John F. Manning

Metabolism of explosive compounds by sulfate-reducing bacteria.

Abstract. The metabolism of various explosive compounds—1,3,5-trinitrobenzene (TNB), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX)—by a sulfate-reducing bacterial consortium, Desulfovibrio spp., was studied. The results indicated that the Desulfovibrio spp. used all of the explosive compounds studied as their sole source of nitrogen for growth. The concentrations of TNB, RDX, and HMX in the culture media dropped to below the detection limit (<0.5 ppm) within 18 days of incubation. We also observed the production of ammonia from the nitro groups of the explosive compounds in the culture media. This ammonia served as a nitrogen source for the bacterial growth, and the concentration of ammonia later dropped to <0.5 mg/L. The sulfate-reducing bacteria may be useful in the anaerobic treatment of explosives-contaminated soil.

Metabolism of 2,4,6-trinitrotoluene by aPseudomonas consortium under aerobic conditions

An aerobic bacterial consortium was shown to degrade 2,4,6-trinitrotoluene (TNT). At an initial concentration of 100 ppm, 100% of the TNT was transformed to intermediates in 108 h. Radiolabeling studies indicated that 8% of [14C]TNT was used as biomass and 3.1% of [14C]TNT was mineralized. The first intermediates observed were 4-amino-2,6-dinitrotoluene and its isomer 2-amino-4,6-dinitrotoluene. Prolonged incubation revealed signs of ring cleavage. Succinate or another substrate—e.g., malic acid, acetate, citrate, molasses, sucrose, or glucose—must be added to the culture medium for the degradation of TNT. The bacterial consortium was composed of variousPseudomonas spp. The results suggest that the degradation of TNT is accomplished by co-metabolism and that succinate serves as the carbon and energy source for the growth of the consortium. The results also suggest that this soil bacterial consortium may be useful for the decontamination of environmental sites contaminated with TNT.

Aerobic gram-positive and gram-negative bacteria exhibit differential sensitivity to and transformation of 2,4,6-trinitrotoluene (TNT)

Abstract. A systematic evaluation of the ability of different bacterial genera to transform 2,4,6-trinitrotoluene (TNT), and grow in its presence, was conducted. Aerobic Gram-negative organisms degraded TNT and evidenced net consumption of reduced metabolites when cultured in molasses medium. Some Gram-negative isolates transformed all the initial TNT to undetectable metabolites, with no adsorption of TNT or metabolites to cells. Growth and TNT transformation capacity of Gram-positive bacteria both exhibited 50% reductions in the presence of approximately 10 μg TNT ml−1. Most non-sporeforming Gram-positive organisms incubated in molasses media amended with 80 μg TNT ml−1 became unculturable, whereas all strains tested remained culturable when incubated in mineral media amended with 98 μg TNT ml−1, indicating that TNT sensitivity is linked to metabolic activity. These results indicate that the microbial ecology of soil may be severely impacted by TNT contamination.

Biological transformation of 2,4,6-trinitrotoluene (TNT) by soil bacteria isolated from TNT-contaminated soil

Abstract Four Pseudomonas spp. were isolated from a soil consortium enriched from soil contaminated with 2,4,6-trinitrotoluene (TNT). All four species extensively transformed TNT. The rate of transformation varied among species. In isolate 4, 100% of TNT (100 ppm) was transformed in 4 days. The TNT transformation was achieved by the four isolates through a co-metabolic process with a succinate co-substrate. The four isolates produced NO 2 − from TNT. The maximum NO 2 − production, observed for isolate 1, was equal to 30% of the NO 2 − available from the nitro groups of TNT. For other isolates the NO 2 − production varied from 10 to 16%. The radiolabeling studies showed signs ring cleavage. Isolate 3 used 13% of 14 C-TNT to make cellular material, and isolate 4 converted 6% of 14 C-TNT to biomass. The production of 14 C-CO 2 was observed for all four isolates, but the amount of 14 C-CO 2 produced was quite low: isolate 4 produced 14 C-CO 2 from approximately 1% of 14 C-TNT. The rate of degradation of TNT intermediates was very slow, reflecting possible difficulties in metabolizing the intermediates of TNT to CO 2 . The main intermediates were identified as 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene.

Anaerobic biodegradation of explosives and related compounds by sulfate-reducing and methanogenic bacteria : a review.

In recent years, research on microbial degradation of explosives and nitroaromatic compounds has increased. Most studies of the microbial metabolism of nitroaromatic compounds have used aerobic microorganisms. Ecological observations suggest that sulfate-reducing and methanogenic bacteria might metabolize nitroaromatic compounds under anaerobic conditions if appropriate electron donors and electron acceptors are present in the environment, but this ability had not been demonstrated until recently. Few review papers exist, and those deal mainly with aerobic bacterial degradation of explosives; none deals with anaerobic bacteria. In this paper, we review the anaerobic metabolic processes in the degradation of explosives and nitroaromatic compounds under sulfate-reducing and methanogenic conditions.

