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Title: Aerobic biotransformation and mineralization of 2,4,6-trinitrotoluene

Abstract

Respirometric mineralization studies of 2,4,6-trinitrotoluene (TNT) were conducted with microorganisms isolated from a site contaminated with munitions waste in Illinois. Nine aerobic bacterial species were isolated under a carbon- and nitrogen-limited condition and tentatively identified as: one Pseudomonas species; one Enterobacter species; and seven Alcaligenes species. Experiments were performed using each of the nine organisms individually and with a consortium of all nine bacterial species. The aerobic microorganisms were cultured in a sterile nutrient solution with glucose and 20 mg/L TNT. Mineralization was determined using uniformly ring-labeled {sup 14}C-TNT in a respirometer that trapped the evolved CO{sub 2}. Biodegradation behavior was characterized based on oxygen consumption, distribution of {sup 14}C activity, and high-performance liquid chromatography (HPLC) analysis of TNT and its transformation products.

Authors:
; ;  [1]
  1. Texas A and M Univ., College Station, TX (United States). Dept. of Civil Engineering
Publication Date:
Sponsoring Org.:
Department of the Army, Washington, DC (United States)
OSTI Identifier:
490983
Report Number(s):
CONF-950483-
ISBN 1-57477-008-X; TRN: IM9729%%234
Resource Type:
Conference
Resource Relation:
Conference: 3. international in situ and on-site bioreclamation symposium, San Diego, CA (United States), 24-27 Apr 1995; Other Information: PBD: 1995; Related Information: Is Part Of Bioremediation of recalcitrant organics; Hinchee, R.E.; Anderson, D.B. [eds.] [Battelle Memorial Inst., Columbus, OH (United States)]; Hoeppel, R.E. [ed.] [Naval Facilities Engineering Services Center, Port Hueneme, CA (United States)]; PB: 378 p.; Bioremediation, Volume 3(7)
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; 45 MILITARY TECHNOLOGY, WEAPONRY, AND NATIONAL DEFENSE; BIODEGRADATION; TNT; AEROBIC CONDITIONS; SITE CHARACTERIZATION; ILLINOIS; REMEDIAL ACTION; LIQUID COLUMN CHROMATOGRAPHY

Citation Formats

Bae, B.H., Autenrieth, R.L., and Bonner, J.S.. Aerobic biotransformation and mineralization of 2,4,6-trinitrotoluene. United States: N. p., 1995. Web.
Bae, B.H., Autenrieth, R.L., & Bonner, J.S.. Aerobic biotransformation and mineralization of 2,4,6-trinitrotoluene. United States.
Bae, B.H., Autenrieth, R.L., and Bonner, J.S.. 1995. "Aerobic biotransformation and mineralization of 2,4,6-trinitrotoluene". United States. doi:.
@article{osti_490983,
title = {Aerobic biotransformation and mineralization of 2,4,6-trinitrotoluene},
author = {Bae, B.H. and Autenrieth, R.L. and Bonner, J.S.},
abstractNote = {Respirometric mineralization studies of 2,4,6-trinitrotoluene (TNT) were conducted with microorganisms isolated from a site contaminated with munitions waste in Illinois. Nine aerobic bacterial species were isolated under a carbon- and nitrogen-limited condition and tentatively identified as: one Pseudomonas species; one Enterobacter species; and seven Alcaligenes species. Experiments were performed using each of the nine organisms individually and with a consortium of all nine bacterial species. The aerobic microorganisms were cultured in a sterile nutrient solution with glucose and 20 mg/L TNT. Mineralization was determined using uniformly ring-labeled {sup 14}C-TNT in a respirometer that trapped the evolved CO{sub 2}. Biodegradation behavior was characterized based on oxygen consumption, distribution of {sup 14}C activity, and high-performance liquid chromatography (HPLC) analysis of TNT and its transformation products.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = 1995,
month =
}

