Bioremediation of 2,4,6-Trinitrotoluene under Field Conditions

In situ bioremediation of the nitroaromatic explosive. 2,4,6-trinitrotoluene (TNT) provides a cost-effective alternative for cleaning up contaminated ...
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Environ. Sci. Technol. 2007, 41, 1378-1383

Bioremediation of 2,4,6-Trinitrotoluene under Field Conditions PIETER VAN DILLEWIJN,† A N T O N I O C A B A L L E R O , ‡ J O S EÄ A . P A Z , † M . M A R G O N Z AÄ L E Z - P EÄ R E Z , † J O S EÄ M . O L I V A , § A N D J U A N L . R A M O S * , † Estacio´n Experimental del Zaidı´n, Consejo Superior de Investigaciones Cientı´ficas, Apdo. Correos 419, E-18008 Granada, Spain, and Unio´n Espan ˜ ola de Explosivos S.A., Avda del Parteno´n 16, 5a planta, Campo de las Naciones E-28042, Madrid, Spain

In situ bioremediation of the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) provides a cost-effective alternative for cleaning up contaminated sites. Here we compare the effectiveness of several bioremediation techniques: natural attenuation, bioaugmentation with TNT-degrading Pseudomonas putida JLR11, phytoremediation with maize (Zea mays L.) and broad beans (Vicia faba L.), and rhizoremediation with maize and broad beans inoculated with P. putida JLR11. Experiments in spiked hydroponic medium demonstrated that inoculation with bacteria did not affect TNT levels. On the other hand, axenic plants were able to remove 32% to 38% of the TNT from the medium. However, when plants were inoculated with bacteria, TNT disappeared to an even greater extent (80% to 88%), a result that advocates a role for P. putida JLR11 in rhizoremediation. In field experiments neither natural attenuation nor bioaugmentation with P. putida JLR11 affected TNT levels to a significant degree. However, the extractable TNT content in rhizosphere soil associated to maize roots decreased by more than 96% in 60 days regardless of inoculation. This indicates that under these field conditions, the effect of phytoremediation by maize overshadowed any effect of rhizoremediation by P. putida JLR11.

Introduction Vast sites worldwide are contaminated with the polynitroaromatic explosive 2,4,6-trinitrotoluene (TNT) because of improper handling, processing, or disposal and military activities. The toxicity and mutagenic effects of this recalcitrant xenobiotic have led environmental agencies to declare the removal of this pollutant a high priority. A number of methods have been devised to remediate TNT polluted soil and water. These usually consist of transporting contaminated material elsewhere for decontamination with various physicochemical or biological techniques (1-3). The search for cost-effective as well as ecologically viable alternatives has led to increased investigation in the use of strategies based on bioremediation, especially those in which the contaminant can be dealt with in situ. * Corresponding author phone: +34 958 181608; fax: +34 958 135740; e-mail: [email protected]. † Estacio ´ n Experimental del Zaidı´n, CSIC. ‡ Present address: Go ¨ teborg University, Cmb Microbiologi Box 462, 405 30 Go¨teborg, Sweden. § Unio ´ n Espan ˜ ola de Explosivos S.A. 1378

