
TNT transformation products are affected by the growth conditions of Raoultella terrigena Harald Claus Æ Nina Perret Æ Tobias Bausinger Æ Gregor Fels Æ Johannes Preuß Æ Helmut Ko¨nig
Received: 22 September 2006 / Revised: 24 October 2006 / Accepted: 24 October 2006 / Published online: 30 November 2006 Springer Science+Business Media B.V. 2006
Abstract High concentrations of 2,4,6-trinitrotoluene (TNT) and related nitroaromatic compounds are commonly found in soil and groundwater at former explosive plants. The bacterium, Raoultella terrigena strain HB, isolated from a contaminated site, converts TNT into the corresponding amino products. Radio-HPLC analysis with [14C]TNT identified aminodinitrotoluene, diaminonitrotoluene and azoxy-dimers as the main metabolites. Transformation rate and the type of metabolites that predominated in the culture medium and within the cells were significantly influenced by the culture conditions. The NAD(P)H-dependent enzymatic reduction of nitro-substituted compounds by cell-free extracts of R. terrigena was evaluated in vitro.
H. Claus (&) H. Ko¨nig Johannes Gutenberg-University Mainz, Institute of Microbiology and Wine Research, J. J. Becherweg 15, 55099 Mainz, Germany e-mail:
[email protected] N. Perret G. Fels Department of Chemistry, University of Paderborn, Warburger Str. 100, 33098 Paderborn, Germany T. Bausinger J. Preuß Johannes Gutenberg-University Mainz, Institute of Geography, J. J. Becherweg 21, 55099 Mainz, Germany
Keywords azoxy dimers bioremediation nitroreductase trinitrotoluene Abbreviations ADNT DANT NB DNB TNB DNT TNT TN-2,2¢-azoxy
aminodinitrotoluene diaminonitrotoluene nitrobenzene dinitrobenzene trinitrobenzene dinitrotoluene 2,4,6-trinitrotoluene 4,4¢,6,6¢-tetranitro-2,2¢azoxytoluene TN-4,4¢-azoxy 2,2¢,6,6¢-tetranitro-4,4¢azoxytoluene TN-2,4´-azoxy 4,4¢,6,6¢-tetranitro-2,4¢azoxytoluene TN-2¢,4-azoxy 4,4¢,6,6¢-tetranitro-2¢,4azoxytoluene NA nitroaniline DNA dinitroaniline NP nitrophenol
Introduction As a result of the manufacture and widespread use of explosives, pesticides, dyes and pharmaceuticals, large amounts of nitroaromatic compounds are released into the environment. A serious
123
412
Biotechnol Lett (2007) 29:411–419
2004). A variety of bacteria reduce the nitro groups of TNT stepwise to the corresponding amines (R-NH2), generating the isomeric ADNTs and DANTs as the predominant metabolites (Kro¨ger et al. 2004). Reactive nitroso (R-NO) and hydroxylamino (R-NHOH) compounds are the intermediates of the reductive sequence (Vorbeck et al. 1998; Zaripov et al. 2004), which can further react to condensated dimmers and acetylated derivates of TNT (Heiss and Knackmus 2002; Kro¨ger et al. 2004). In addition, some of the nitroso intermediates may also be covalently bound to the cell matrix (Leung et al. 1995) (Fig. 1). These reactions characterize the co-metabolic pathway of TNT degradation by Raoultella terrigena strain HB, recently isolated from a contaminated former explosive production site (Claus et al. 2006a). Radioactivity measurements demonstrated that about 10–20% of the [14C]TNT initially present remained in the culture supernatant, whereas the residual 80–90% was tightly associated with the cellular pellet. This entrapment may be used for the treatment of TNT-contaminated waters (Claus et al. 2006b). However, occasionally we observed that the nutrient concentration in the culture media determined not only the transformation velocity, but also the nature and amounts of soluble and cellbound metabolites (Claus et al. 2006a). In order to find out the optimum transformation conditions,
