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Groundwater at many military factory, munition storage and maneuver sites is contaminated by explosives chemicals that were released into the subsurface. The 2,4,6-trinitrotoluene (TNT) is among the most common explosive pollutants. In this study, two TNT-degrading strains, isolated from TNT-contaminated soils and wastewater sludge, were applied for TNT biodegradation. Based on the 16S rDNA sequence analyses, these two bacterial strains were identified as Achromobacter sp. and Klebsiella sp. via biochemical and DNA analyses. Microcosm study was conducted to evaluate the feasibility and efficiency of TNT biodegradation under aerobic conditions. Results indicate that TNT degradation by-products were detected in microcosms (inoculated with Achromobacter sp. and Klebsiella sp.) with cane molasses addition. Klebsiella sp. and Achromobacter sp. used TNT as the nitrogen source and caused completely removal of TNT. Two possible TNT biodegradation routes could be derived: (1) part of the TNT was transformed to nitrotoluene then transformed to nitrobenzene followed by the nitro substitute process, and trinitrobenzene, dinitrobenzene, and nitrobenzene were detected; and (2) TNT was transformed via the nitro substitute mechanism, and dinitrotoluene followed by nitrotoluene isomers were detected. The initial TNT degradation involved the reduction or removal of the nitro substitute to an amino derivative or free nitrite. Results show that the second route was the dominant TNT biodegradation pathway. The produced by-products were also degraded without significant accumulation during the degradation process. These findings would be helpful in designing a practical system inoculated with isolated TNT degradation strains for the treatment of TNT-contained groundwater.

In subsurface environment, small-scale heterogeneities usually cause the reduction of the applicability of in situ remedial techniques. Biogeochemical heterogeneities and preferential groundwater flow paths create complex hydrogeologic conditions at most contaminated sites. A thorough understanding of the resulting three-dimensional distribution of contaminants is a necessity prior to determining a need for remediation. In this study, a gasoline spill site was selected to examine the effects of aquifer heterogeneities and geochemical variations on petroleum hydrocarbon biodegradation via different oxidation-reduction process. At this site, two multilevel sampling wells were installed to delineate the lateral (5 m) and vertical (0.5 m) distribution of contaminant concentrations and different biogeochemical parameters. Two 5-cm (I.D.) continuous soil cores [from 4 to 8 m below land surface (bls)] were collected within the gasoline plume to evaluate the distribution of the microbial population in soils. Results show that high microbial activities were observed in soil samples based on the following evidences: (1) high petroleum hydrocarbon degradation rate, and (2) high microbial biomass. Each soil section was used for chemical extraction, microbial enumeration, and grain size distribution. Results show that the soil sections with more permeable sediment materials corresponded with higher biomass (total anaerobes > 2 x 106cells/g) and significant contaminant degradation. However, those sections with less permeable sediments contained lower microbial population. Results indicate that the subsurface microorganisms were distributed unevenly in the aquifer, and some regions were devoid of microorganisms and biodegradation activities. Spatial distribution of microorganisms, soil materials, and biogeochemical characteristics in the subsurface soils control the extent and kinetics of contaminant biodegradation. Thus, using blended aquifer materials for measurement of in situ biodegradation rates may not achieve representative results.