by Sarah Monti, Ph.D., LC-MS Sr. Product Specialist, and Ethan Hain, Ph.D., LC-MS Product Coordinator, Shimadzu Scientific Instruments
Per- and polyfluoroalkyl substances (PFAS) are often called “forever chemicals” because of their rugged stability in the environment. These man-made compounds, used for decades in nonstick cookware, firefighting foams, textiles, and coatings, resist natural degradation due to the strength of the carbon–fluorine (C-F) bond.1,2 The persistence and prevalence of PFAS has led to widespread contamination of soil and groundwater,3 which is of substantial concern due to the toxicity and bioaccumulation of these contaminants.1,4,5
The popular 2019 film “Dark Waters” raised public awareness regarding the dangers and prevalence of PFAS contamination, but environmental remediation remains a significant challenge.6-9 The most effective current techniques remove PFAS from the environment but do not destroy the toxic chemicals.6-8 The strong C-F bond requires extreme and often hazardous conditions, such as temperatures exceeding 1000 °C, for chemical or physical degradation.6-8 The success of bioremediation for non-toxic remediation of pollutants including hydrocarbons, insecticides, explosives, etc. is encouraging, but the man-made C-F bond is an imperfect target for naturally occurring microbial enzymes.10
Fortunately, technological innovations are providing progress using genetically engineered microorganisms (GEMs) for PFAS bioremediation.10 Genetically engineered Pseudomonas putida demonstrated 52% PFOA removal efficiency in a pilot study.11 Recent work at Princeton University, led by Peter Jaffé, has delivered a breakthrough in enzymatic PFAS destruction: the discovery that a specialized bacterium, Acidimicrobium sp. strain A6, can degrade PFAS under anaerobic conditions12. A6 possesses enzymes that can cleave the strong C–F bond12. In controlled laboratory reactors, A6 degraded up to 60% of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) over 100 days.12 Genomic analysis revealed the presence of a reductive dehalogenase gene (rdhA).12 The enzyme from this gene catalyzes reductive defluorination, in which electrons are transferred to PFAS molecules, replacing fluorine atoms with hydrogen.12 The process releases fluoride ions into solution, measurable proof that C–F bonds are broken. Unfortunately, naturally occurring A6 has a slow growth pattern and requires specific growth conditions.12 Using genetic engineering to place the key rdhA gene into an organism better suited for bioremediation conditions offers great hope for safe and efficient PFAS bioremediation.
Sensitive Detection of PFAS at Low Concentration
While genetic engineering can help in improving remediation efforts, detection difficulties are another major challenge, especially because the bioaccumulation of PFAS means that very low concentrations of PFAS in the environment concentrate up the food chain to clinically relevant concentrations within organisms.3 This requires the detection of very dilute contaminant mixtures in the environment.13 Liquid chromatography mass spectrometry (LC-MS) is a gold standard technique for PFAS detection. Regulatory methods for PFAS analysis often involve complex extractions for concentration and purification of samples, which cost significant time and labor. There are a few regulatory methods for direct injection methods which streamline this process; however, these present their own challenges as they can be less sensitive or push the LC-MS to its limit.
Shimadzu’s LCMS-8065XE triple-quadrupole mass spectrometer is engineered for direct injection of complex environmental samples while maintaining sub-nanogram detection limits. This instrument provides accuracy and sensitivity for PFAS detection, promoting use of simpler workflows to increase the efficiency of PFAS detection. Improving throughput boosts the return on investment for organizations performing PFAS testing, reducing costs and increasing accessibility.
Figure 1: Percent increase in sensitivity of 29 PFAS analytes using the LCMS-8065XE compared to previous generation LC-MS.
The LCMS-8065XE incorporates several technologies working synergistically to achieve these goals. The StreamFocus ion source effectively removes liquid from the sample, allowing more of the analytes of interest to be guided into the mass spectrometer while reducing contamination. The UFsweeper IV collision cell uses ion optics to accommodate the increased number molecules being transferred through the ion path to the higher sensitivity detector. These technologies enable an increase in sensitivity of up to 4700% for PFAS analytes (Figure 1) and detection limits below 1 ng/L for 29 PFAS analytes simultaneously using a streamlined, direct injection method.
Designing a Lab Workflow for PFAS Bioremediation
To study the PFAS GEM’s activity and generate reliable data, researchers can follow a structured workflow:
- Prepare the specimen in optimal conditions;
- Confirm presence of the species by qPCR by targeting rdhA genes;12
- Set up control reactors (e.g., no organism, no PFAS, or heat-killed inoculum);
- Spike experimental cultures with target compounds such as PFOA or PFOS;
- Incubate for experimental timeframe and collect regular liquid aliquots while maintaining culture;
- Prepare samples by removing biomass by filtration or centrifugation and storing them at 4 °C;
- Monitor fluoride release by ion-chromatography, such as EPA 300/300.1;14
- Use LC-MS/MS to track decrease in starting PFAS concentration by direct injection using the LCMS-8065XE;
- Plot PFAS and fluoride concentrations versus time to determine degradation rates and microbial activity.
This workflow provides a direct link between microbial activity via fluoride analysis, and mineralization of PFAS by LC-MS/MS analysis.
Last thoughts
The environmental and public health crisis posed by PFAS contamination demands both innovative biology and advanced analytics. The discovery of Acidimicrobium sp. A6 by Peter Jaffé’s team at Princeton proves that microbes can, in fact, break the “unbreakable” carbon–fluorine bond. Although challenges remain in scaling and accelerating this process, the foundation for biological PFAS remediation has been established, and utilizing GEMs can be the final piece of the puzzle.
Equally critical is the ability to monitor degradation at trace levels with high confidence. Using a high-sensitivity liquid chromatography mass spectrometer to deliver the sensitivity, robustness, and intelligence is necessary for direct-injection PFAS analysis. By eliminating extensive sample prep, analysts can accelerate research workflows and preserve the subtle chemistry of biodegradation.
Together, microbial innovation and mass spectrometry form a complementary toolkit: biology to safely destroy PFAS, and instrumentation capable of detecting even minute traces of the toxins. These technologies hold the potential to transform PFAS remediation from a costly challenge into a sustainable reality, clearing our waters for future generations.
About the authors
Sarah Monti, Ph.D., is an analytical chemist/biochemist with experience utilizing and adapting analytical chemistry and biochemistry techniques to expand their practical applications. She enjoys analyzing data and communicating the information to audiences of widely varying backgrounds. In her role as an LCMS Senior Product Specialist at Shimadzu Scientific Instruments, she indulges in these passions working with Shimadzu's complete range of flagship LCMS instruments during application development, customer training and troubleshooting sessions for clients.
Ethan Hain, Ph.D., is the LCMS Product Coordinator at Shimadzu Scientific Instruments. He obtained his doctorate in chemical and biochemical engineering at the University of Maryland Baltimore County for his work investigating the occurrence, source and toxicity of contaminants of emerging concern in the Chesapeake Bay. Ethan previously worked as an environmental engineer in the Regulatory Review and Engineering Branch of the Office of Groundwater and Drinking Water at the EPA and as an LCMS Product Specialist for SSI. Ethan enjoys cooking, baking and watching football with his wife and son.