Jun 23, 1995 - a laboratory scale as a bioremediation technology for degradation or immobilization of 2,4,6-trinitrotoluene. (TNT) in contaminated soils.
Appl Microbiol Biotechnol (1996) 44:795-800
J. B r e i t u n g • D . B r u n s - N a g e l D . G e m s a • E. y o n L i i w
© Springer-Verlag 1996
• K. S t e i n b a c h • L. K a m i n s k i
Bioremediation of 2,4,6-trinitrotoluene-contaminatedsoils by two different aerated compost systems
Received: 23 June 1995/Accepted: 3 July 1995
Two composting systems were compared on a laboratory scale as a bioremediation technology for degradation or immobilization of 2,4,6-trinitrotoluene (TNT) in contaminated soils. The first compost was aerated from the beginning whereas the second compost was only aerated after an anaerobic prephase of 65 days. In the first compost system the TNT concentration declined rapidly by 92% but, at the end, TNT could be partially recovered. During the anaerobic prephase of the second compost system, TNT was almost completely converted to aminodinitrotoluenes, which during the subsequent aeration almost entirely disappeared. In addition, the second compost generated less toxic material than the first one as confirmed by inhibition of bioluminescence of Vibrio fischeri. These data show that microbiological TNT-degradation systems can be successfully designed which are prerequisite for an efficient bioremediation of contaminated soils.
Abstract
Introduction 2,4,6-trinitrotoluene (TNT) is an explosive used in conventional weaponry. Unfortunately previous manufacture and handling has resulted in a heavy soil and sediment contamination at munition plants. Since TNT is a highly toxic and mutagenic compound, soil decontamination is strongly indicated. For decontamination,
J. Breitung ( ~ ) • D. Bruns-Nagel. L. Kaminski D. Gemsa E. von L6w Bereich Umwelthygiene, Institut ftir Immunologic, Medizinisches Zentrum fiir Hygiene, Pilgrimstein 2, D-35037 Marburg, Germany. Fax: 06421 282309 K. Steinbach Department of Organic Chemistry, Philipps University, D-35037 Marburg, Germany
composting appears to provide a cautious and ecologically more adequate method than incineration. The composting procedure was preferred by several investigators as an efficient method to detoxify TNTcontaminated soils (Osmon and Andrews 1978; Kaplan and Kaplan 1982; Isbister et al. 1984; Williams et al. 1992). In the composting studies of Isbister et al. (1984), the TINT concentration in composts was found to decline rapidly by 85% within 6 weeks, and only insignificant quantities of monoaminodinitrotoluenes were detected. By the use of ring-labelled [14C] TNT it was shown that n o 1¢CO2 was generated, indicating that no ring cleavage occurred. Most of the radioactivity was bound to non-extractable "compost solids" and increased with compost age and stability. Similarly, Osmon and Andrews (1978) and Williams et al. (1992) found a rapid decrease of the TNT concentration during composting. Only in the study by Williams et al. (1992) were minor amounts of mono- and diaminonitrotoluenes detected. Kaplan and Kaplan (1982) detected 22% of the radioactivity mainly in the insoluble humin matrix and in humic and fulvic acid after 92 days of composting. In all previous studies, no effort was made to examine a possible resolubilization of the contaminants from the humin matrix under more drastic conditions. Such an approach could indicate that nitrotoluenes are not that tightly bound to the humin matrix and thus an ecotoxicological risk may still persist. Along the same lines, no data on contaminant concentrations in leachates of composts have as yet been provided. Furthermore, in most previous studies the indispensable biotoxicological tests were not performed to ascertain that a definite detoxification process had taken place during composting. The present study provides evidence that the customary compost systems may not be without ecotoxicological risks. We will present an approach for a more secure compost procedure for detoxifying TNT-containing soils.