Conference:
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  • TNT is the explosive most widely used by the military, but current technologies for treating munitions production wastewater are costly and produce a secondary waste, spent GAC. Bioremediation holds promise as a more effective and less costly treatment strategy. An anaerobic fluidized-bed granular activated carbon (GAC) bioreactor in series with an activated sludge reactor was used to treat 2,4,6-trinitrotoluene (TNT). A wastewater solution of 100 mg/L 2,4,6-TNT, as well as carbonate buffer and nutrient solutions, were fed to the anaerobic reactor. Ethanol and ammonia were supplied for microbial growth at 540 mg/L and 20 mg/L, respectively. The mixed anaerobic culturemore » completely transformed the TNT to unknown products. Attempts to detect these products by gas chromatography (GC) and high-performance liquid chromatography (HPLC) were unsuccessful. The effluent from this reactor was treated further in an activated sludge system. The TNT transformation products were partially oxidized in the aerobic stage, resulting in recovery of 91% of the total influent nitrogen as ammonia, nitrate, and biomass. This corresponds to recovery of 82% of influent TNT nitrogen.« less
  • A strain of Clostridium bifermentans isolated from a munitions-supplemented enrichment was able to remove both TNT (2,4,6-trinitrotoluene) and RDX (1,3,5-triaza 1,3,5-trinitrocyclohexane) from its growth media. Biotransformations of TNT and RDX by cometabolism in a nutrient rich medium reduced the removal time from several days to a few hours, as compared to a nutrient limited medium. Redox potential (Eh) of the media had important effects on the biological and abiological transformations of the munition compounds.
  • The biotransformation of 2,4,6-trinitrotoluene (TNT) by Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-N-acetylamido-4,6-dinitrotoluene and its p isomer, 2-formylamido-4,6-dinitrotoluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitrotoluene and 4-N-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4-N-acetylhydroxy-2,6-dinitrotoluene and 4-N-acetoxy-2,6-dinitrotoluene (as acetylated HADNT),more » and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines, while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days.« less
  • The transformation of 2,4,6-trinitrotoluene (TNT) was investigated in cultures of the cyanobacterium Anabaena sp. by conducting a series of batch assays. 2,4,6-Trinitrotoluene was added to Anabaena sp. cultures in single and consecutive additions, at various initial concentrations, to determine its transformation kinetics, to identify products formed, to evaluate potential toxicity, and to determine the effect of light deprivation on the TNT transformation process. 2,4,6-Trinitrotoluene disappearance occurred only in the presence of Anabaena sp. cultures maintained under a normal 16-h photoperiod. Toxicity leading to culture chlorosis and death was observed in batch systems with an initial TNT concentration greater than 10more » mg/L. A low rate and extent of TNT disappearance was observed in light-deprived cultures, which were inhibited even at low TNT concentrations. At pH values between 7.5 and 8.5, azoxy-tetranitrotoluene isomers were detected in both the culture medium and solvent extracts of biomass and accounted for only 20 and 4.4% of the initially added TNT moles, respectively. At a culture pH range between 5.6 and 5.9, achieved by aeration with a 5% CO{sub 2}/air mixture, hydroxylaminodinitrotoluene equimolar to the TNT addition was produced and then depleted from the culture medium with prolonged incubation. Although TNT reduction in Anabaena sp. cultures occurred, yielding low levels of azoxy-tetranitrotoluene isomers or hydroxylaminodinitrotoluene, uptake and other transformation reactions of TNT and/or its transformation products by Anabaena sp. may have taken place. Based on a less than 15% observed increase of biomass concentration over the relatively short incubation periods and by considering the mean biomass concentration constant, the TNT disappearance rate followed pseudo-first-order kinetics. The biomass carbon-normalized TNT disappearance rates in Anabaena sp. cultures were about three orders of magnitude higher than previously reported TNT disappearance rates obtained in batch cultures of aquatic plants.« less
  • The effects of 2,4,6-trinitrotoluene (TNT) on indigenous soil populations and pure bacterial cultures were examined. The number of colony-forming units (CFU) appearing when TNT-contaminated soil was spread on 0.3% molasses plates decreased by 50% when the agar was amended with 67 {mu}g TNT mL{sup -1}, whereas a 99% reduction was observed when uncontaminated soil was plated. Furthermore, TNT-contaminated soil harbored a greater number of organisms able to grow on plates amended with greater than 10 {mu}g TNT mL{sup -1}. The percentage of gram-positive isolates was markedly less in TNT-contaminated soil (7%; 2 of 30) than in uncontaminated soil (61%; 20more » of 33). Pseudomonas aeruginosa, Pseudomonas corrugate, Pseudomonasfluorescens and Alcaligenes xylosoxidans made up the majority of the gram-negative isolates from TNT-contaminated soil. Gram-positive isolates from both soils demonstrated marked growth inhibition when greater than 8-16 {mu}g TNT mL{sup -1} was present in the culture media. Most pure cultures of known aerobic gram-negative organisms readily degraded TNT and evidenced net consumption of reduced metabolites. However, pure cultures of aerobic gram-positive bacteria were sensitive to relatively low concentrations of TNT as indicated by the 50% reduction in growth and TNT transformation which was observed at approximately 10 {mu}g TNT mL{sup -1}. Most non-sporeforming gram-positive organisms incubated in molasses media amended with 80 {mu}g TNT mL{sup -1} or greater became unculturable, whereas all strains tested remained culturable when incubated in mineral media amended with 98 {mu}g TNT mL{sup -1}, indicating that TNT sensitivity is likely linked to cell growth. These results indicate that gram-negative organisms are most likely responsible for any TNT transformation in contaminated soil, due to their relative insensitivity to high TNT concentrations and their ability to transform TNT.« less