MadSci Network: Microbiology

Re: Have there been any studies done on TNT-eating bacteria?

Date: Wed Oct 20 16:29:51 1999
Posted By: Michael Benedik, Faculty Biology and Biochemistry, University of Houston
Area of science: Microbiology
ID: 940105432.Mi

There have been dozens of studies done to isolate bacteria which can degrade TNT. It turns out that TNT is a good source of Nitrogen that bacteria can use. Most organisms can not use free N2 from the air, but must have "fixed" nitrogen like ammonia (NH3) or some compound with an ammonia group. This is why we often have nitrogen or ammonia rich fertilizer for plants.

However TNT is sufficiently different from most ammonia sources that most bacteria are unable to degrade it, but there are some that can.

Bioremediation of TNT turns out to be not very different from bioremediation of lots of other compounds. Finding bacteria capable of performing the reaction is usually simple. There are probably a half dozen or more different bacteria that can degrade TNT. Designing a setup in the laboratory that makes it work efficiently requires more effort, but usually is straightforward as well. The problem comes with cleaning up an environmental site. Any site out in nature is not well controlled like the laboratory. There are many other competing bugs, the weather changes all the time, and most important, other nutrients are limiting. This is usually the limiting factor. Lets say we have an infinite amount of TNT the bugs can use as a nitrogen source, but if they don't also have a carbon energy source they won't grow. So often times bioremediating a natural site literally requires determining what other growth components are limiting at that location and supllying them in a cost effective manner. This has proven to be the challenge.

Genetic engineering has led to the construction of a bacterial strain of Pseudomonas that can use TNT both as a nitrogen and carbon source. This might eventually be useful for natural sites. Also investigators have put the genes for TNT degradation into plants. These plants can now grow on TNT contaminated soil and will (slowly) degrade the available TNT. Below is the abstract for that paper.

[Moderator's Note: You can locate articles on the subject at PubMed. Try a query of TNT bacteria, for instance.]

Nature Biotechnology 1999 May;17(5):491-4
Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase.
French CE, Rosser SJ, Davies GJ, Nicklin S, Bruce NC
Institute of Biotechnology, University of Cambridge, UK.

Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems. To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives. Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds. Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings. This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil.

And here are abstracts from 2 recent papers showing the current state of research in the microbial field of TNT remediation.

Current Microbiology 1998 Jan;36(1):45-54
Biotransformation patterns of 2,4,6-trinitrotoluene by aerobic bacteria.
Kalafut T, Wales ME, Rastogi VK, Naumova RP, Zaripova SK, Wild JR
Department of Biochemistry and Biophysics, The Texas A&M University System, College Station, TX 77843-2128, USA.

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp. , and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO2 from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO2 from the nitroaromatic ring.

Applied and Environmental Microbiology 1993 Jul;59(7):2171-7 Initial-phase optimization for bioremediation of munition compound-contaminated soils.
Funk SB, Roberts DJ, Crawford DL, Crawford RL
Department of Bacteriology and Biochemistry, University of Idaho, Moscow 83843.

We examined the bioremediation of soils contaminated with the munition compounds 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5- triazine, and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine by a procedure that produced anaerobic conditions in the soils and promoted the biodegradation of nitroaromatic contaminants. This procedure consisted of flooding the soils with 50 mM phosphate buffer, adding starch as a supplemental carbon substrate, and incubating under static conditions. Aerobic heterotrophs, present naturally in the soil or added as an inoculum, quickly removed the oxygen from the static cultures, creating anaerobic conditions. Removal of parent TNT molecules from the soil cultures by the strictly anaerobic microflora occurred within 4 days. The reduced intermediates formed from TNT and hexahydro-1,3,5-trinitro-1,3,5-triazine were removed from the cultures within 24 days, completing the first stage of remediation. The procedure was effective over a range of incubation temperatures, 20 to 37 degrees C, and was improved when 25 mM ammonium was added to cultures buffered with 50 mM potassium phosphate. Ammonium phosphate buffer (50 mM), however, completely inhibited TNT reduction. The optimal pH for the first stage of remediation was between 6.5 and 7.0. When soils were incubated under aerobic conditions or under anaerobic conditions at alkaline pHs, the TNT biodegradation intermediates polymerized. Polymerization was not observed at neutral to slightly acidic pHs under anaerobic conditions. Completion of the first stage of remediation of munition compound-contaminated soils resulted in aqueous supernatants that contained no munition residues or aminoaromatic compounds.

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