Spectroscopic studies using absorption spectroscopy (AS), photofragmentation spectroscopy (PF-LIF), and cavity ringdown spectroscopy (CRDS) were preformed on 2,4,6-Trinitrotoluene. The NO detection of the energetic material (EM) was done using combinations of the previous procedures. Calculations for the absorption coefficient and cross sections were obtained. The procedure of photofragmentation required heating of the sample to generate an absorption curve and a cross section. Absorption spectroscopy, which covered a range of 195-300 nm, also corresponded with the use of heating the sample to obtain the two values. Cavity ringdown spectroscopy investigations were done on the sample at room temperature. A higher accuracy for the level of detection was obtained using a combination of photofragmentation at various wavelengths and cavity ringdown spectroscopies.

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Microbial mats are natural heterotrophic and autotrophic communities dominated by cyanobacteria (blue-green algae). These constructed mats are durable, tolerant to a variety of toxins and resilient under changing environmental conditions. This research demonstrates that microbial mats provide an effective remediation treatment for 2,4,6 Trinitrotoluene (TNT) in water and soil. It showed that TNT is removed to undetectable limits after 5 days of treatment under any of the following conditions: light/dark; total light; total dark. This work also shows that in the presence of an inorganic material (lead), mats were able to remove both contaminants efficiently, thus making the microbial mat a good choice for mixed waste remediation. Kinetic studies performed during the first five hours of microbial treatment showed a pseudo first order reaction indicating that TNT removal is initially proportional to the concentration of TNT. The major metabolites detected after 24 hour of treatment were 4-amino-2,6- dinitrotoluene, 2-amino-4,6-dinitrotoluene, and 2,4-diamine-6-nitrotoluene. These metabolites have a toxicity level similar to TNT. However, mat extracts and growth medium concentrates taken after 24 hours treatment of TNT showed little or no toxicity. The lack of toxicity demonstrated by treated mat extracts and media concentrates suggest that these metabolites are not the final metabolic products. The chemical nature of these metabolites suggests that the chemical mechanism of biotransformation involves reduction of the nitro groups at the ortho and para position of the TNT structure. Results obtained from light and dark experiment suggest that photooxidation or photodegradation is not an important mechanism for degradation of TNT by mats. Results show that live mats likely degrade TNT via a biotransformation process. In comparison, heat killed mats show a much slower removal of TNT than live mats. TNT was the only species found in the water column and extracts of heat killed mats, which indicates that TNT is removed by a passive absorption process, but no evidence of biodegradation was observed.

