The Department of Defense (DoD) has identified explosives and energetic compounds as contaminants of concern at numerous locations, including ammunition depots, production facilities, and live-fire training installations. Nitroaromatic and nitramine compounds such as trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are explosives that are soluble at low concentrations and pose a threat to soil, sediment, and groundwater. Off-site migration of contaminated groundwater may prove to be a direct threat to human health and the environment.

The broad objective of this research project was to characterize the role of Fe(III) reduction, extracellular electron shuttling, and the organisms that mediate these processes in cyclic nitramine biodegradation. The project was a basic science investigation, designed to identify the factors that influence RDX (and HMX within some experiments) degradation in anaerobic, subsurface aquifer material. The results will be used to design in-situ or ex-situ bioremediation strategies predicated on extracellular electron transfer and Fe(III)-reducing microbial physiology. The specific technical objectives of this research project were to (1) demonstrate that humic substances or alternate electron shuttling compounds added to RDX-contaminated aquifer material stimulate Fe(III) reduction and promote RDX reduction; (2) determine if humic substances (natural and synthetic) transfer electrons directly to RDX; (3) quantify the rate of RDX reduction catalyzed by Fe(II) versus reduced humic substances; and (4) identify the dominant microbial community associated with humics and Fe(III)-mediated RDX reduction.

Technical Approach

Fe(III)- and humic substance-reducing microorganisms significantly transform organic and inorganic compounds, including contaminants. Humic substances (i.e., humics) are naturally occurring compounds that transfer electrons from microbial respiratory enzymes to solid phase Fe(III). Humics also transfer electrons directly to a variety of compounds. Humics-mediated electron transfer from microbial respiration to Fe(III), other metals, or contaminants is referred to as electron shuttling. Nitroaromatic compounds are reported to be terminal electron acceptors for anaerobic microbial respiration. However, these processes may be inefficient in situ due to the poor distribution of nitroaromatic-reducing microorganisms and the overall kinetics of the individual reactions. Recent evidence indicates that Fe(II) can abiotically transfer electrons to nitroaromatic compounds, thereby altering their distribution and toxicity. These experiments were performed in pure phase or with pure cultures under artificial laboratory conditions.

The experiments performed during this project tested the following hypotheses:

  • Adding humic substances to nitroaromatic-contaminated aquifer material will stimulate Fe(III) reduction and the Fe(II) produced will consequently transfer electrons to RDX;
  • Reduced humic substances will transfer electrons directly to RDX in aquifer material;
  • Direct electron transfer from humic substances reduces RDX faster than electron transfer from reduced humics to Fe(III) → Fe(II) to RDX;
  • Synthetic humic substances will reduce RDX as effectively as natural, purified humic substances; and
  • Geobacteraceae will be enriched in situ during humics-mediated RDX reduction.


Humic substance-mediated bioremediation of RDX is a simple, in-situ alternative to bioremediation in anaerobic subsurface environments, aerobic bioremediation, pump-and-treat, and zero-valent iron. Regulatory acceptance for this technology is likely given that humic substances are natural compounds, Fe(III)- and humics-reducing microorganisms are ubiquitous, and all amendments are soluble. This technology may also be well-suited for co-mingled plumes, which are common at DoD sites. (Project Completed – 2007)