ecological problem worldwide is the presence of 2,4,6-trinitrotoluene (TNT) in soils, groundwater and surface waters at sites where this explosive formerly was manufactured, loaded or demilitarised (Preuß and Haas 1987; Lenke et al. 2000; Fuller et al. 2004; Lewis et al. 2004; Preuß 2006). During the production and/or photo chemically transformation of TNT also significant amounts of trinitrobenzene (TNB) arise and enter the environment (Davies et al. 1997). TNT and also some of its primary metabolites are found to be toxic and mutagenic to aquatic and terrestrial organisms (Meada et al. 2000). Consequently, there is an urgent need for remediation to clean up contaminated sites to ensure environmental quality and safety. Presently, several methods of treating TNT contaminated soils have been developed (Lenke et al. 2000; Rodgers and Bunce 2001; Heiss and Knackmuss 2002; Fuller et al. 2004; Lewis et al. 2004; Schrader and Hess 2004) but all are very cost intensive. Carbon adsorption has often been used for effective treatment of ground- and surface-water from contaminated sites (Wujcik et al. 1992). However, the spent carbon constitutes a toxic and explosive waste (Schmidt et al. 1998). A recent study demonstrated that TNT degradation by bacteria may be an economically and realistic alternative for in situ bioremediation and for the cleanup of contaminated sites (Fritsche et al. 2000; Hawari et al. 2000; Zhao et al.
Fig. 1 Formation of dimers from TNT degradation products, and proposed covalent binding of TNT metabolites to cellular material
CH3
CH3
O2N
NO2
NO2
NO2 O2N [2H]
N
HO NH
TNT
CH3
O 2N [2H]
O2N
CH3 NO2 O2N [2H]
NH
OH
HX-protein
O
NO2 CH3
O 2N
NO2
O 2N
O H3C
N N
CH3 H3C
N
X Protein
OH O 2N
NO2
tetranitro-4,4'-azoxytoluene
123
O2N
protein-bound TNT-derivative
NO2
NH2
4-ADNT
Biotechnol Lett (2007) 29:411–419
we studied the effects of growth parameters (concentrations of TNT and glucose, pH, temperature) on the reaction rate and nature of transformation products. We also investigated the capacity of cell-free extracts to transform nitroaromatic compounds.
Materials and methods Microorganisms and chemicals The origin of R. terrigena strain HB and TNT used for degradation experiments has been previously described (Claus et al. 2006a). [14C]TNT (uniformly ring labelled; specific activity 2.2 mCi/ mmol), 4-ADNT and 1,3,5-TNB were synthesized according to described procedures (Meyer 1909; Sitzmann 1974; Nielsen et al. 1979; Kro¨ger and Fels 2000). 1,2-DNB, 1,4-DNB, 2,6-DNT and 3nitrophenol were purchased from Sigma-Aldrich Inc. (Munich, Germany). 1,3-DNB and 2,4-DNT were obtained from Merck Inc. (Darmstadt, Germany) and 2-nitrophenol from Riedel-de Hae¨n Inc. (Seelze, Germany). Analytical methods TNT and its main metabolites were analysed by HPLC and Radio-HPLC as described recently (Claus et al. 2006a). Culture conditions for bacterial transformation of nitroaromatic compounds For transformation studies, the mineral salt medium of Kalafut et al. (1998) was used with NH4Cl (7 mM) as nitrogen source. Nitroaromatic compounds were added at between 40 lM and 400 lM to the mineral salt medium before autoclaving. Phosphate and glucose solutions were prepared separately and added sterile to the residual components. For 14C studies, uniformly ring labelled [14C]TNT (33.3 kBq/ml), was added to the medium. Glucose was at either 0.3% or 3% (w/v). The pH of the culture medium was adjusted between 5.0 and 8.0 using 200 mM Na2HPO4 buffer. R. terrigena was precultured in
413
Standard I nutrient broth for 16 h at 30C on a shaker before inoculation into mineral salt media. These cultures were incubated for up to 7 days under aerobic conditions on a rotary shaker at temperatures between 10C and 37C. At regular intervals, aliquots were taken to determine transformation products. At the end, cells and insoluble material were separated by centrifugation at 40,000 · g for 30 min. The bacterial cell mass was washed twice with phosphate buffered saline solution (pH 7.4), extracted with acetonitrile for 16 h at 30C and centrifuged as above. The resulting fractions (supernatant, washings, acetonitrile extract) were analysed by HPLC. Preparation of cell-free extracts for enzymatic assays Raoultella terrigena HB was grown in 500 ml Standard-I nutrient broth (Merck, Darmstadt, Gemany) supplemented with TNT (44 lM) for 24 h at 30C. Cells were harvested by centrifugation (20 000 · g), washed twice in 100 mM sodium phosphate buffer (pH 7.0) and disrupted by at least two passages through a French pressure cell. The suspension was centrifuged at 40 000 · g at 4C for 30 min and the supernatant stored at –20C. The protein concentration of the cell-free extract was determined using a BCA protein assay kit (Uptima, France). Enzyme assays Nitroreductase activity was determined according to Kim and Song (2005). The assay mixture contained 22 lM of the corresponding substrate and 300 lM NAD(P)H in 100 mM Na/K phosphate buffer (pH 5.9). The reaction was started by adding 100 ll cell extract to 900 ll assay mixture and the enzymatic activity was measured by following the initial decrease of NADPH absorbance at 340 nm. In order to identify enzymatic transformation products, a similar assay mixture containing 117 lM TNT was incubated for 24 h at 30C. The reaction was stopped by heating at 100C for 1 min and metabolites identified by HPLC as described above.
123
414
Results Analysis of TNT and its metabolites Under standard conditions (0.3% glucose, pH 7.0, 30C) TNT was completely removed within 4 h by growing cultures of R. terrigena strain HB. Metabolites were identified using HPLC and radio-HPLC (Fig. 2). In the culture supernatants 2-ADNT and 4-ADNT were detected along with small amounts of 2,4-DANT, TN-2,2¢-azoxy and TN-4,4¢-azoxy. In contrast to the culture supernatant, the main transformation products found in the cell extracts were azoxy dimers. The radiochromatogram of the extract identified three peaks, two of which could be assigned to TN-2,2¢azoxy and TN-4,4¢-azoxy, respectively, in a ratio of 1:10. The third peak (Fig. 2c) most probably can be attributed to either the TN-2,4¢-azoxy or the TN-2¢,4-azoxy, or a mixture of these condensation products. The production of the dimers is likely the result of spontaneous hydroxylaminonitroso condensation reactions (Fig. 1). Effect of temperature and pH on TNT transformation
Biotechnol Lett (2007) 29:411–419
(Table 3). However the nature of metabolites varied considerable. At low glucose concentrations (0.3%) ADNTs were found as main transformation products in culture supernatants as predominant metabolites, whereas under high glucose conditions mainly DANTs were identified instead. In cell extracts high concentrations of TN-azoxys could be found at low glucose concentrations, but dramatically lower concentrations were detected at high glucose concentrations. These data suggest that an increased glucose concentration shifted the degradation products from preferentially ADNTs to mostly DANTs and that dimerization from hydroxylamino- and nitroso-intermediates to azoxy-compounds predominantly occurs at low glucose concentration. Increasing TNT concentrations at low amounts of glucose resulted in decreasing concentrations of 4-ADNT in the supernatant, whereas TN-4,4-azoxy in the cell extract slightly increased. In contrast, high glucose and TNT concentrations favoured the formation of 2,4-DANT, but TN-4,4-azoxy remained at low concentrations. Transformation of nitroaromatic compounds by cell-free extracts
Maximum transformation rate coincided with the optimum growth rate of R. terrigena strain HB at 37C and was low at 10C (Table 1). With respect to transformation products, low temperatures favoured the production of ADNTs, whereas more DANTs were found at higher temperatures. The influence of pH on TNT transformation is given in Table 2. Again the transformation rate followed the growth rate, which was low at pH of 5.0 and optimum between pH 7.0 and 8.0. The appearance of 4-ADNT reached a maximum at days 1–2 of incubation, which was followed by a slight decrease up to the end of incubation time. The 2,4-DANT concentrations increased with time and pH in the culture supernatants. Very low concentrations of 2,6-DANT were observed at pH 7–8, whereas no 2,6-DANT was detected at pH 5–6.