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The in situ bioremediation techniques tested thus far include natural attenuation, bioaugmentation, phytoremediation, and combinations such as rhizoremediation. Natural attenuation is a passive process in which the contaminated terrain is left undisturbed. Remediation in this strategy depends on the natural capacity of indigenous plants and microorganisms to eliminate the contaminant from the terrain. Additionally, natural attenuation depends on other processes such as photodegradation (surface soil), slow hydrolysis, reactions with metals, sorption, and migration. Costs related to this strategy are low, as usually only monitoring is required. Bioaugmentation consists of the addition of microbes or microbial products (enzymes) to the contaminated matrix to degrade the contaminant (4, 5). Microbes have an arsenal of nitroreductases (1) which reduce the nitro side groups of TNT to produce reduced transformation products or products with fewer nitro substituents (6). Both reactions are of interest because reduced forms of TNT such as hydroxylamino- or amino-dinitrotoluenes bind irreversibly with humic acids and soil organic and inorganic material, thereby lowering bioavailability (7). An additional advantage of this approach is that aromatic compounds with fewer nitro substituents are more easily degraded (8). Therefore, the addition of bacteria to sites where natural attenuation is not a viable remediation strategy could assist in the removal of TNT from soil and water. However, the major challenge faced by this technology is the persistence and effectiveness of the microorganisms used. For phytoremediation (9), plants are used to eliminate the contaminant. Plants appear to deal with TNT like a “green liver”, whereby the contaminant is detoxified and sequestered within plant tissues rather than mineralized to carbon dioxide and nitrogen (10, 11). Detoxification occurs by transformation of the chemical followed by conjugation to plant metabolites such as glucose and then sequestration of the resulting macromolecules into vacuoles or polymers such as lignin (12, 13). The result is that most TNT and its derivatives accumulate in the roots, with smaller amounts transported to the stem and to the leaves (12, 14). The effectiveness of phytoremediation depends on the bioavailability of TNT and on the capacity of the plant to tolerate and take up the contaminant (10, 11). In rhizoremediation plant roots stimulate rhizosphere microbial communities to degrade the contaminant by aerating the soil and releasing nutrients through root exudates (15). Also, the root network provides niches to protect the bacteria against desiccation and other abiotic and biotic stresses. In addition, rhizospheric microbial populations can help the plant tolerate higher concentrations of a contaminant or stimulate plant growth. Therefore inoculation of plants with efficient TNT-degrading microbes is another technique which might be implemented to increase remediation effectiveness (16). A good candidate for bioaugmentation and rhizoremediation is the Pseudomonas putida strain JLR11 (17). This strain is closely related to P. putida strain KT2440, an excellent root colonizer, and is capable of growing with TNT as the sole nitrogen source to form a panoply of metabolic products (17). Furthermore, this bacterium has been shown to be capable of respiring TNT under anaerobic conditions (18). The fact that it efficiently degrades TNT anaerobically is of interest because the oxygen gradient decreases dramatically in soil within centimeters of the surface. P. putida JLR11 is capable of assimilating nitrogen from TNT via nitrite and ammonium (19), and a number of enzymes which attack 10.1021/es062165z CCC: $37.00

 2007 American Chemical Society Published on Web 01/18/2007

TNT have been identified, such as the nitroreductase PnrA (20). In this study we investigated the effectiveness of several bioremediation strategies in a field site contaminated with TNT. We compared the results obtained with natural attenuation, bioaugmentation, phytoremediation, and rhizoremediation.

