IAP-24-040

Impact of perfluoroalkyl substance (PFAS) on soil structure and microorganisms

Perfluoroalkyl and Polyfluoroalkyl Substances (PFAS) are a family of human made chemicals used by industry as part of stain- and water-resistant fabrics, cleaning products, paints, fire-fighting foams and in cookware. They are pollutants of increasing concern as they are now commonly found in waterbodies largely due to industrial waste emissions. While there are over 4700 of PFAS developed, only a small number have been extensively studied, with some, such as perfluorooctane sulfonic acid (PFOS) and Perfluorooctanoic acid (PFOA) now banned for commercial use by international convention due to links to cancer and organ damage. They have also been associated with negative impacts on the development of children. However, these PFAS are still present in the environment through its “legacy” use and accumulate in fish, birds, plants and animals. For example, perfluorooctane sulfonic acid (PFOS), a PFAS, used as a surfactant in a range of industrial sectors and consumer products is found in an ever-growing number of water bodies, agricultural soils and plants and bioaccumulate in many organisms, including mammals.

The large diversity of remaining PFAS and their impact on soil health and microorganisms remains underexplored. Therefore, changes to microbial growth and function within soils by any PFAS is of concern as they perform beneficial and detrimental functions to both plants and animals. PFAS shape bacterial communities, reducing the biodiversity but favouring microbes that can breakdown PFAS. We can exploit those microbes to breakdown PFAS in situ through a process known as biotransformation which will be more effective, while reducing both cost and wastes generated.

The overall AIM of this project is to determine the effects of PFAS on soil structure and microbiome. The key OBJECTIVES (O) of this work are:
O1) to identify suitable candidate PFAS from literature, using the “arrowhead” approach (representative PFAS together with its salts and precursors).
O2) to measure physical, chemical and enzymatic differences in soils spiked with PFAS.
O3) to determine microbial biomass and identify the microorganisms enriched from soils under PFAS conditions.
O4) to biotransform PFAS using well characterised soil microorganisms enriched from soils adapted to PFAS conditions.

To achieve the abovementioned objectives, the student will:

O1) Use an extensive literature search to identify suitable candidate PFAS or use patterns in the EU “married up” with any monitoring data in the water environment from the Environment Agency, as a surrogate for soil data.

O2) Collect soil samples from agricultural lands, woodlands and grasslands in Scotland and spike with candidate PFAS (O1). Physical-chemical-enzymatic soil analysis will be performed on the obtained soils, including temperature, pH, total elemental composition, particle-size distribution, aggregate stability, cellobiohydrolase, β-glucosidase, phosphatase and N-acetylglucosaminidase, etc.

O3) Spike soils with candidate PFAS (O1) and flush with eluate measured for levels of microbial biomass using the MicroBIOMETER kit. Soil microbes will also be metabarcoded for the 16S or 18S rRNA genes and sequenced (MiSeq) to characterize microbial communities to a genus level. Interesting PFAS-adapted bacteria or yeasts will be isolated by Soil Extract Medium (SEM) or Luria–Bertani(LB) agar plates with appropriate antibiotics or antifungals, and characterised biochemically and microscopically.

O4) Evaluate the biotransforming capabilities of the bacteria or yeast isolated and characterised in O2 through GC/MS analysis. PFAS-adapted bacteria or yeasts will be exploited for the potential for biotransformation using a microcosm study with clean and sterile soil spiked with commercial PFAS.

Year 1

Conduct a literature review and design an appropriate research strategy for work within the laboratory (microbiology/cell biology);
Sample collection from soils in farms and woodlands in Scotland;
Isolate and analyse microbial samples;
Undergo training in metabarcoding and MiSeq sequencing;
Undergo training in GC/MS in Heriot-Watt University;
Undergo training in to perform physical-chemical-enzymatic analysis on the obtained soils;
Attend and present at one local conference.

Year 2

Repeat Year 1’s work for reproducibility and to observe sample variation: Sample collection from soils from different farms and woodlands in Scotland; Isolate and analyse microbial samples & sequence analysis of interesting microbial samples;
Culturing and/or maintaining a variety of PFAS-resistant microorganisms;
Microcosm study of PFAS-resistant microorganisms;
Attend and present at one local/overseas conference;
Begin chromatographic/spectrometric analysis of PFAS, PFAS degradation products and native organic matter in soils and microcosms.

Year 3

Expand on Year 2’s work by culturing and/or maintaining a wider variety of PFAS-resistant microorganisms and furthering GC/MS analyses;
Microcosm study of PFAS-resistant microorganisms;
Attend and present at one local/overseas conference.

Year 3.5

Thesis finalisation and paper writing (although it is anticipated that these activities will be ongoing throughout the PhD).

The analytical techniques required for this study are already established at the University of Stirling and include: SDS-PAGE, western blotting, microbiological / biochemical assays, geolocation of soils with a Global Positioning System (GPS) equipment, total elemental composition of soil using an Aqua Regia digestion and Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Other physical (bulk density, particle-size distribution, aggregate stability), chemical (TOC and C:N) and enzymatic (cellobiohydrolase, β-glucosidase, phosphatase and N-acetylglucosaminidase) parameters. Training will be provided by Dr. Clare Wilson and Mr Ian Washbourne (University of Stirling). Complementary training on gas chromatography mass spectrometry (GC/MS) techniques will be provided at Heriot Watt University using protocols, methods and instrumentation already established at Heriot-Watt University.

The student will receive training in “Linux for Genomics” & “RNA-seq Data Analysis” from Edinburgh Genomics (https://genomics.ed.ac.uk/ ). S/he will be trained in experimental design and data analysis related to the project work. Furthermore, s/he will attend courses on Effective Research, Scientific Writing, Statistics for Environmental Evaluation (and use of R) and Presentation Skills.

The student will also benefit from wider interaction within research groups at Stirling and Heriot-Watt Universities and interested parties in the Scottish Government. They will be expected to present the results of their research annually at the highly popular BES Student Symposium as well as at IAPETUS events and workshops. The student will also be expected to present their work at local and international conferences.