In vitro transformation assays were performed with bacterial cell-free extracts and NADPH as source of the reducing equivalent. After 24 h TNT was completely consumed and the same metabolites were identified as in the culture experiments (Table 4). Kinetic studies confirmed that the reductive enzymes had high activities for TNT (Table 5). The two DNT isomers were reduced to different extents, preferentially the para-nitro-compound. 1,3,5-TNB was a similar good substrate as TNT. With all DNBs tested, the cell extract exerted identical relative activities of about 13%. The activity with 2-methyl-5-nitroaniline was about 15% and with the 3-nitroisomer only 3.5%. 4-ADNT was not reduced, whereas nitrophenols served as substrates for the cellular extracts even though they were not converted by whole cells (data not shown).
Effect of TNT and glucose concentration
Discussion
TNT from 40 lM to 400 lM was completely transformed at both glucose concentrations tested
Previously we described an R. terrigena strain HB, which eliminated TNT by the nitro-group
123
Biotechnol Lett (2007) 29:411–419 250
200
TNT
TNT
A
cps
150
100
Retention Time [min]
50
0 0
500
1000
1500
2000
2500
3000
3500
4000
4500
time [sec]
160
B
140
DANT
100 cps
ADNT's
120
80 60 DANT
ADNT's
Retention Time [min]
40 20 0 0
500
1000
1500
2000
2500
3000
3500
4000
4500
3500
4000
4500
time [sec] 160
80
TN-2,2’-azoxy
100
Retention Time [min]
60
TN-4,4’-azoxy
TN-2,2’-azoxy
120
TN-4,4’-azoxy
C
140
cps
Fig. 2 Radio-HPLC(dotted lines) and HPLCUV- chromatograms (insert with solid lines) of culture supernatants (Fig. 2B) and cell extracts (Fig. 2C) of R. terrigena strain HB after six days growth in a minimal medium + TNT. Supernatant of sterile control (8.7 kBq/ml [14C]TNT, Fig. 2A)
415
40 20 0 0
500
1000
1500
2000
2500
3000
time [sec]
123
416
Biotechnol Lett (2007) 29:411–419
Table 1 Effect of temperature on TNT transformation in cultures of R. terrigena HB Incubation conditions
Growth (OD 600 nm)
10C 24 48 72 20C 24 48 72 30C 24 48 72 37C 24 48 72
0.16 0.24 0.64 0.40 0.74 1.02 0.50 0.75 1.09 0.60 0.93 0.93
h h h h h h h h h h h h
2,4,6-TNT and metabolites in the culture supernatant [lM] 2,4,6TNT
242,4ADNT ADNT DANT
2,6DANT
3,5DANT
TN-4,4¢azoxy
TN-2,2¢azoxy
348 165 69 74 0.5 – 8.6 – – 0.4 – –
2.2 3.6 5.1 5.4 3.5 0.4 6.3 0.6 0.3 3.0 0.2 0.5
– – 0.1 – 1.1 4.3 – 1.6 1.8 – – 0.6
1.2 1.4 0.8 1.8 – – 0.8 – – 0.2 – –
– 0.3 – 1.3 0.8 – 1.3 – – 0.5 – 0.1
– 0.2 – 0.9 0.1 – 1.1 – – – – 0.1
3.4 9.1 20.5 16.2 33.5 8.85 22.1 16.8 6.2 14.8 10.2 19.1
– – 0.8 – 10.2 62.0 0.5 30.0 33.4 0.4 3.8 16.9
The sterile minimal salt medium contained 3 g l–1 glucose and 348 lM TNT Variation between replicate samples was less than 10% Table 2 Effect of pH on TNT transformation in cultures of R. terrigena HB Incubation conditions Growth (OD 600 nm) 2,4,6-TNT and metabolites in the culture supernatant [lM] 2,4,6-TNT 2-ADNT 4-ADNT 2,4-DANT 2,6-DANT TN-4,4¢-azoxy pH 5.0 24 48 72 pH 6.0 24 48 72 pH 7.0 24 48 72 pH 8.0 24 48 72
h h h h h h h h h h h h
0.11 0.14 0.64 0.20 0.58 1.02 0.58 0.78 1.09 0.61 0.76 0.93
302.34 – – 56.76 – – 52.06 – – – – –
0.34 1.19 0.62 2.19 1.81 – 1.92 – – 2.67 – –
1.62 10.10 9.66 8.99 19.47 5.56 11.52 1.78 2.95 41.35 9.83 4.63
– – 0.60 – 2.00 2.82 – 4.91 11.63 2.63 16.84 15.82
– – – – – – – 0.31 0.59 0.24 0.67 0.92