Experimental Section Chemicals, Organisms, and Culture Conditions. TNT was obtained from Unio´n Espan ˜ ola de Explosivos (Madrid, Spain) and 4-amino-2,6-dinitrotoluene from AccuStandard (New Haven, Connecticut). P. putida JLR11 was grown routinely at 30 °C in M9 minimal medium supplemented with 0.5% (wt/vol) glucose or 10 mM sodium benzoate as a carbon source (21). Hydroponic Assay. Seeds of maize (Zea mays L.) and broad beans (Vicia faba L.) were surface sterilized and germinated as described previously (22). Tubes were filled with 10 mL of M9 minimal medium lacking NH4Cl and spiked with 300 µM TNT. Four conditions were tested: medium, medium with germinated seedlings (one seedling per tube), medium inoculated with approximately 106 cells of P. putida JLR11 per milliliter, and medium inoculated with the same number of bacterial cells and with seedlings. The tubes were placed in a growth chamber during 1 week under controlled conditions (60% humidity, 14:10 h light:dark photoperiod, 24:18 °C day:night temperature). The precise concentrations of TNT and its transformation products were determined with the methods described below at setup and termination of the experiment. The number of viable bacterial cells was determined when appropriate at the start and finish of the experiment by counting colony forming units (CFU) after serial plating on selective M9 minimal medium with 10 mM benzoate and kanamycin (25 µg/mL) as described previously (22). Microcosm Assays. Sterile maize seedlings were inoculated or not with approximately 106 P. putida JLR11 cells per entire seedling (22). These were then planted in sterilized potting material (Compo Sana Universal) and grown for 10 days. Then the plantlets were transplanted to plant pots containing 600 g of sterilized soil originating from the Estacio´n Experimental del Zaidı´n, Granada, Spain, which had been spiked to approximately 50 mg TNT/kg soil. This loamy silt soil is classified as Humic Haploxerept (23) with the following characteristics: 38% sand, 43% silt, and 19% clay; pH 7.9; organic matter content of 2.1%; and 8% CaCO3 content. Sampling consisted of analyzing rhizosphere soil (soil closely associated with roots) from roots from four selected plants. Concentrations of TNT and its transformation products were determined as described below, and the number of viable bacterial cells was determined by counting CFUs as described above by serially diluting soil slurries onto plates containing selective medium. Field Experiment. The trial was performed inside a plot within the grounds of a demilitarization plant operated by Fabricaciones Extremen ˜as, S.L. (FAEX), a subsidiary company of the Unio´n Espan ˜ ola de Explosivos at El Gordo, Ca´ceres, Spain (Long. 5°20′30′′, Lat. 39°52′02′′). The soil is a loamy sand classified as a Dystric Xerorthents (23) with the following characteristics: 82% sand, 8% clay, and 10% silt; pH 4.7; organic matter content of 0.8%; and 0% CaCO3 content. The plot consisted of a 300 m2 pit that was tilled mechanically to disperse the contaminant as uniformly as possible (Figure 1). To study the effects of natural attenuation or bioaugmentation, 20 cm diameter plant pots were partially buried in the central area of the experimental plot. For natural attenuation, pots were filled with unamended soil from the plot. For bioaugmentation, pots were filled with soil into which a bacterial suspension in 50 mM phosphate buffer

FIGURE 1. Contour map of the TNT concentrations (mg TNT/kg soil) in the experimental plot before planting maize. The map was constructed by applying the Kriging method to bulk soil sample data with Surfer 8.02 software. The axes are given in meters. had been thoroughly mixed to ensure homogeneous distribution of the bacterial cells to, approximately, 106 CFU of P. putida JLR11 per gram of soil. To determine the effect of rhizoremediation or phytoremediation, sterile germinated maize seedlings were inoculated or not with approximately 106 cells of P. putida JLR11 per seedling as above. Seedlings were then planted in sterilized potting material and grown for 1 week. The plantlets were transported to the experimental area and transplanted into the contaminated soil (Figure 1). Plants in the plots were sprinkler irrigated once a week. The plot was equipped against foraging animals and flooding and kept free of weeds. Maize plants were planted at the end of April 2004. Sampling consisted of analyzing bulk soil (soil not associated with roots) taken at a depth of 10 cm, or rhizosphere soil (soil closely associated with roots) from roots from selected plants. Concentrations of TNT and transformation products were determined as described below, and the number of viable bacteria was counted as CFU per gram of soil as described above. Analytical Methods. To determine TNT and transformation product concentrations in bulk or rhizosphere soil we used a modified version of the US-EPA 8330 method. To 2 g of soil we added 10 mL of acetonitrile, and the mixture was shaken vigorously overnight. After the mixture was allowed to settle, 0.5 mL supernatant was collected and mixed with 0.5 mL of a CaCl2 solution (5 g/L). The mixture was centrifuged and the supernatant passed through a GHP Acrodisc 0.45 µm syringe Filter (Pall) before analysis by high performance liquid chromatography (HPLC). Extraction of TNT and its transformation products from plant tissues with acetonitrile was performed according Pavlostathis et al. (24). For HPLC, a Hewlett-Packard model 1050 chromatograph equipped with a diode array detector and a 5 µm C18RP column (Novapak C18, 150 × 3.9 mm, Waters S.A., Barcelona) was used. Samples were run for 5 min in a 60% (vol/vol) acetonitrile:water solution at a flow rate of 0.7 mL/min with the detector set at 230 nm. For quantification, samples were compared with a reference curve made with solutions containing known amounts of pure TNT. Statistical analyses and standard errors (p < 0.05) were calculated using either Excel 2003 or Graph Pad InStat 3 programs with data from at least three samples. For statistical comparisons between samples, Kruskal-Wallis tests (nonparametric ANOVA) were performed and for those between data pairs, one-tailed Mann-Whitney tests. VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. TNT and 4ADNT concentrations and the persistence of P. putida JLR11 in hydroponic assays initially spiked with approximately 0.3 mM TNT. TNT (filled boxes) and 4ADNT (gray boxes) concentrations are compared after 7 days in each treatment: medium only; medium inoculated with P. putida JLR11; medium with a maize seedling; medium with a broad bean seedling; inoculated medium with a maize seedling; and inoculated medium with a broad bean seedling. The number of CFU/mL of P. putida JLR11 (white boxes) is compared between the initial inoculation size and the different inoculated treatments after 7 days. Error bars indicate standard error (p < 0.05, n g 3). Kruskal-Wallis test for each data group is P < 0.0001 (significantly different).

