Conversion of 2,4,6-trinitrotoluene to a mixture of biodegradable compounds by dissolution in alkaline solution

Abstract

An efficient and low-cost method for conversion of TNT by alkaline degradon to a mixture of products and the subsequent mineralization of such products by microbes.

Description

This application is a non-provisional application claiming benefit of provisional application No. 60/035,984 filed Jan. 22, 1997 entitled Conversion of TNT to a Mixture of Biodegradable Compounds.

FIELD OF THE INVENTION

The present invention relates generally to eliminating environmentally hazardous TNT. More specifically, the method relates to chemically modifying TNT; thus, allowing it to be mineralized microbiologically.

BACKGROUND

The 2,4,6 isomer of trinitrotoulene (TNT) and its environmental transformation products are the most important munitions-derived pollutants encountered at military installations. Large amounts of these compounds have been released into the environment during manufacturing and disarming of ordnance. Remediation of sites contaminated with explosives is required by current statutes, and cleanup criteria have often been set on the order of 25 mg/kg for TNT. It is estimated that the United States probably has in excess of a billion tons of soil contaminated by TNT at >25 mg/Kg. Incineration of soils costs on the order of $1,200/cubic yard. Biological methods for cleanup of this pollutant would be cheaper but have been slow in the making because of the difficulty microbial systems have in metabolizing TNT. Metabolism of aromatic compounds requires cleavage of the ring system, and the chemical structure of TNT makes this difficult. It is postulated that the electrophilic nature and the orientation of the nitro groups prevents biological mineralization. Rieger and Knackmuss, "Basic Knowledge and Perspectives on Biodegradation of 2,4,6,-Trinitrotoulene and Related Nitroaromatic Compounds in Contaminated Soil". in: Bioremediation of Nitroaromatic Compounds. Edited by J.. C. Spain. #49 in the series Environmental Science Research. (Plenum Press, New York, N.Y., 1995), report that oxygenase reactions are "unknown" for trinitro compounds due to the electron withdrawing power of the three nitro groups. The electron withdrawing effect of the nitro groups makes the ring electron deficient and makes electrophilic attack by hydroxylase enzymes difficult. Inhibition of hydroxylation of the ring effectively eliminates TNT as a microbial nutrient and explains the remarkable persistence of this environmental pollutant.

Although the electron withdrawing effect of the three nitro groups may be sufficient in itself to protect TNT from microbiological attack, it is believed that the orientation of the nitro groups is the primary factor in this resistance. During synthesis of TNT, the initial nitration of the toluene ring occurs preferentially at the ring positions ortho or para to the methyl substituent. Additional nitrations of the ring become more difficult because of the "deactivation" of the ring caused by the cumulative electron-withdrawing effect of each nitro group. An additional result of this deactivation is that new nitro groups are added ortho and para to the methyl group and meta to each other. ##STR1## The resultant meta spacing of the three nitro groups ensures that any additional substituents added to the ring will also be selectively positioned meta to each other. However, biological cleavage of aromatic rings requires emplacement of phenolic substituents ortho or para to each other, Dagley, S. "Microbial Degradation of Organic Compounds in the Biosphere." American Scientist, 63:681-689 (1975); Simpson and Evans "The Metabolism of Nitrophenols by Certain Bacteria." The Biochemical Journal, 55:24 (1953). Thus, even if hydroxylations of the aromatic ring were to occur, the required spacing of the phenolic hydroxyl groups could not be achieved without subsequent isomerization. It is likely that the meta orientation of TNT and its resistance to hydroxylation results in inhibition of biological cleavage and these factors are responsible for the persistency of TNT as a contaminant in natural environments.

On the other hand, TNT can be biologically mineralized under laboratory conditions. The probable reaction sequence begins with reduction of two or more of the nitro groups to primary amines. In subsequent steps, the amino groups undergo transamination reactions that yield a ring structure with meta substituted phenolic substituents. In later steps, the methyl carbon is removed and rearrangements occur that move the phenolic groups to an ortho configuration. The ring is then oxidatively cleaved. See Selivansvskaya et al. "Terminal steps in Prepatory Metabolism of 2,4,6,-Trinitrotoulene in Pseudomonas flourescens." Mikrobiologia, 56:6, 1040-1041 (1986) as cited in Walsh, Environmental Transformation Products of Nitroaromatics and Nitramines. US Army Corps of Engineers Cold Regions Reseach & Engineering Laboratory, Special Report 90-2, ##STR2## Although mineralization of the ring carbons of TNT has been demonstrated in the laboratory, it appears that TNT is not utilized as a nutrient by microorganisms in natural environments. This most likely results from the fact that the required sequence of reduction, transamination, isomerization, and oxidation is disfavored if other food sources are available.