0.04 0.04 0.04 3.41 0.76 0.11 0.17 0.04 – 1.02 – –
The sterile minimal salt medium contained 3 g l–1 glucose and 303 lM TNT Variation between replicate samples was less than 10%; concentration of TN-2,2¢-azoxy was below detection limit Table 3 Effect of different glucose and TNT concentrations on transformation products in cultures of R. terrigena HBa Glucose in minimal medium [%]
3
TNT in minimal media [lM] Metabolites detected [%] in
40 240 Supernatant
400
40 240 400 Cell extract
40 240 Supernatant
400
40 240 Cell extract
400
2-ADNT 4-ADNT 2,4-DANT 2,6-DANT azoxy dimmers
– 1.0 16.4 – –
0.0 0.1 34.7 1.4 –
– 0.0 – – 0.6
– 9.6 1.7 – –
0.2 3.3 0.2 0.0 –
–
0.2 1.7 0.0 – 42.8
a
0.3
0.0 0.1 22.5 0.9 –
– 0.6 0.3 – 3.1
0.0 0.5 0.4 – 3.4
0.1 4,0 0.3 0.0 –
After 7 days at 30C and pH 7.0; Variation between replicate samples was less than 10%
123
0.6 – – 32.0
– 0.9 0.0 – 37.1
Biotechnol Lett (2007) 29:411–419
417
Table 4 Transformation of TNT by a cell extract of R. terrigena HB Transformation products (lM) Assaya TNT + NADPH (control) TNT + NADPH + cell extract a
2,4,6-TNT 117 4.9
2-ADNT – 1.9
4-ADNT – 20.6
2,4-DANT – 1.5
TN-4,4¢-azoxy – 3.5
Assay was incubated at 37C for 24 h; reaction was stopped by heating at 100C for 1 min
Table 5 Activity of a cell extract from R. terrigena HB with different nitroaromatic compounds Substratea
Activityb (units ml–1)
2,4,6-TNT 2,4-DNT 2,6-DNT 1,3,5-TNB 1,3-DNB 1,4-DNB 2-Methyl-3-nitroaniline 2-Methyl-5-nitroaniline 2-NP 3-NP 4-ADNT
1.00 0.52 0.13 0.90 0.13 0.13 0.04 0.15 0.20 0.15 0.00
a
Stock solutions (2.2 mmol) of substrates in methanol were diluted 1:100 in K/Na phosphate buffer (pH 5.9) in the final spectrophotometric assay
b
One unit corresponds to a decrease of 0.1 of absorbance at 340 nm per minute
reduction pathway (Claus et al. 2006a). The explosive was mainly converted to polymeric azoxy condensation products, which accumulated within the bacterial cells and less to soluble ADNT and/or DANTs. By balancing the degradation reaction using 14C-TNT, we found that some of the material could not be extracted from the cell pellet but was rather covalently bound to the organic matrix (Leung et al. 1995). This entrapment may be exploited for the microbial remediation of TNT-contaminated waters (Claus et al. 2006b). In order to optimise this process we investigated the effects of culture conditions on the TNT transformation in more detail. Although TNT elimination was observed at all incubation temperatures tested, pH 8.0 and 37C may regarded as optimum with respect to the transformation velocity. Similar conditions have been found for the biodegradation of TNT by Pseudomonas
putida (Park et al. 2003). TNT was completely eliminated at all concentrations tested, however the amount of glucose in the mineral salt media had a significant impact on the quantitative and qualitative distribution of metabolites in the supernatants and cells. At low glucose conditions (0.3%) mainly ADNTs were detected, along with the formation of larger amounts of tetranitroazoxytoluenes. In contrast, at a ten-fold higher glucose concentration (3% glucose), 2,4-DANT was the almost exclusively detectable metabolite in the culture medium, accompanied by only minor amounts of azoxy-dimers in the cell pellet. One explanation is that at high glucose