A laboratory study of the bioremediation of 2,4,6-trinitrotoluene-contaminated soil using aerobic/anoxic soil slurry reactor

The successful operation of an aerobic/anoxic laboratory-scale soil slurry reactor showed that soil contaminated with 2,4,6-trinitrotoluene (TNT) and hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) could be treated in batches or semicontinuously. Batch treatment resulted in the transformation of TNT. Semicontinuous treatment resulted in complete degradation of TNT. In addition to removing TNT, the slurry reactor also removed contaminants such as trinitrobenzene, 2,4-dinitrotoluene, RDX, and octahydro-l,3,5,7-tetranitro-l,3,5,7-tetraazocine (HMX). Radiolabeled TNT incubated with reactor biomass showed that 23% of [{sup 14}C]TNT was mineralized, 27% was converted to biomass, and 8% was adsorbed onto the soil. The rest of the [{sup 14}C]TNT was accounted for as metabolites, including a ring cleavage product identified as 2,3-butanediol. Increasing the frequency of soil addition from once to two or three times weekly did not affect the TNT removal rates. The soil slurry reactor also maintained the bacterial population fairly well, needing only 0.3% molasses as a cosubstrate.

Biotransformation of 2,4,6-trinitrotoluene (TNT) by co-metabolism with various co-substrates: A laboratory-scale study

Abstract Previous studies on the biotransformation of 2,4,6-trinitrotoluene (TNT) have shown that many aerobic bacterial consortia can transform TNT by co-metabolism. In this study various co-substrates have been used with the main objective of finding an inexpensive carbon source for large-scale biotreatment of TNT. Succinate, citrate, malic acid, acetate, glucose, sucrose, and molasses were used as carbon sources for an aerobic bacterial consortium transforming TNT. The results indicated that, among the various carbon sources studied, the cultures that received molasses at a concentration of 0·3% transformed 100 ppm of TNT within 12 h of incubation at ambient temperature, whereas the cultures with other carbon sources took more than 100 h to transform 100 ppm of TNT. The major intermediates identified were 4-amino-2,6-dinitrotoluene and its isomer, 2-amino-4,6-dinitrotoluene. Studies with [ 14 C]TNT provided no significant evidence that TNT was mineralized to CO 2 . The bacterial consortium was composed of various microorganisms, primarily Gram-negative rods. Molasses is an inexpensive carbon source that can be used in large-scale application for the biotreatment of TNT-contaminated soil and water.

Biotransformation of explosives by anaerobic consortia in liquid culture and in soil slurry

Abstract A laboratory study was conducted to study the feasibility of removing explosives in contaminated soil under anaerobic conditions. Anaerobic enrichment cultures were prepared from soil samples under various electron-accepting conditions, namely, sulfate-reducing, methanogenic, and nitrate-reducing conditions. The sulfate-reducing condition was very effective in removing all of the explosive compounds from the soil. The sulfate-reducing consortium removed 100% of 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitrobenzene (TNB) within 10–15 days of incubation and removed 75 to 95% of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), within 21 days of incubation. The consortium used explosive compounds as the nitrogen source, however, it did not use these compounds as the sole carbon source. The various metabolites obtained from TNT metabolism were 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT), 2,4-diamino-6-nitrotoluene (2,4- d -6-NT), and 2-methyl pentanoic acid. This sulfate-reducing consortium was further studied for its usefulness in removing TNT at the contaminated site. The results showed that the consortium can remove TNT under 5% and 10% soil slurry conditions. This laboratory study demonstrated that under anaerobic conditions, sulfate-reducing bacteria can be useful in the bioremediation of contaminated soil with TNT and other explosives.

Use of Trinitrobenzene as a Nitrogen Source by Pseudomonas vesicularis Isolated from Soil

Abstract. An aerobic Gram-negative bacterium identified as Pseudomonas vesicularis was isolated from soil contaminated with 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitrobenzene (TNB). This bacterium used TNB as the sole source of nitrogen. The TNB was metabolized within 80 h of incubation. The major metabolites produced were dinitroaniline, dinitrobenzene (DNB), nitroaniline, nitrobenzene (NB), and ammonia. The concentrations of DNB and NB produced in the culture medium were nearly stoichiometric. The ammonia concentration in the culture medium increased during the course of incubation. The end product of TNB metabolism was NB, which did not undergo further degradation even after long incubation time. This bacterium could be used in a syntrophic culture system with other NB-degrading bacteria to remove TNB completely from soil and water at contaminated sites.

In situ bioremediation of explosives-contaminated soil: A soil column study

In situ bioremediation of soil contaminated with explosives was studied using columns packed with contaminated soil. Several operating strategies were investigated, including continuous flooding of the soil column with dilute molasses or succinate solution, and periodic operating cycles consisting of flooding followed by draining and aeration. Two control columns were also used, which were flooded with deionized water. All of the soil columns that received molasses solution degraded 2,4,6-trinitrotoluene (TNT) and other explosive contaminants present in the soil. However, the most efficient removal of contaminants in terms of TNT removal rates was achieved by the soil column that was operated periodically with flooding with molasses solution, followed by draining and aeration. The columns that received succinate as co-substrate did not perform well. In control columns with deionized water, there was no removal of TNT. Results of this study will aid in improving the design and operation of field-scale bioremediation systems.