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Materials and methods Preparation of composts Composts used in both experiments were compose d of 3.5 kg highly contaminated soil (up to 20g TNT/kg dry weight) and of 3kg chopped sugar-beet (approx. 2 cm long and 0.5 cm diameter). The soil was obtained from the former ammunition plant Werk Tanne in the area of Clausthal-Zellerfeld in Lower Saxony, Germany. The soil was sieved through a wire basket with 8-mm meshes and was thoroughly mixed with chopped sugar-beet. The compost mixture was placed in a Dewar vessel (101). At the bottom of the vessel a perforated polyethylene tube (1 cm diameter) was installed for aeration of the compost pile via a membrane blower (approx. 0.3m 3air/h). Another polyethylene tube (0.5cm diameter) was placed at the bottom to withdraw the leachates. Both tubes were covered with glass wool to prevent obstruction by soil particles. Two differently treated compost systems were employed. Compost 1 was aerated throughout the whole experiment. Compost 2 was flooded with 31 tap-water at the beginning of the procedure for 65 days (this stagnant water is designated as compost water) and then, after the compost water had been withdrawn, aerated for another 97 days. The resulting compost water from the anaerobic prephase of compost 2 was collected and further treated in a 10-1 biofermenter (Janke & Kunkel, Staufen, Germany) up to day 162. The compost water was aerated (0.5 m3/h) and stirred continuously (150 rpm) at 25°C in the biofermenter. The overall compost moisture was maintained at approximately 60% during the aerobic phase of both composts.
Preparation of extracts from compost and liquids For analyses of nitrotoluene content, compost material and liquids were extracted as described below. At the times indicated, five parallel samples of compost material (each 5 g wet weight) were extracted with 15 ml methanol for 15 min via continuous ultrasonification (Bandelin Sonorex Super, Berlin, Germany) at room temperature in 50-ml centrifuge tubes. The methanolic extract was centrifuged at 5000 rpm for i0 min to remove soil particles. For gas chromatography/electron capture detector (GC/ ECD) analyses, the extracts were diluted in toluene. In addition to this standard method of extraction, we used a more drastic method to determine the resolubility of nitrotoluenes from the humin matrix. The material was pretreated with 2ml 8 M HC1 for 24h at 25°C and then 13 ml methanol was added. The following steps comprised the standard extraction. Aliquots of 1 ml compost leachates, generated during the aerobic phase of both composts, and of the compost water from the anaerobic prephase of compost 2 were extracted with 1 ml toluene by 5 rain horizontal shaking at 150 rpm. Thereafter, the toluene supernatant was analysed by GC/ECD.
Merck/Hitachi L-5000 LC controller, Hitachi, Tokyo, Japan) on a Nucleosil 120-5 C18 column (length 250mm; diameter 3mm) at 25°C (column thermostat: Gynkotek, Munich, Germany). The initial solvent composition was 70% water 30% methanol, which was held for 10rain. Then a linear gradient to 50% water and 50% methanol was run over 5min. This solvent ratio was held for 20rain. After that a linear gradient up to 100% methanol was run over 35 min. Finally, the column was equilibrated for 20 min with the initial solvent composition of 70% water and 30% methanol. The flow rate was set to 0.4ml/min. Aliquots of 201al were injected by an automatic sampler (Gyna 50; Gynkotek, Munich, Germany). Detection was performed with a diodearray detector UVD 340; Gynkotek, Munich, Germany). The detector was set at 230nm to detect the nitroaromatic compounds. Peaks were scanned from 200 nm to 400 nm for compound identification. HPLC analyses were mainly performed to determine the diamino and azoxy compounds in the compostextracts since they were not readily detectable by GC/ECD.
Determination of physical parameters Compost temperature was recorded at intervals of several days via a standard electronic thermometer. For determination of compost dry weight, 10 g material was dried at 105°C for 24 h. pH and redox in the liquid phase were monitored via an ion activity meter (Philips PW 9413, Einhoven, The Netherlands) and a Schott CG 840 pH meter connected to a Pt 42 A redox electrode (Schott, Mainz, Germany).
Bioluminescence toxicity assay Ecotoxicological monitoring was conducted by measuring the inhibition of the bioluminescence by water extracts of the composts using the marine luminescent bacterium Vibrio fischeri NRRL-B11177 as test organism. The water extracts of the composts were prepared using the protocol of the German standard methods for the examination of water, waste water and sludge (Beuth 1991). The inhibition of bacterial bioluminescence was monitored by a temperature-controlled photomultiplier at 15°C (LUMIStox; Dr. Lange, Berlin, Germany). The GL2o values were used to determine the acute toxicity in leachates, compost water and water extracts of compost material. The GL2o value is defined as the dilution step of a sample with which the light emission of the bacteria is inhibited less than 20% in the tests (LUMIStox manual; Dr. Lange, Berlin, Germany).