It follows from the above that conversion of TNT to a compound that contains only two nitro groups and "unsubstituted" ring carbons in the ortho or para configuration would greatly enhance its potential for microbial degradation. In addition, if the compound that was formed already had the two phenol substituents in place and properly orientated, metabolism of TNT would be accelerated. ##STR3##

The present invention relates to converting TNT to a mixture of dinitro compounds that contain two or three hydroxy substituents and then degrading these reaction products with microbial systems.

SUMMARY OF THE INVENTION

Thus, an object of the invention is to provide a method for eliminating munitions derived pollutants.

Another object of the invention is to render TNT susceptible to microbial attack.

Still another object of the invention is to use microbial systems to mineralize degradation products generated from TNT.

These and other objects of the invention are obtained as set forth in the detailed description of the invention below.

DETAILED DESCRIPTION OF THE INVENTION

The sensitivity of nitroaromatic compounds has been known for years. Urbanski, Chemistry and Technology of Explosives Volume 1. Pergamon Press. New York, N.Y. (1985), summed up the situation in stating: "Alkalis when reacted with trinitrotoluene (Note: this refers to symmetrical 2,4,6-TNT) very easily effect a considerable change in the substance, yielding red or brown colored addition products containing metal. Inorganic acids separate from these products as well as an organic substance which is no longer trinitrotoluene. Numerous investigations carried out to elucidate the structure of this substance have given no definite answer as yet."

The U.S. Army Technical Manual of Military Explosives (TM 9-1-1300-214) states that bases can convert symmetrical (2,4,6) TNT to highly reactive trinitrobenzyl anions. The anions are alleged to be short-lived and give rise to several products that are demethylated and contain only one phenolic substituent. The products reported in TM 9-1300-214 are 2,4,5-trinitrophenol and 3,5-dinitrophenol. We have seen evidence for the existence of the latter compound as a minor component present in the products produced by the alkaline degradation of TNT. The mechanism for demethylation may be by displacement of the methyl group by a potassium salt Feuer, H. The Chemisry of the Nitro and Nitrso Groups., (Interscience Publishers, a division of John Whiley & Sons New York N.Y., 1969). If sodium hydroxide were substituted for potassium methoxide, a similar compound would be expected, and the acidity of the resultant phenol would generate the demethylated species. ##STR4## The above is a reasonable pathway, but the principal products observed after alkaline dissolution of the invention were dinitro toluenes having two or more hydroxyl groups.

Procedure

In the present process the conversion of TNT to a mixture of biodegradable compounds requires only the dissolution of the TNT in an aqueous alkaline solution, preferably a potassium metal solution, preferably potassium hydroxide having a pH greater than 11.5, and preferably between 11.5 and 13. More preferably, the pH is 12.3. At 12.3, the conversion of TNT to biodegradable compounds is completed in 12 to 18 hours. Visual indication of the reaction is seen in the transformation of the solution from a red to a deep bronze color. The by-products or reaction products of TNT subjected to alkaline dissolution exhibit the following characteristics:

1. Products elute before TNT, and before both of the dinitrocresols (DNCs) standards on reversed phase HPLC. Therefore, the product is probably more polar than these compounds.

2. Changes in the concentration of the HPLC mobile phase or substitution of water for the mobile phase does not affect the elution time. Therefore, the product is probably ionic or has some ionic character.

3. The retention time of the product(s) in reversed phase HPLC is lengthened substantially by reacting the product in acetyl chloride or acetic anhydride/pyridine reagent. Thus, the products are probably alcoholic or phenolic.

4. The HPLC retention times are not affected by reacting the product with absolute ethanol and HCl gas. Thus, the compound is not a benzoate.