Results and Discussion Bioaugmentation, Phytoremediation, and Rhizoremediation under Laboratory Conditions. To compare the different applications to remediate TNT contamination, laboratory tests were undertaken in hydroponic medium consisting of phosphate buffer and salts present in minimal medium supplemented with 300 µM TNT as the potential sole carbon and nitrogen source. In these experiments natural attenuation was simulated with minimal medium only, bioaugmentation with medium inoculated with 106 CFU/mL P. putida JRL11, phytoremediation with axenic maize or broad bean seedlings in the medium, and rhizoremediation with seedlings inoculated with bacteria. Assays were run in triplicate. Maize and broad bean plants were used for these experiments because of their vigorous growth and resistance to TNT (at least 213 mg TNT/kg soil for maize (25) and at least 500 mg TNT/kg soil for Phaseolus vulgaris (26)). The results (Figure 2) indicate that after 1 week only 3% of the TNT disappeared from the untreated medium. When maize or broad bean seedlings were used under axenic conditions, 32% to 38% of the TNT was removed from the medium, respectively. When only bacteria were added, only 10% of the TNT disappeared from the medium. However, 80% to 88% was removed when maize or broad bean was inoculated with P. putida JLR11. The TNT transformation products detected in the medium were 4-amino-2,6-dinitrotoluene (4ADNT) and only traces of 2-amino-4,6-dinitrotoluene, but not diaminonitrotoluenes nor azoxynitrotoluenes. In reference to the initial concentration of TNT, after 7 days less than 0.5% of the TNT converted into 4ADNT could be detected in the medium inoculated with P. putida JLR11. In medium containing maize or beans, 3% or 2% of the TNT converted to 4ADNT could be detected, respectively. In the case of inoculated maize, 6.5% of the TNT converted into 4ADNT was present in the medium, while in the case of inoculated beans this value was 3%. Therefore, it appears that maize plants biotransform and exude more ADNTs into the medium than beans. When colonized with P. putida JLR11, the amount of ADNTs in the medium increased even more. 1380

