Anaerobic Biodegradation of 2,4,6-Trinitrotoluene and other Nitroaromatic Compounds by Clostridium Acetobutylicum

Title: Anaerobic Biodegradation of 2,4,6-Trinitrotoluene and other Nitroaromatic Compounds by Clostridium Acetobutylicum
Investigator: J.Hughes , F. Rudolph , G. Bennett
Institution: Rice University

Introduction

Nitroaromatic compounds are widely used in the synthesis of dyes, pesticides, pharmaceuticals and munitions and have become common contaminants of soil, sediments, and water. Nitroaromatics are often remarkably recalcitrant in the environment and bioremediation of contaminated materials has been only marginally successful due to incomplete metabolism and the production of unwanted toxic intermediates. Predicting the fate of these compounds in the environment, or development of bioremediation systems is hindered by the lack of information regarding fundamental aspects of metabolism. For example, applications of bioremediation technologies (composting, conventional waste water treatment, and anaerobic slurry reactors) to TNT contaminated materials are currently underway without knowing end products of metabolism, or factors which may influence product formation.

The purpose of this project is to investigate the anaerobic biotransformation of TNT and other nitroaromatics under highly controlled conditions using cultures of Clostridium acetobutylicum. Research in our laboratory has demonstrated the ability of C. acetobutylicum to rapidly transform many nitroaromatics. The high rates of transformation and absence of "typical" intermediates/endproducts may represent a significant advance in our understanding of anaerobic nitroaromatic metabolism. These studies are capitalizing on an existing library of information on C. acetobutylicum allowing for a fundamental analysis of anaerobic nitroaromatic degradation. Results will be beneficial to studies of anaerobic bioremediation processes and the fate of nitroaromatics in anaerobic environments aquatic sediments in particular. The long-term benefit will include the identification of genes responsible for contaminant transformation and the development of probes to assess nitroaromatic transformation in situ.

Background

Reduction of the aryl-nitro groups is an important fate process in anaerobic environments. Reducing the nitro group to an amine decreases toxicity and increases the ability to aerobically degrade the aromatic ring; however intermediate nitroso- and hydroxylamino-forms are mutagenic and have complex chemistries that affect their environmental fate. While reduction of the aryl nitro-group appears to be a fortuitous process, Clostridia are known to be particularly suited for catalyzing nitro-reduction [1, 10, 41, 43] even when no additional electron withdrawing groups are present. Research in our laboratory has demonstrated the ability of C. acetobutylicum to transform a range of nitroaromatics TNT in particular without the accumulation of aryl amines [24]. The focus of our studies has been to 1) investigate intermediates/end products of this metabolism, 2) evaluate the effects of environmental conditions on transformation, and 3) determine enzymes/genes involved in the process.

Outputs to Date

Pucik, L. E., and J. B. Hughes, "Capillary Electrophoretic Separation of TNT and TNT-Transformation Products," Journal of Capillary Electrophoresis -- accepted.
T. A. Khan, R. Bhadra, and Hughes, J. B., "Transformation of TNT and Related Nitroaromatics by Clostridium acetobutylicum," Journal of Industrial Microbiology -- accepted.
T. A. Khan (1996), "Transformation of TNT and Related Nitroaromatics by Clostridium acetobutylicum," M. S. Thesis. Rice University
Pucik, L. (1996) "The Fate of TNT Reduction Products in Aerobic Microbial Systems," M. S. Thesis. Rice University

Summary of Results

Initial studies have focused on the rate and extent of TNT reduction in C. acetobutylicum cultures assuming that aminated nitrotoluenes would represent common intermediate/end products. These studies produced two interesting results with implications in bioremediation and/or fate processes. First, aminated nitrotoluenes were not detected during TNT transformation. Instead hydroxylamino-compounds constituted the only aromatic transformation intermediates observed. The presence of these compounds in experimental systems may result from several factors

A "unique" form of metabolism.
Experimental conditions
- the pH of the growth medium is 4.1 to 5.2 under most culture conditions; and
- reduced sulfur, that rapidly catalyzes the abiotic reduction of TNT to the "stable" diaminonitrotoluenes [41], is absent from the growth medium employed in these studies).
The use of "different" analytical methods. Through the development of HPLC methods, amines and hydroxylamines are easily separated. The EPA-approved HPLC technique (Method 8330 or GC/MS used in many ongoing studies generally yield confounding information on these intermediates, and it is often assumed that the compound observed is an amine. To our knowledge we have been the first group to demonstrate the complication in the analysis of 2,4-diamino-6-nitrotoluene.

Hydroxylamines are interesting functional groups because of their ability to react chemically under environmental conditions. Potential reactions include covalent binding to soil, condensation with nitroso-groups forming azoxy dimers, or rearrangement to form aminated phenols (the Bamberger rearrangement). These reactions can reduce bioavailability or result in a substituted phenol that may be subject to mineralization. In experimental systems, products are highly water soluble (many precipitate in methanol), and ongoing studies are characterizing this polar fraction.