concentrations an excess of reduction equivalents is produced by aerobic metabolism. As six electrons, provided by NAD(P)H, are needed for the complete reduction of one nitro group in TNT (see Fig. 1; Vorbeck et al. 1998; Heiss and Knackmus 2002), the surplus of NAD(P)H may be used for the reduction of a further nitro group. Farmore, high amounts of NAD(P)H will preclude the accumulation of nitroso-dinitritoluenes, thus preventing azoxy dimer formation (Williams et al. 2004). It is most likely that these reactions are catalyzed by nitroreductases, which are widely distributed in gram-negative bacteria and are related to the old yellow flavoenzyme of yeast (French et al. 1998; Klausmeier et al. 2001; Williams et al. 2004; Kim and Song 2005). The efficiency of these enzymes depends on the redox-potential of the substrate, which is particularly obvious for TNT and 4-ADNT. The oneelectron redox potential for TNT has been shown to be 167 mV as compared to –82 mV for the 2,4-DANT (Kim and Song 2005). This probably is the reason, why the DANTs are dead-end products of metabolism (Heiss and Knackmuss 2002; Lewis et al. 2004). It is widely
123
418
accepted that because of the electron deficiency in trinitroaromatic compounds an initial reductive metabolization step is very favourable and that, for instance in TNT, this process predominantly starts at the para-nitro-group (Pak et al. 2000, Riefler and Smets 2002; Kim and Song 2005). However, some bacterial strains produce mainly the ortho-derivate (2-ADNT), which may be due to differences in substrate specificity of the degrading enzymes (Oh et al. 2003; Maeda et al. 2006). In addition to nitro-group reduction, the nitroreductases of Enterobacter cloacae, Escherichia coli, Pseudomonas fluorescens and Pseudomonas putida catalyse reduction of TNT aromatic ring by hydride addition yielding orange-coloured hydride- and dihydride Meisenheimer complexes under the release of nitrite (French et al. 1998; Williams et al. 2004; Khan et al. 2004; Cabellero et al. 2005). However, the metabolites produced by cultures and cell extracts indicate, that the enzymes from R. terrigena prefer the nitro-group reduction pathway similar to its close relative Klebsiella sp. (Kim and Song 2005). Apart from TNT, cell cultures of R. terrigena HB transformed DNTs and nitrobenzenes (data not shown). The comparison of microbial transformation of whole cells as opposed to a cell-free extract suggests that nitrophenolic compounds are substrates for the reducing enzymes, but that they presumably cannot pass the bacterial cell membrane and/or act as metabolic inhibitors. In contrast, nitrobenzenes were as good substrates for whole cells and cell extracts. Conclusively, our results have shown that R. terrigena strain HB eliminates low and high TNT concentrations from water samples and that the efficiency of the process can be regulated by controlling temperature, nutrient and pH conditions. In addition to TNT the bacterium may be useful for the treatment of other nitroaromatic wastes as well. Acknowledgements The authors appreciate a financial support by the ‘‘Zentrum fu¨r Umweltforschung der Johannes Gutenberg-Universita¨t’’. H. C. wants to thank Magdalena Krol (University of Posen, Poland) for conducting some of the microbiological experiments.