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In light of our data for bacterial persistence (Figure 2), it appears that the poor transformation ability of medium inoculated with P. putida JLR11 could be due, for a large part, to the poor survival of the bacteria in this medium (1000-fold decrease). This is not unexpected as TNT is the only carbon and nitrogen source available under these conditions, and P. putida JLR11 cannot use TNT as a carbon source (9). In contrast, Figure 2 also shows a significant population increase (by more than 2 orders of magnitude) in P. putida JLR11 in the presence of the plant, which indicates that the nutrients and carbon provided by plant exudates dramatically enhanced P. putida JLR11 proliferation. The plant on its own eliminated TNT from the medium but the increased disappearance of TNT when bacteria were also present and proliferating suggests that the bacteria were stimulated by the plant to degrade TNT, although we cannot rule out that plant uptake of TNT was stimulated by bacteria. In the literature, stimulation of plants by bacteria has only been reported in relation to plant growth or resistance to pathogens. In comparison to the results with the untreated control that simulated natural attenuation, these findings show that the order of effectiveness in the removal of TNT was rhizoremediation > phytoremediation > bioaugmentation. Moreover, in assays with spiked medium containing only plant exudates, the addition of P. putida JLR11 led to a decrease in the TNT concentration within days (data not shown). These observations suggest a role for P. putida JLR11 in rhizoremediation. Several of the maize plants used in the assays were analyzed for TNT or its transformation products; however, only low amounts could be detected in plants grown in hydroponic assays with medium saturated with TNT crystals (approximately 0.5 mM TNT). Here, 0.0454 mg TNT/g root wet weight and 0.0197 mg 4ADNT/g root wet weight could be detected in the roots of maize plantlets but none in shoots. Diamino derivatives and other transformation products were not detected, and possible conjugates were too polar to be separated and analyzed with the methods used. On the whole, these findings are in agreement with other reports showing

FIGURE 3. Disappearance of TNT in a field assay. TNT concentrations under natural attenuation (open diamond), under bioaugmentation (filled diamond), and in the rhizosphere of uninoculated maize (open triangle) and inoculated maize (filled triangle). Error bars indicate standard error (p < 0.05, n g 3). Kruskal-Wallis tests: natural attenuation, bioaugmentation P g 0.3393 (not significantly different); phytoremediation, rhizoremediation P e 0.0063 (significantly different). One-tailed Mann-Whitney test: no significant differences (P g 0.0571) between inoculated and uninoculated plants or between inoculated or uninoculated pots. Only significant differences seen (P ) 0.0286) between plants and pots at T ) 60 and sometimes at T ) 85 and 120 days. that plants can accumulate TNT and derived products in their roots. Although we did not observe TNT in the shoot under these conditions, it has also been reported that under high concentrations of soil contamination, TNT has been found in the stover and kernels of maize (25), suggesting that the extended exposure to the contaminant also leads to accumulation in these tissues. Bioaugmentation, Phytoremediation, and Rhizoremediation under Field Conditions. On the basis of the above results, the efficiency of the various bioremediation strategies were compared in situ to determine which could be implemented to clean up a contaminated field site. Sampling for TNT content throughout the plot before initiating the experiment showed that the TNT concentration was not homogeneous (Figure 1), with concentrations ranging from 5 to 420 mg TNT/kg soil. These large variations in TNT concentrations are not uncommon in polluted sites, as have been observed by other authors at other locations (3, 27). The strategies used in the experiment consisted of (1) partially buried plant pots filled with displaced soil to simulate natural attenuation; (2) partially buried plant pots filled with displaced soil inoculated with P. putida JLR11 to simulate bioaugmentation; (3) maize plants transplanted into the experimental plot to simulate phytoremediation; and (4) maize plants inoculated with JLR11 and transplanted into the experimental plot to simulate rhizoremediation. Considering the large variations seen between samples, natural attenuation in partially buried pots appeared to cause only a small but not significant (Mann-Whitney test P ) 0.1838 between initial and end TNT concentrations) decrease in TNT concentrations over 120 days (Figure 3). The only transformation product detected in any of the soil samples regardless of treatment was 4ADNT. In the case of natural attenuation, 4ADNT concentrations remained stable at approximately 1.5 mg/kg soil throughout the experiment. Likewise, in bulk soil samples taken outside the planted area, no significant changes were seen in the end concentrations of TNT or its only detectable transformation product, 4ADNT. It has been shown that stimulation of indigenous microbial communities by tilling or grading can enhance TNT transformation (27, 28). However, this did not seem to be the case in the experiments described here. The results obtained with bioaugmentation with P. putida JLR11, regarding TNT elimination and presence of TNT transformation products (Figure 3), were similar to those observed for natural attenuation (Mann-Whitney test P g