5. The compound will not form an ester with benzoic acid. Dinitrocresol standards readily undergo this reaction.

6. The products form a hard insoluble resin when dried that will not dissolve in any common organic solvent. The resin will, however, dissolve if HCl is bubbled through the solution. This behavior is common in phenoxide salts.

7. The compound will produce products of much longer HPLC retention time when subjected to reactions that normally generate ethers from phenolic precursors. However, the reactions produced "ether" in low yield.

8. The product has a strong bronze color that disappears at pH<3. This disappearance is accompanied by a sharp increase in the partition coefficient between ethyl acetate and water. There is an additional abrupt increase in the partition coefficient when the pH is dropped below 1. This behavior is consistent with a quinone compound which possesses strong electron withdrawing (deactivating) substituents.

9. The octanol water partition coefficient of the product is 0.04. The octanol water partition coefficient of the product after esterification (acetic anhydride/pyridine) is 0.84. (Determined by partition of C.sup.14).

10. Mass spectra conducted by chemical ionization demonstrated that a component having a molecular mass of 212 was probably the molecular ion of the predominant compound generated from TNT subjected to the alkaline process. Additional species were found with probable masses of 184, 200, 169, 155, and 228.

11. After neutralization by washing with H.sub.2 O, the dissolution product of TNT can be subjected to microbial attack by microbes capable of using the nitrogen sources of dinitrotoluenes having two or more hydroxyl groups. One such microbe is Rhodococcus erythropolis HL 24-2, which was originally isolated as a 2,4-dinitrophenol-degrading bacterium.

As alluded to above the degradation product of the invention can be subjected to microbial digestion by any microbes capable of digesting dinitrotoluenes having two or more hydroxyl groups. Microbes capable of digesting dinitrotoluenes having two or more hydroxyl groups include Rhodococcus erythropolis HL24-1 and 24-2 and mixtures thereof (see Lenke, et al Applied and Environmental Microbiology, September 1992, Vol. 58, pp 2928-2932. Herein incorporated by reference).

Biodegradation

As discussed above the criterion for success in converting TNT to more biodegradable products requires that phenol hydroxy groups be emplaced or be capable of emplacement at positions ortho or para from each other, and the ring must contain no more than two nitro groups. The products produced by the alkaline dissolution of the invention meet these requirements. The initial microbial testing of products made from ring-labeled C.sup.14 -TNT demonstrated a rapid loss of radiolabel from solution accompanied by a less than stoichiometric evolution of C.sup.14 --CO.sub.2 when subjected to microbial decay in the presence of microbes capable of digesting dinitrotoluenes.

Variations within the spirit and scope of the invention described are we will be apparent to those of ordinary skill in the art. Any examples presented are for illustration and do not serve to limit the invention. The meets and bounds of the invention are found with reference to the claims set forth below and equivalents thereof.

Claims

1. A method of degrading 2,4,6-trinitrotoluene which comprises:

subjecting 2,4,6-trinitrotoluene to alkaline dissolution to generate a reaction product; and

subjecting the reaction product to microbial digestion, wherein microbial digestion is conducted in the presence of Rhodococcus erythropolis HL24-1 or Rhodococcus erythropolis HL24-2.

2. The method of claim 1, wherein the alkaline dissolution is conducted in the presence of an alkaline solution having a pH between 11.5 and 13.

3. The method of claim 1, wherein the alkaline dissolution is conducted in the presence of potassium hydroxide.

4. A method of degrading 2,4,6-trinitrotoluene which comprises:

subjecting 2,4,6-trinitrotoluene to alkaline dissolution to generate a reaction product, wherein the reaction product comprises dinitrotoluenes having two or more hydroxyl groups; and

subjecting the reaction product to microbial digestion by a microbe capable of digesting dinitrotoluenes having two or more hydroxyl groups.

5. The method of claim 4, wherein the alkaline dissolution is conducted in the presence of an alkaline solution having a pH between 11.5 and 13.

6. The method of claim 4, wherein the alkaline dissolution is conducted in the presence of potassium hydroxide.

7. A product susceptible to microbial attack produced by a method comprising:

contacting 2,4,6-trinitrotoluene with an alkali to generate a reaction product incapable of forming an ester with benzoic acid; and

neutralizing the reaction product by washing the same with water to obtain a product susceptible to microbial attack.

8. The product of claim 7, wherein the alkali comprises potassium hydroxide.