Results Chemical analyses of compost and liquid extracts Compost and leachates extracts were analyzed for nitrotoluenes by GC/ECD (Hewlett Packard 5890 series II plus, Amsterdam, Netherlands). The injection volume was 1 btl. The temperature of the injector and detector was 250°C. A 30-m-Iong DB 5 column (0.25 ~tm film) from J&W Scientific (Folsom, Calif., USA) was used and heated isotherm to 230°C. Samples were also analysed by high-performance liquid chromatography (HPLC) (Merck/Hitachi 655A-11 liquid chromatograph;
Two different composts containing highly cont a m i n a t e d soil w e r e c o m p a r e d t o e x a m i n e d e g r a d a t i o n o r f i x a t i o n o f T N T a n d its m e t a b o l i t e s t o t h e h u m i n matrix. The chief difference between the two composts w a s t h a t o n e ( d e s i g n a t e d c o m p o s t 1) w a s c o n t i n u o u s l y a e r a t e d f o r 28 d a y s w h e r e a s t h e o t h e r c o m p o s t (design a t e d c o m p o s t 2) w a s first f l o o d e d w i t h t a p - w a t e r f o r 65 d a y s a n d , a f t e r t h e w a t e r h a d b e e n d r a i n e d off, a e r a t e d f o r a n o t h e r 97 d a y s .
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Fig. 4 Time course of nitrotoluene concentration in the liquid phase of compost 2. Until the 65th day, compost 2 was flooded with water. Thereafter, the compost water was drained off and separately treated in a biofermenter. At the times indicated, 1-ml aliquots were withdrawn, extracted with 1-ml toluene and nitrotoluenes were subsequently analysed by GC/ECD
Fig. 5 Kinetics of the biotoxicity decrease in the liquid phase of compost 2. GL2o values were determined by the bioluminescence test, initially in the water of compost 2 and, after 65 days, in the separated and biofermenter-treated compost water. GL2o is the dilution step at which the light emission of V.fischeri is inhibited less than 2O%
compost 2 was further monitored up to day 134. Figure 5 shows that the compost water lost most of its toxicity when incubated in the biofermenter. The decrease in toxicity closely parallelled the decrease of nitroaromatic compounds as shown in Fig. 4.
model humic substances may be characterized as a complex containing an inner core structure and outer edges, which are connected to non-humic organic compounds. In this complex, ample space is left to accommodate and integrate small molecules such as TNT. Therefore, it seems likely that a drastic treatment with acid could liberate 25% of the non-covalently bound and unmetabolized TNT. At the present stage of research, the fate of the remaining 75% of the TNT is unknown. An exact investigation would require the use of ring-labelled [ 1 4 C H ] TNT, an experimental approach that is planned for the near future. Until now, no firm bindings of aromatic nitro compounds to humic substances have been reported. Hence, it can be concluded that TNT has first to be converted into its amino derivatives for a covalent integration into the humus matrix. Theoretically this notion is not surprising since the binding of an amino group to humin matrices is more feasible than that of a nitro group (Hsu and Bartha 1974; Helling and Krivonak 1978; Bollag and Myers 1992). In line with this, Parris (1980) reported a covalent binding of aromatic amines to humates via reactions with carbonyls and quinones. In general, the humic and fulvic acids of humus may be regarded as a heterocondensate with molar masses of approximately 20 000-50 000 g/mol, 35%-92% being compounds such as quinones, phenols, aromatic acids, N-heterocycles,
Discussion
In the present study, two different composting procedures were chosen to study immobilization of TNT or its metabolites since, as shown previously, mineralization of TNT in liquid cultures by naturally occurring microorganisms has been shown to be incomplete (Fernando et al. 1990; Boopathy et al. 1993, 1994; Preuss et al. 1993; Michels and Gottschalk 1994; Vorbeck et al. 1994). In the aerated compost 1, TNT declined rapidly by 92%, but approximately 25% of its original concentration could be recovered by an acidic treatment. In contrast, the TNT decrease in the anaerobic prephase of compost 2 was slower but was accompanied by the striking appearance of the metabolites 4-ADNT and 2ADNT, which only declined in the subsequent aerobic phase. The reason why TNT disappeared in compost 1 may be explained on the basis of the model of humic substances proposed by Ziechmann (1977). According to the
800 diverse proteins and polysaccharides, which m a y provide an abundance of different structures and reactive groups for chemical interactions with aminonitrotoluenes. The binding itself is thought to be catalysed either by microbial enzymes such as peroxidases (Klibanov et al. 1980) or chemically via metal ions as catalysts (Shindo and H u a n g 1984). O u r results represent a promising starting point for future improvements, which have to focus on three main aspects. First, there must be an optimization of the anaerobic prephase in compost 2 to shorten the time of T N T reduction to its amino derivatives and, in addition, a faster metabolism of these c o m p o u n d s in the subsequent aerobic phase. Second, the toxicity experiments have to be expanded by using a variety of cellular, including h u m a n target systems to analyse the entirely unknown toxicity and mutagenicity of T N T metabolites. Third, the microbial organisms mediating T N T metabolism in the soil have to be m o r e closely identified. O n these premises, the detoxification of T N T and its metabolites by compost-based bioremediation m a y represent a convenient and manageable procedure to remove poisonous matter from contaminated soils. Acknowledgements This work was supported by the Bundesministerium ffir Forschung und Technik (BMFT), by the Umweltministerium Land Niedersachsen and by the Industrieverwaltungsgesellschaft (IVG). Project management was done by Industrieanlagen Betrielosgesellschaft (IABG).