0.6857, not significantly different). Although TNT removal was more pronounced, it was not quite significant (between initial and end TNT concentrations: Mann-Whitney test P ) 0.0545). Moreover, the concentration of 4-ADNT also remained stable at approximately 2.8 mg 4ADNT/kg soil throughout the experiment. That the concentration of TNT and its transformation products changed little was probably a result of the limited survival of P. putida JLR11 in bulk soil. Throughout the duration of the field assay the number of viable JLR11 cells per gram of soil decreased by up to 4 orders of magnitude below the initial number (6.3 ( 1.5 × 105 CFU/g soil). The poor persistence of P. putida JLR11 in bulk soil has been observed previously in the field with the P. putida strain KT2440 (29). In that study, the survival of KT2440 decreased within 50 days from 106 to 102 bacteria/g soil. The poor persistence of P. putida strains in bulk soil may be attributed not only to the lack of nutrients but also to the sensitivity of this strain to environmental factors such as desiccation (J. Mun ˜ oz-Rojas, personal communication). In any case, the poor survival of P. putida JLR11 probably explains the similarity of the results observed between natural attenuation and bioaugmentation in the field, because it also underlies the similarities seen when these conditions were simulated under hydroponic conditions. On the other hand, significant decreases in TNT concentrations were observed in the rhizosphere of maize plants, especially after 2 months (Figure 3). After this time, more than 96% of the extractable TNT disappeared regardless of whether plants were inoculated or not with P. putida JLR11. Similarly, the concentration of 4-ADNT in the rhizosphere of either inoculated or uninoculated plants dropped to approximately 0.3 mg ADNT/kg soil after 2 months. Because P. putida JLR11 successfully colonized the rhizosphere of maize and maintained a stable population (oscillating between 103 to 105 CFU/g soil) throughout the experiment, this effect on TNT concentrations cannot be attributed to poor P. putida JLR11 persistence. These findings suggest that in this experiment the effect of phytoremediation by maize overshadowed any effect the bacteria might have on TNT removal. Other indigenous bacteria with the capacity to degrade TNT and colonize the rhizosphere of maize could be degrading TNT in the rhizosphere of uninoculated maize more efficiently than P. putida JLR11. In fact, bacteria with TNT degrading activity were isolated from the same plot (Agathos et al., personal communication), and one species belonging to the genus Pseudomonas was found (Schink et al., personal communication). This is not surprising because Pseudomonas species are found worldwide in soil and water environments (30). Neither plant material or unextractable TNT or its transformation products bound to the soil matrix were analyzed as the TNT used was not radioactively labeled. Therefore, mass-balance studies were not undertaken, and it is unknown how much TNT was sequestered in these tissues or soil fractions. Most likely, the majority of the TNT was sequestered within plant tissues as deduced from the hydroponic assays and the fact that the soil of the experimental plot has very low levels of clay and organic material to which TNT and its transformation products can covalently bind (31). The possibility that some or all of the unaccountable TNT was lost due to the effect of irrigation or dilution of TNT down the soil column cannot be ruled out. However, we observed that TNT levels in bulk soil samples taken outside the planted area but within the contaminated plot and which had received the same irrigation treatment remained unchanged. Therefore, irrigation probably had little effect on general TNT concentrations. To ascertain whether other plants exert the same effect as maize, a second field experiment was performed with VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. TNT concentrations in the rhizosphere of maize (closed circles) or maize inoculated with P. putida JLR11 (open boxes) under microcosm conditions. Error bars indicate standard error (p < 0.05, n ) 4). Kruskal-Wallis test: P g 0.0067 (significantly different). One-tailed Mann-Whitney test shows P > 0.15 (not significantly different) between all data pairs at each time point. broad beans. Once more, TNT concentrations decreased rapidly in the rhizosphere of inoculated and uninoculated plants, although TNT removal was not as efficient as in the previous field trial performed with maize (86% for inoculated plants and 62% for uninoculated plants). Again, no significant differences were seen due to inoculation with P. putida JLR11 (P > 0.05 in one-tailed Mann-Whitney tests between data at each time point); however, the survival of the bacteria was poor in the rhizosphere (102 CFU/g rhizosphere soil at end of experiment). Both the lower efficiency of TNT removal and the poor persistence of P. putida JLR11 could have been due to low seasonal temperatures, as the experiment was performed late in the year (autumn 2004). To confirm the similarities observed between inoculated and uninoculated maize in the field trial, microcosm studies were undertaken using a different soil type and sterilization of the soil before spiking with TNT. By using sterilized soil, the effect of TNT degrading indigenous bacteria is eliminated. Furthermore, by using pots, the effect of irrigation can be negated. Moreover, a different soil was used with double the clay and organic material content than the soil of the experimental plot in the field trial. Not surprisingly, the survival of P. putida JLR11 in the rhizosphere remained high (above 105 CFU/g soil) throughout the assay. Although it appears that the TNT concentration in the rhizosphere of plants inoculated with P. putida JLR11 did not decrease as fast as in that of uninoculated plants (Figure 4), no significant differences could be seen between data sets (Mann-Whitney one-tailed pair tests P g 0.1714). In the microcosm experiment the decrease in TNT concentrations was more rapid than that in the field. This and the fact that the quantities of the only detectable TNT transformation product, 4ADNT, remained stable below 0.7 mg 4ADNT/kg soil throughout the experiment could possibly be due to greater interactions between TNT and its transformation products with the increased amount of clay and organic content in the sterilized soil. Nevertheless, both field and microcosm results indicate that phytoremediation by maize had a greater effect on TNT concentrations in soil than rhizoremediation by P. putida JLR11. This is contrary to that expected from hydroponic assays and from a previous report for a bacteria-plant combination (16) in soil contaminated with at least 17 g TNT/ kg soil. In either case, the bioavailability of TNT should be higher than in the soil experiments described here. This suggests that rhizoremediation with P. putida JLR11 might have had more effect if the soil used would have contained higher concentrations of TNT. Nevertheless, the results indicate that phytoremediation is the most effective strategy in the experimental design we used to test TNT treatment in soil. Although care must be taken when extrapolating these results to other field locations 1382