123
Biotechnol Lett (2007) 29:411–419
References Caballero A, Esteve-Nu´n˜ez A, Zylstra GJ, Ramos JL (2005) Assimilation of nitrogen from nitrite and trinitrotoluene in Pseudomonas putida JLR11. J Bact 187:396–399 Claus H, Bausinger T, Lehmler I, Perret N, Fels G, Dehner U, Preuß J, Ko¨nig H (2006a) Transformation of 2,4,6-trinitrotoluene (TNT) by Raoultella terrigena. Biodegradation DOI 10.10007/s10532-005-9033-7 Claus H, Bausinger T, Lehmler I, Dehner U, Preuß J, Ko¨nig H (2006b) Bakterielles Einschrittverfahren zum TNT-Abbau. German Patent No. 10359610, 5 Jan 2006 Davies EP, Boopathy R, Manning J (1997) Use of trinitrobenzene as a nitrogen source by Pseudomonas vesicularis isolated from soil. Curr Microbiol 34:192–197 French CE, Nicklin S, Bruce NC (1998) Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and a pentaerythitol tetranitrate reductase. Appl Environ Microbiol 64:2864–2868 Fritsche W, Scheibner K, Herre A, Hofrichter M (2000) Fungal degradation of explosives: TNT and related nitroaromatic compounds. In: Spain JC, Hughes JB, Knackmus HJ (eds) Biodegradation of nitroaromatic compounds and explosives. CRC Press, Boca Raton, pp 213–237 Fuller ME, Hatzinger PB, Rungmakol D, Schuster RL, Steffan RJ (2004) Enhancing the attenuation of explosives in surface soils at military facilities: combined sorption and biodegradation. Environ Tox Chem 23:313–324 Hawari J, Beaudet S, Halasz A, Thiboutot S, Ampleman G (2000) Microbial degradation of explosives: biotransformation versus mineralization. Appl Microbiol Biotechnol 54:605–618 Heiss G, Knackmus HJ (2002) Bioelimination of trinitroaromatic compounds: immobilisation versus mineralization. Curr Opin Microbiol 5:282–287 Kalafut T, Wales ME, Rastogi VK, Naumova RP, Zaripoca SK, Wild JR (1998) Biotransformation patterns of 2,4,6-trinitrotoluene by aerobic bacteria. Curr Microbiol 36:45–54 Khan H, Barna T, Harris RJ, Bruce NC, Barsukov I, Munro AW, Moody PCE, Scutton NS (2004) Atomic resolution structures and solution behaviour of enzyme-substrate complexes of Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase. J Biol Chem 29:30563–30672 Kim HY, Song HG (2005) Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp C1 and enzymatic transformation of 2,4,6-trinitrotoluene. Appl Microbiol Biotechnol 68:766–773 Klausmeier RE, Appleton JA, DuPre ES, Tenbarge K (2001) The enzymology of trinitrotoluene reduction (Reprinted). Int Biodeterior Biodegrad 48:67–73 Kro¨ger M, Fels G (2000) 14C-TNT synthesis revisited. J Labelled Cpds Radiopharm 43:217–227
Biotechnol Lett (2007) 29:411–419 Kro¨ger M, Schumacher ME, Risse H, Fels G (2004) Biological reduction of TNT as part of a combined biological-chemical procedure for mineralization. Biodegradation 15:241–248 Lenke H, Achtnich C, Knackmus HJ (2000) Perspectives of bioelimination of polynitroaromatic compounds. In: Spain JC, Hughes JB, Knackmus HJ (eds) Biodegradation of nitroaromatic compounds and explosives. CRC Press, Boca Raton, pp 91–126 Leung KH, Yao M, Stearns R, Chiu S-H L (1995) Mechanism of bioactivation and covalent binding of 2,4,6-trinitrotoluene. Chemico-Biological