In recent years, various advanced oxidation processes have been explored for perfluorooctanoic acid (PFOA) degradation. Yet factors influencing their efficacy and degradation mechanism are not fully understood. The objective of this proof-of-concept project was to evaluate a novel method to remediate per- and polyfluoroalkyl substances (PFAS) based on bacterial strains that are capable of producing superoxide at high rates and assessing their capacity to degrade model PFAS. Specifically, this project explored the potential of superoxide (including that generated by bacterial hyper-producers of extracellular superoxide) to degrade PFAS. Specific tasks included:

  1. Determine the potential for superoxide to degrade and defluorinate PFAS, such as PFOA.
  2. Identify and isolate bacteria with high superoxide production activity and explore factors to enhance their superoxide production.
  3. Evaluate the ability of these superoxide hyper-producing bacteria to degrade PFAS.

Technical Approach

Superoxide has been reported to degrade and defluorinate PFOA, and some prior publications suggest that it may play a critical role in the degradation of other PFAS. Furthermore, it was also recently discovered that many heterotrophic bacteria produce extracellular superoxide, with production rates spanning several orders of magnitude between different species. Moreover, superoxide production can be enhanced by the presence of cofactors (e.g., up to 100-fold using NADH), and its reactivity can be increased by certain solid matrices (with high surface area), redox mediators, or low polarity solvents. Therefore, the team hypothesized that bacteria capable of producing high concentrations of superoxide can be used as a basis for PFAS bioremediation strategies. Accordingly, the team first assessed PFAS degradation under various conditions in a low complexity, controlled system using chemically- and enzymatically-generated superoxide. The team then planned to screen heterotrophic bacteria to identify the highest superoxide-producing strains (hyper-producers), and assess their ability to degrade PFAS (PFOA, perfluorooctanesulfonic acid (PFOS), 8:2 fluorotelomer alcohol) under conditions identified as maximally enhancing superoxide production and reactivity. Finally, PFAS degradation by superoxide hyper-producers would be assessed using PFAS-spiked aquifer material.


For Objective 1, the team conducted a series of experiments to assess whether superoxide (produced from various sources) could directly transform PFOA. These efforts were reported in an Environmental Science & Technology Letters paper, with the conclusion that superoxide alone is not sufficient to degrade or defluorinate PFOA. Further tests were conducted to assess this potential remediation technology, and the team found that superoxide was able to degrade other organic compounds (e.g., bisphenol A [BPA], trichloroethene [TCE]). Overall, superoxide alone did not appear suitable for developing standalone remediation strategies for PFOA, but could be useful for other commonly co-occurring priority pollutants.

For Objective 2, the team developed a superoxide production assay that could be used to easily screen for the presence of this capability in bacteria. The team first acquired a model superoxide producer, Pseudomonas putida strain GB-1, to use for method development and as a benchmark. The team then developed an assay based on the observation that microbial superoxide production is involved in manganese oxidation. Oxidized manganese (Mn(III+)) is very reactive, and can be quantified using the colorimetric indicator Leucoberbelin blue I. After successfully demonstrating this assay using GB-1, the team tested various environmental samples and have identified some superoxide-producing bacteria. Preliminary tests have demonstrated that superoxide-producing bacteria can be easily and routinely isolated from environmental sources, which highlights the potential for developing bioremediation strategies based on the use of indigenous superoxide hyperproducers.

For Objective 3, since the team showed that superoxide alone could not degrade PFOA, the team also evaluated several other microbial enzymes that had been reported in the literature to degrade PFOA or PFOS. The team tested a laccase-mediator system and a peroxidase-mediator system under various optimized conditions. Additionally, the team tested the xanthine oxidase-hypoxanthine system in conjunction with MnCL2 to promote manganese oxide formation. However, none of these systems demonstrated a significant removal of either PFOA or PFOS after one week, reflecting a common problem with some PFAS degradation papers: the lack of reliable reproducibility.


This work resolved a controversy in the literature regarding the role of superoxide in degrading PFOA, which will help advise and guide the remediation community in developing more effective treatment approaches that do not rely on production of such ROS. These results were published in a rigorous academic journal, Environmental Science & Technology Letters, highlighting the relevance of these results.

Additionally, the identification and characterization of superoxide hyper-producing bacteria will facilitate the development of both in situ and ex situ bioremediation strategies for other priority groundwater pollutants (e.g., TCE), which could lead to significantly reduced treatment costs. This research also increases our understanding of potential natural attenuation routes (via extracellular reactive species) and how biostimulation might be used to enhance their efficacy and increase biodegradation rates. This strategy should be evaluated for the remediation of a wide range of recalcitrant chemicals of concern. Furthermore, the knowledge that superoxide and hydroxyl radicals cannot directly degrade PFOA advises the remediation community to explore other strategies besides reactive oxygen species for degradation. (Project Completion - 2021)


Javed, H., C. Lyu, R. Sun, D. Zhang, and P.J.J. Alvarez. 2020. Discerning the Inefficacy of Hydroxyl Radicals during Perfluorooctanoic Acid Degradation. Chemosphere, 247:125883.

Javed, H., J. Metz, T.C. Eraslan, J. Mathieu, B. Wang, G. Wu, A. L. Tsai, M. S. Wong, and P.J.J. Alvarez. 2020. Discerning the Relevance of Superoxide in PFOA Degradation. Environmental Science & Technology Letters, 7(9):653–658.


Javed, H. 2020. Mechanistic Insights on the Merits and Limitations of Advanced Treatment Processes for Removal of Contaminants of Emerging Concern (Ph.D. Dissertation). Rice University.