References Beuth Verlag (1991) Normenausschug Wasserwesen (NAW) im DIN Deutsches Institut f/St Normung e.V. Bollag J-M, Myers C (1992) Detoxification of aquatic and terrestical sites through binding of pollutants to humic substances. Sci Totai Environ 117/118:357-366 Boopathy R, Kulpa CF, Wilson M (1993) Metabolism of 2,4,6trinitrotoluene (TNT) by Desulfovibrio sp. (B strain). Appl Microbiol Biotechnol 39:271~275 Boopathy R, Manning J, Montemagno C, Kulpa C (1994) Metabolism of 2,4,6-trinitrotoluene by a Pseudomonas consortium under aerobic conditions. Curr Microbiol 28:131 137
Breitung J, Bruns-Nagel D, L6w Ev, Steinbach K, Kaminski L, Haas R, Gemsa D (1995) Mikrobielle Sanierung von 2,4,6-Trinitrotoluol (TNT) kontaminierten B6den. UWSF-Z. Umweltchem. ()kotox. 7:i95-200 Fernando T, Bumpus JA, Aust SD (1990) Biodegradation of TNT (2,4,6-trinitrotoluene) by Phanarochaete chrysosporium. Appl Environ Micorbiol 56:1666 1671 Helling CS, Krivonak AE (1978) Physiochemical characteristics of bound dinitroaniline herbicides in soil. J Agric Food Chem 26:1156 1163 Hsu TS, Bartha R (1974) Biodegradation of chloroaniline humus complexes in soil and in culture solution. Soil Sci 118:213-219 Isbister JD, Anspach GL, Kitchens JF, Doyle RC (1984) Composting for decontamination of soils containing explosives. Microbiologica 7:47-73 Kaplan DL, Kaplan AM (1982) Thermophilic biotransformations of 2,4,6-trinitrotoluene under simulated composting conditions. Appl Environ Microbiol 44:757-760 Klibanov AM, Alberti BN, Morris J, Felshin LM (1980) Enzymatic removal of toxic phenols and anilins from waste waters. J Appl Biochem 2:414-421 Michels J, Gottschalk G (1994) Inhibition of the lignin peroxidase of Phanarochaete chrysosporium by hydroxylamino-dinitrotoluene, an early intermediate in the degradation of 2,4,6-trinitrotoluene. Appi Environ Microbiol 60:187 194 Osmon JL, Andrews CC (1978) The biodegradation of TNT in enhanced soil and compost systems. (U.S. Army Armament Research and Development Command, ARLCD-TR-77032, Dover, N.J. Technical Information Service Publication no. ADE 400073) National Technical Information Service~ Springfield. Va Parris GE (1980) Covalent binding of aromatic amines to humates. 1. Reactions with carbonyls and quinons. Environ Sci Technol 14:1099 1106 Preuss A, Fimpel J, Diekert G (1993) Anaerobic transformation of 2,4,6-trinitrotoluene (TNT). Arch Microbiol 159:345-353 Shindo H, Huang PM (1984) Catalytic effectsof manganese (IV),iron (III), aluminium and silicon oxide on the formation of phenoiic polymers. Soil Sci Soc Am J 48:92~934 Vo}beck C, Lenke H, Fischer P, Knackmus H-J (1994) Identification of a Hydride-Meisenheimer complex as a metabolite of 2,4,6trinitrotoluene by a Mycobacterium strain. J Bacteriol 176:932-934 Williams RT, Ziegenfuss PS, Sisk WE (1992) Composting of explosives and propellant contaminated soils under thermophilic and mesphilic conditions, J Ind Microbiol 9:137-144 Ziechmann W (1977) Zwischenmolekulare Krfifte und die Struktur von Huminstoffen. Z Pflanzenern~ihr Bodenkd 140:151-157