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due to differences in soil characteristics everywhere, here both the field and microcosm experiments in which different soils were used under different treaments gave similar conclusions. Both maize and broad bean plants grow vigorously and have dense root systems, moreover these crop species are suitable for temperate and tropical regions where intensive cultivation is possible. TNT and its transformation products can concentrate in roots, and when soil concentrations of the contaminant are high this toxic compound can be found in the stover and kernels of maize (25) and in the stems and seed pods of “bush” beans (12, 32). As a result, the crop is not suitable for consumption and whole plants need to be removed. Therefore, controlled elimination of plants is also necessary in this type of remediation strategy. As far as we know this is the first report in which current techniques used for in situ remediation are compared under field conditions. The results indicate that phytoremediation with maize or bean give the best results to eliminate TNT in soil even without inoculation with P. putida JLR11. Although highly contaminated soil was not tested, these crops are potentially useful alternatives to remediation surface soils contaminated with medium levels (i.e., hundreds of mg/kg soil) of TNT.

Acknowledgments This study was supported by a grant from the European Commission (MADOX QLRT-2001-00345). We acknowledge the valuable technical assistance of Germa´n Fernande´z and employees at the FAEX plant, El Gordo, Ca´ceres. Also we thank K. Shashok for improving the use of English in the manuscript and AÄ ngel Iriarte for soil characterization.

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Received for review September 11, 2006. Revised manuscript received December 5, 2006. Accepted December 12, 2006. ES062165Z

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