Concern over 1,4-dioxane contamination in groundwater is driven by several factors, including the extensive use and improper disposal of 1,4-dioxane in industrial processes, the high mobility of 1,4-dioxane in water, the recalcitrance of 1,4-dioxane to degradation in the environment, and the classification of 1,4-dioxane as a probable human carcinogen. Current 1,4-dioxane remediation technologies generally entail ex situ pump-and-treat procedures that are not cost effective and are unable to remove co-contaminants such as tetrachloroethene (PCE) and trichloroethene (TCE).

This project consisted of two phases, a proof-of-concept (Phase I) and an extended follow-on study (Phase II). The objectives of Phase I were to: (1) design a microbially-driven Fenton reaction that autocatalytically generated hydroxyl radicals and degraded 1,4-dioxane, TCE, and PCE singly or in combination as co-contaminants at circumneutral pH without the need for continual addition of H2O2 or ultraviolet irradiation to regenerate Fe(II), (2) optimize the 1,4-dioxane degradation rates by varying the duration and frequency of the aerobic and anaerobic incubation periods, and (3) determine the pathway for 1,4-dioxane degradation by identifying the transient intermediates produced during the microbially-driven Fenton reaction for 1,4-dioxane degradation. The Phase I effort was completed in 2014.

The objective of Phase II was to develop alternative in situ remediation technologies based on the microbially-driven Fenton reaction for degradation of 1,4-dioxane, PCE, and TCE. In Phase II, the development of ex situ remediation technologies entailed a series of modifications to upscale and optimize the batch reactor configurations developed in Phase I. In addition, a series of flow-through column experiments were conducted to identify strategies for controlling the microbially-driven Fenton reaction in situ and optimizing contaminant degradation processes with contaminated sediments manipulated under simulated field conditions.

Technical Approach

The microbially-driven Fenton reaction was subsequently employed to drive a HO. radical-generating Fenton reaction that degraded 1,4-dioxane, tetrachloroethene, and trichloroethene. In the microbially-0driven Fenton degradation system, S. oneidensis batch cultures were provided with lactate as electron donor and Fe(III) as anaerobic electron acceptor and exposed to alternating aerobic-anaerobic conditions. During the aerobic phase, S. oneidensis reduced oxygen to hydrogen peroxide, while during the anaerobic phase, S. oneidensis reduced Fe(III) to Fe(II). During the aerobic-to-anaerobic transition period, the produced Fe(II) and hydrogen peroxide interacted chemically via the Fenton reaction to form Fe(III), HO- ion, and HOŸ radical which in turn can degrade contaminants. This approach was tested in liquid batch reactors, fed-batch reactors, and solid-state flow-through reactors. Further manipulation of Fenton reaction rates was examined by deleting reactive oxygen species (ROS) related genes in S. oneidensis. H2O2 is a byproduct of incomplete metabolism of oxygen during aerobic respiration, and a key reactant during the Fenton reaction. By manipulating select genes, including catalases and peroxidases directly implicated in ROS defense in S. oneidensis, the project team endeavored to enhance the rate of contaminant degradation.



During Phase I, the project team successfully designed a microbially-driven Fenton reaction that autocatalytically generated hydroxyl radicals and degraded 1,4-dioxane, TCE, and PCE either singly or in combination as co-contaminants. In comparison to conventional (purely abiotic) Fenton reactions, the microbially-driven Fenton reaction operates at circumneutral pH and does not require addition of exogenous H2O2 or ultraviolet irradiation to regenerate Fe(II) as Fenton reagents. The 1,4-dioxane degradation process is driven by the Fe(III)-reducing facultative anaerobe S. oneidensis. Batch cultures amended with lactate, Fe(III), and 1,4-dioxane were subsequently exposed to alternating aerobic and anaerobic conditions. During the aerobic period, S. oneidensis produced H2O2 via microbial aerobic respiration, while during the anaerobic period S. oneidensis produced Fe(II) via microbial Fe(III) reduction. During the transition from aerobic-to-anaerobic conditions, H2O2 and Fe(II) interacted chemically via the Fenton reaction to produce hydroxyl radicals that completely degraded 1,4-dioxane at source zone concentrations (10 mM) in 53 hours with optimal aerobic-anaerobic cycling frequencies of 3 hours. Acetate and oxalate were detected as transient intermediates during the microbially-driven Fenton degradation of 1,4-dioxane, an indication that conventional and microbially-driven Fenton degradation processes follow similar reaction pathways. The microbially-driven Fenton reaction also degraded TCE and PCE both singly and in combination with 1,4-dioxane as co-contaminant. The microbially-driven Fenton reaction thus provides the foundation for further development of alternative ex situ and in situ remediation technologies to degrade 1,4-dioxane, TCE, PCE, and potentially PFAS. These results were published in the Phase I Final Report.

During Phase II, it was observed during the aerobic-to-anaerobic transition period, the microbially-driven Fenton reaction oxidatively degraded source-zone concentrations of 1,4-dioxane, tetrachloroethene, and trichloroethene. This method was applied to attempt degradation of perfluorooctanoic acid, but it remained recalcitrant to the microbially-driven Fenton reaction. Batch reactor experiments demonstrated that 1,4-dioxane degradation by the microbially-driven Fenton reaction may not be as efficient with ferrihydrite than ferric citrate as terminal electron acceptor. As organic ligands are known to affect the Fenton reaction, it is likely that stabilization of Fe(II) by ligands in solution as well as participation of the organic ligand in dark ROS production affects the generation of hydroxyl radicals that are involved in organic contaminant degradation. Furthermore, comparison of S. oneidensis H2O2 scavenging with ROS mutants and other Shewanella species demonstrates S. oneidensis as the weakest H2O2 scavenger tested. Perhaps the ability of S. oneidensis to degrade contaminants is due to poor ROS scavenging causing excessive extracellular hydroxyl radicals.


The microbially-driven Fenton reaction provides a foundation for development of alternate ex situ and in situ remediation technologies to degrade chemicals of concern at DoD sites. In addition, the microbially-driven Fenton reaction may be applied as an alternate remediation technology for a broad range of other emerging chemicals of concern susceptible to degradation by hydroxyl radicals generated by the Fenton reaction, and thus may benefit remediation efforts throughout the United States. (Project Completed – 2021)  


Sekar, R., M. Taillefert, and T.J. DiChristina. 2016. Simultaneous Transformation of Commingled Trichloroethylene, Tetrachloroethylene, and 1,4-Dioxane by a Microbially Driven Fenton Reaction in Batch Liquid Cultures. Applied and Environmental Microbiology, 82(21):6335–6343.