2,4,6-trinitrotoluene (TNT) is a toxic and recalcitrant pollutant contaminating soils and groundwater. Therefore, characterization of microbial populations of TNT-contaminated soils and isolation of bacteria degrading this pollutant are of primordial importance. Comparison of hybridizations of 16S rRNA derived from uncontaminated and TNT-contaminated soil samples required the development of a functional ANOVA model. Specifically, a statistical tool was necessary to compare dissociation curves obtained from thermal dissociation analysis of RNA hybridizations to DNA microarrays, and to determine if the dissociation curves significantly differed. To test and validate the model, we used dissociation curves from in vitro transcribed 16S rRNA amplified from two environmental samples hybridized to a phylogenetic microarray. Detection and rejection of outlier curves was important for appropriate discrimination between curves. The identification of significantly different curves was more efficient with the model than approaches relying on measurements at a single temperature. This functional ANOVA analysis was used to improve discrimination between hybridizations of two soil microbial communities. Following hybridization of in vitro transcribed 16S rRNA derived from an uncontaminated and a TNT-contaminated soil sample to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and mismatch (MM) variants, thermal dissociation was used to analyze the nucleic acid bound to each PM-MM probe set. Functional ANOVA of the dissociation curves generally discriminated PM-MM probe sets when values of Td (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greater than Td. Comparison of signal intensities measured prior to dissociation analysis from hybridizations of the two soil samples revealed significant differences in domain-, group-, and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridizations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary. These differences in hybridizations between the two soil samples were likely the result from the presence of TNT. The effect of TNT on soil microbial communities was further investigated with additional uncontaminated and TNT-contaminated soil samples using 16S rRNA PCR-DGGE and cultivation-dependent techniques. In all contaminated soil samples, the amount of DNA extracted was lower than in the uncontaminated ones. Analysis of bacterial diversity by DGGE showed a predominance of Pseudomonadaceae and Xanthomonadaceae in the TNT-contaminated soil samples compared to the uncontaminated ones. Caulobacteraceae were also present in several contaminated soil samples. The culturable microflora of these soils was studied by plate counts on agar supplemented with dilute nutrient broth. The number of CFUs was lower in a TNT-contaminated soil inoculum than in an uncontaminated one. In the former, most of the CFUs belonged to Pseudomonadaceae, and to a lesser extent, to Caulobacteraceae. In addition to the above contaminated soil samples, a pristine soil was artificially contaminated with different concentrations of TNT and incubated for 4 months. The amount of DNA extracted decreased in the highly contaminated soil samples (1.4 and 28.5 g TNT/kg soil). After 7 days of incubation of these soil samples, there was a clear shift of their original flora to a population dominated by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae and Caulobacteraceae. When the TNT concentration was lower (140 mg TNT/kg soil), a moderate shift in the bacterial population was observed. These results indicate that TNT affects soil bacterial diversity and richness by selecting for a narrow range of bacterial species that belong mostly to Pseudomonadaceae and Xanthomonadaceae. TNT-contaminated soil samples probably contained TNT-degrading bacteria. In order to isolate bacteria that can denitrate TNT, enrichment cultures were carried out with TNT as sole nitrogen source and in the absence of oxygen. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum, in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. Concomitantly, TNT-reduced compounds were detected as well as unidentified polar metabolites. The concentration of nitrite released from TNT was proportional to the concentration of ferrihydrite in the medium. The release of nitrite was lower when the concentration of initially spiked TNT was reduced by one order of magnitude. Under these conditions, the concentration of nitrite peaked and then its concentration slowly decreased and production of ferrous ions was detected. A decrease of nitrite concentration and production of ferrous ion were observed when TNT was omitted and nitrite and ferrihydrite were provided. These results suggest that nitrite-reducing conditions were initially achieved, followed by iron-reducing conditions. When grown aerobically on a chemically defined medium, P. aeruginosa strain ESA-5 produced a greenish extracellular compound. This product was identified as phenazine-1-carboxylic acid (PCA). When purified PCA was incubated with TNT in the presence of NADH, nitrite was released. The concentration of nitrite released was dependent on the concentration of NADH and PCA. Denitration also occurred with two TNT-related molecules, 2,4,6-trinitrobenzaldehyde and 2,4,6-trinitrobenzyl alcohol. The release of nitrite was coupled with the formation of two polar metabolites and mass spectrometry analyses indicated that each of these compounds had lost two nitro groups from the trinitroaromatic parent molecule. The results obtained with the PCA mediated denitration of TNT in the presence of inhibitors of oxygen reactive species suggested the involvement of superoxide (O2.-). When exogenous PCA was added to a P. aeruginosa ESA-5 liquid culture containing TNT as sole nitrogen source, bacterial growth was significantly enhanced compared to cultures containing TNT without PCA.

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Contamination of soils and groundwater with nitroaromatic compounds such as 2,4,6-trinitrotoluene (TNT) and dinitrotoluenes (DNTs) has drawn considerable attention due to widely distributed contamination sites and substantial efforts for cleanup. Two isomers of DNT, specifically 2,6-dinitrotoluene (2,6-DNT) and 2,4-dinitrotoluene (2,4-DNT), occur as soil and groundwater contaminants at former TNT production sites. The discovery of bacteria that use DNT isomers as electron donors has encouraged bioremediation at contaminated sites. Current work is extending the existing engineered bioremediation to naturally occurring in situ biodegradation and focuses on the application of natural attenuation (NA) as a remediation strategy for residual DNT at contaminated sites. More specifically this research evaluated factors influencing in situ bioremediation of DNTs and TNT in surface soils, vadose zones, and saturated medium. Applications involving surface soils and vadose zones investigated the potential of water infiltration to promote in situ bioremediation. Studies in saturated media were more applicable to NA. Factors that were also considered in studies conduced included: 1) the presence and distribution of degrading microbes in field soils (Barksdale, WI); 2) the dissolution and bioavailability of contaminants in historically contaminated soils; and 3) the effect of mixtures of contaminants (i.e., DNTs and TNT) on biodegradation processes. This research provided information useful for practitioners considering an in situ bioremediation NA as a remedial solution for contaminated sites. Under the condition simulating downflow of surface waters or rainwater, the rapid rate of DNT degradation could be facilitated by the availability of oxygen in the soil gas without concern of toxicity (i.e., nitrite evolution and pH drop) and addition of nutrients. As a result, in situ bioremediation or NA should be strongly considered as a remedial option for Barksdale soils and similar sites where relatively low concentrations of DNT isomers are present as contaminants. At TNT contaminated sites TNT was not mineralized by indigenous microorganisms despite oxidative biotransformation, and mixed culture capable of growth on DNT also could not develop the mineralization of TNT during DNT degradation. This suggests that the mixtures of contamination did not improve the potential for in situ TNT bioremediation.