Due to the rapid reduction of TNT and monohydroxyl-forms, 2,4-dihydroxylamino-6-nitrotoluene accumulates rapidly in experimental systems. This is subject to further metabolism that does not result in the accumulation of the trihydroxylaminonitrotoluene. The initial product(s) is light and oxygen sensitive. 14C-studies have demonstrated that after the dihydroxylamine-level, the products of reduction are highly polar and not separated by reverse-phase HPLC or by GC/MS. We have developed TLC procedures to isolate product(s) and are currently using preparative-scale low-pressure normal-phase column chromatography, in conjunction with large-scale fermentation runs and NMR/CIMS, to produce, isolate, and identify this product(s).

The second major finding to date is the influence of the metabolic activity of the culture on the initial stages of nitro-reduction. C. acetobutylicim, like many other fermentative bacteria, will grow on sugars, but changes products and electron transfer processes as culture conditions change. For this organism, the final products of sugar metabolism are organic acids (acidogenesis) unless the pH drops below ~ 4.5. Then the bacteria will produce acetone and butanol (solventogenesis). Interestingly, the formation of hydroxylamines, and their subsequent transformation to "polar products" is profoundly influenced by this switch in metabolism. Specifically, the rate and extent of transformation drops rapidly as the organism enters solventogenic growth. These findings are the basis of a manuscript accepted for publication [24].

This result has practical applications in the operation of anaerobic bioremediation systems for treatment of nitroaromatics. Rapid rates of transformation may be limited to the growth phase of the reactor operation, which may present a significant operational challenge. In the presence of excess carbohydrates, solventogenesis can be begin within hours after start-up, while dissolution/desorption processes may require significant time periods. In batch systems, it may be possible to sustain acidogenic conditions through careful regulation of carbohydrate additions. Alternatively, a continuous flow system where acid levels do not induce solventogenesis could sustain high levels of acidogenesis for extended periods. A third possibility is to maintain a balanced mixed-culture where produced acids and H2 are metabolized by non-fermentative bacteria in a manner analogous to anaerobic digestion.

Ongoing Studies

In addition to work investigating the "polar residue," studies have been conducted to investigate the influence of temperature on the rate and extent of transformation observed. These experiments were conducted in anaerobic serum bottles inoculated with C. acetobutylicum under a range of temperatures (20°C to 37°C) and at contaminant levels of 20 to 50 mg/L. The primary focus of these experiments was to observe the influence of reduced temperature on the accumulation of hydroxylamines. Reducing the temperature resulted in a decrease of the observed TNT transformation rate (and subsequently the production of hydroxylamines). Despite the lower temperature, there was no apparent change in the eventual extent of transformation observed; however, a reduction in the biotransformation rate may be more significant in systems containing sediments, where competing chemical reactions (e.g., irreversible binding) may then be favored.

Also, studies have been conducted to investigate the transformation of nitroaromatics other than TNT. A partial list of nitroaromatics used in these studies include: nitrobenzene, (o-, m-, and p-) dinitrobenzene, dinitrotoluenes (various isomers), 1,3,5-trinitrobenzene, various fluoro-nitrotoluenes, HMX, and RDX. All are transformed under acidogenic growth conditions, with formation of hydroxylamino intermediates.

In another segment of our ongoing studies, the anaerobic reduction of TNT by C. acetobutylicum will be investigated in slurry systems. TNT-contaminated soils from the Alabama Army Ammunition Plant in Childersburg are available for this purpose, with varying concentrations of TNT from 900-2500 mg/kg. The objectives of these studies will be to evaluate the effects of solids loading, competing physico-chemical processes in the slurry, and of clostridial metabolism on TNT transformation.

To advance pathway analysis and initiate studies of enzymes involved in transformation, cell extracts have been prepared. In brief, the method of preparation developed consists of:

collecting cells by centrifugation followed by one wash step;
treating cells with lysozyme and sonicating;
removing debris by centrifugation.

Cells and extract are subject to anaerobic conditions at all times during the preparation.

The activity of crude extracts has been assayed in 10 ľg/mL TNT solutions with 5mM Tris, pH 7.8 at 25.1°C under anaerobic conditions in the dark. The specific activity for the protein extract was greater than 10 nmol of TNT reduced to the monohydroxylamine form per mg of protein per min. Several peaks during HPLC analysis were observed in cell extracts that have not previously been identified in whole cell studies (whole cell medium has several interfering peaks that do not appear in cell extracts).

Initial steps are in progress to clone the gene encoding enzymes involved in the early step(s) of TNT reduction using a functional selection. A C. acetobutylicum library is currently being constructed in pUC19 and will be maintained in E. coli IVNa F' cells. The library will be plated to minimal medium plates that contain 0.1 mg/mL TNT. Screening for positive clones will be based on the chromogenic property of the reduced form of TNT.

Last Updated: December 23, 1997