Interact 97:37–51 Lewis TA, Newcombe DA, Crawford RL (2004) Bioremediation of soils contaminated with explosives. J Environ Management 70:291–307 Maeda T, Kadokami K, Ogawa HI (2006) Characterization of 2,4,6-trinitrotoluene (TNT)-metabolizing bacteria isolated from TNT-polluted soils in the Yamada Green Zone, Kitakyushu, Japan. J Environm Biotechnol 6:33–39 Meyer J (1909) Verfahren zur Herstellung von symmetrischem Trinitronenzol aus Halogentrinitrobenzol. German Patent No. 1234726, 18 Jul 1909 Nielsen AT, Henry RA, Norris WP (1979). Synthetic routes to aminodinitrotoluenes. J Org Chem 44:2499– 2504 Oh BT, Shea PJ, Drijber RA, Vasilyeva Gk, Sarath G (2003) TNT biotransformation and detoxification by a Pseudomonas aeruginosa strain. Biodegradation 14:309–319 Pak JW, Knoke KL, Noguera DR, Fox BG, Chambliss GH (2000) Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C. Appl Environ Microbiol 66:4742–4750 Park C, Kim T-H, Kim S, Kim S-W, Lee J, Kim S-H (2003) Optimization for biodegradation of 2,4,6-trinitrotoluene (TNT) by Pseudomonas putida. J Biosci Bioeng 95:567–571 Preuß J, Haas R (1987) Die Standorte der Pulver-, Sprengstoff-, Kampf- und Nebelstofferzeugung im ehemaligen Deutschen Reich. Geogr Rundsch 39:578–584
419 Preuß J (2006) Ru¨stungsaltlasten in Deutschland: Die Entstehung bis zum Jahr 1945. In: Hessisches Ministerium fu¨r Umwelt, la¨ndlichen Raum und Verbraucherschutz & HIM-ASG (ed) Boden gut gemacht. Die Sanierung des Ru¨stungsaltstandortes Stadtallendorf. Stadtallendorf: pp 21–51 Riefler PG, Smets BF (2002) NAD(P)H: flavin mononucleotide oxidoreductase inactivation during 2,4,6-trinitrotoluene reduction. Appl Environ Microbiol 68:1690–1696 Rodgers JD, Bunce NJ (2001). Treatment methods for the remediation of nitroaromatic explosives. Wat Res 35:2101–2111 Schmidt TC, Steinbach K, von Lo¨w E, Stork G (1998) Highly polar metabolites of nitroaromatic compounds in ammunition wastewater. Chemosphere 37:1079– 1090 Schrader PS, Hess TF (2004). Coupled abiotic-biotic mineralization of 2,4,6-trinitrotoluene (TNT) in soil slurry. J Environ Qual 33:1202–1209 Sitzmann ME (1974) Chemical reduction of 2,4,6-trinitrotoluene - initial products. J Chem Eng Data 19:179–181 Vorbeck C, Lenke H, Fischer P, Spain JC, Knackmuss HJ (1998) Initial reductive reactions in aerobic microbial metabolism of 2,4,6-trinitrotoluene. Appl Environ Microbiol 64:246–252 Williams RE, Rathbone DA, Scrutton NS, Bruce NC (2004) Biotransformation of explosives by the old yellow enzyme family of flavoproteins. Appl Environ Microbiol 70:3566–3574 Wujcik WJ, Lowe WL, Marks PJ (1992) Granular activated carbon pilot treatment studies for explosives removal from contaminated groundwater. Environ Prog 11:178–189 Zaripov SA, Naumov AV, Suvorova ES, Garusov AV, Naumova RP (2004) Initial Stages of 2,4,6-Trinitrotoluene Transformation by Microorganisms. Microbiol 73:398–403 Zhao JS, Fournier D, Thiboutot S, Ampleman G, Hawari J (2004) Biodegradation and Bioremediation of Explosives. In: Singh A, Ward OP (eds) Soil Biology I, Applied Bioremediation and Phytoremediation. Springer-Verlag, Berlin Heidelberg, pp 55–80
123
