Grantee Research Project Results
2021 Progress Report: Biodegradation of PFASs from Groundwater and Soil
EPA Grant Number: SU840169Title: Biodegradation of PFASs from Groundwater and Soil
Investigators: Goel, Ramesh , Dentinger, Bryn , Diaz, Ruby , Podder, Aditi
Current Investigators: Goel, Ramesh , Dentinger, Bryn , Diaz, Ruby , Nallappan, Namratha
Institution: University of Utah
EPA Project Officer: Spatz, Kyle
Phase: I
Project Period: December 1, 2020 through November 30, 2021 (Extended to November 30, 2023)
Project Period Covered by this Report: December 1, 2020 through November 30,2021
Project Amount: $24,978
RFA: P3 Awards: A National Student Design Competition Focusing on People, Prosperity and the Planet (2020) RFA Text | Recipients Lists
Research Category: P3 Awards , P3 Challenge Area - Safe and Sustainable Water Resources
Objective:
- The objective of this project was to determine what co-cultivation of bacteria and fungi could be used to achieve biodegradation of PFAS such as PFOA and PFOS. No intermediate compounds will be measured during the experiments, only PFOA, PFOS, and fluoride concentrations.
- Investigate under different medium conditions if enzymes produced by co-cultivation of fungi and bacteria can mineralize PFAS.
- Evaluate if the enzyme Manganese peroxidase produced by P. chrysosporium can be used to break down the bond between C-F in PFAS.
- Evaluate the groundwater quality from a site exposed to long-term exposure to PFAS and determine if free fluoride is present in the groundwater.
Progress Summary:
The bacterial strains used in this project were Streptomyces cattleya (SC) and Bacillus amyloliquefaciens (BA), the fungal strains used were Pleurotus ostreatus (PO), Phanerochaete chrysosporium (PC), and Ganoderma lucidum (GL). The initial co-cultivation of bacteria with fungi was performed in Petri dishes using yeast malt extract agar. From the first tests, the following results were obtained:
Bacteria/Fungi | PO | PC | GL |
SC | SC growth enhanced by PO. PO growth inhibited. | SC growth enhanced by PC. | SC inhibits the growth of GL. An inhibition zone was observed, SC tended to grow around GL. GL was more resistant than PC and PO to other environmental fungi like Penicillium. |
BA | PO growth inhibited by BA | BA did not enhance the growth of PC. | GL is growing denser and more compact in the presence of BA. |
The bacteria SC and BA were also co-cultivated, and no inhibition zone was observed between the two bacterial strains.
The PFAs used during this experiment included: PFOA (Perfluorooctanoic acid) and PFOS (Perfluorooctane sulfonic acid). Standards used for quantifying PFOA and PFOS were prepared using methanol, and HPLC-MS/MS was used for quantification. Sep-Pak cartridges were used to perform solid-phase extraction. EPA method 533 was used to determine the concentrations of PFAS present in the analyzed samples. Ion chromatography was used to estimate fluoride concentrations.
Experiments using SC, GL, and co-cultivation of SC/GL did not show any free fluoride. Several growth mediums were tested; Table 1 shows the summary of a co-cultivation result using two different mediums. Experiments with fungi tended to lower the pH, in many instances lower than 2.0. The formation of several organic acids was also observed.
Table 1. Biodegradation experiments
M9 (Area) | Concentration (mg/L) | M11 (Area) | Concentration (mg/L) | |
Control | 617,532 | 786.55 | 654,826 | 835.73 |
SC/GL | 599,617 | 762.92 | 697,104 | 891.48 |
GL | 648,173 | 826.95 | 650,515 | 830.04 |
SC | 579,140 | 735.92 | 805,747 | 1034.75 |
The results presented in table 1 are inconclusive; the concentration of PFOS used for medium 9 was 1052 mg/L and for medium 11 was 1500 mg/L.
Even though polypropylene containers with polypropylene caps were used for the batch experiments, some adsorption is observed just by looking at the concentrations in the control batches. This finding concurs with adsorption into polypropylene, followed by other researchers (Scott et al., 2021). No free fluoride was detected in any of the samples, which indicates no mineralization taking place.
Batch experiments using the Manganese peroxidase enzyme generated by PC were also performed. We used 1-hydroxy benzotriazole (HBT) as a mediator and hydrogen peroxide (H2O2) to activate the enzyme at 27.5 oC and 130 rpm. Batch tests using PFOS were performed to test for mineralization. No fluoride was detected after running the experiments.
Table 2 shows one of the ratios used in one of the enzyme experiments.
Sample | Total Volume (ml) | Milli-Q (ml) | MnP (mg) | H2O2 (ml) | PFOA (ml) @100 mg/L | HBT (mg) |
PF-0 | 15 | 9 | 0 | 1 | 5 | 1 |
PF-1 | 15 | 9 | 0.4 | 1 | 5 | 1 |
PF-2 | 15 | 9 | 0.4 | 1 | 5 | 1 |
PF-3 | 15 | 9 | 0.4 | 1 | 5 | 1 |
PF-4 | 15 | 9 | 0.4 | 1 | 5 | 1 |
The pH of the batches was maintained between 5 and 5.5 by adding 1 N NaOH and 1 N H2SO4 as needed. No free fluoride was detected in the enzyme experiments.
The investigators also collected some groundwater samples from a local site that had used fight fighting foams that contained PFAS. The site had been contaminated with PFAS for over two years. The water was analyzed for fluoride content. No fluoride was detected in the groundwater. It is possible that the bacteria present at the site have not been able to mineralize the compounds.
Future Activities:
No free fluoride was detected from the experiments performed using different mediums, different combinations of microorganisms, and the enzyme Manganese peroxidase. Several researchers have detected intermediate compounds, but no free fluoride have been seen in many biodegradation studies, also reduction of PFOA or PFOS concentrations due to sorption in alignment with the results obtained in this research (Liou et al., 2010; Kwon et al., 2014; Lath et al., 2019)
We used the indication that no free fluoride is an indication that no mineralization has taken place. Concentrations of PFOS and PFAs in the batch reactors in the control experiments were less than the concentrations used; some adsorption into the polypropylene containers used for the experiments were observed. Many of the mediums developed for the experimental batches did not include calcium to prevent the formation of calcium fluoride in the case of potential mineralization of the parent compounds.
Further research is needed to identify and validate materials that limit the adsorption of PFAS into them. Absorption and adsorption of contaminants reduce the concentration of pollutants present in water. A complete mass balance and analysis of all intermediate compounds will be necessary to determine if biodegradation has occurred. No mineralization was observed in any of the experiments.
Supplemental Keywords:
Manganese Peroxidase, Pleurotus ostreatus, Ganoderma lucidum, Phanerochaete chrysosporium; Bacillus amyloliquefaciens, Streptomyces cattleya, biodegradation.Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.