Development of spectroscopic assays for rapid monitoring of estrogen biodegradation

Estrogen hormones are well-established environmental micropollutants which have been linked to endocrine disruption in aquatic organisms in wastewater discharge sites. Biological degradation is the primary wastewater treatment mechanism for estrogen removal. However, treatment efficacy is highly variable and difficult to engineer due to the “black box” nature of biological treatment. Microbial strain selection is a critical impediment towards engineering estrogen biodegradation, since isolating endogenous strains with specific metabolic traits requires lengthy enrichment cultures and is limited to culturable organisms. Furthermore, the highly sensitive and selective chemical trace analysis techniques used to measure estrogen removal are relatively expensive and inefficient. In this thesis, we developed rapid, high-throughput spectroscopic methods designed to monitor estrogen biodegradation. The spectroscopic methods include a fluorometric assay based on the uptake of a fluorescently-labelled estrogen and a colorimetric biosensor using gold nanoparticles (AuNPs) and an aptamer bioreceptor. A synthetic microbial community comprised of characterised estrogen-degrading reference strains was used to evaluate the fitness for purpose of the developed methods. A trace analysis method using conventional chromatography was developed to validate the use of the fluorescent probes with the synthetic microbial community. The biochemical fate and distribution of the BODIPY-estrogen in the estrogen-degrading bacteria – specifically, the biotransformation of BODIPY-estradiol to BODIPY-estrone by Caenibius tardaugens – was used to inform the design of the fluorometric assay. The fluorometric assay utilises a cell impermeable halide quencher to suppress the extracellular fluorescence, and thus, the obtained fluorescence response was attributed to the selective internalisation of BODIPY-estrogen by C. tardaugens. While the fluorometric assay was developed to screen for estrogen-degrading bacteria, the colorimetric aptasensor, which was adapted from published AuNP biosensors and aptamers for this application, was developed to quantify 17β-estradiol (E2) in buffered culture media. The developed aptasensor was evaluated against industry guidelines for ligand-binding assays. While the analytical performance of the aptasensor satisfied the majority of the guidelines’ acceptance criteria, the method suffered from biological interferences by the estrogen-degrading bacteria. The work in this thesis contributes towards expanding the available bioanalytical methods in environmental biotechnology

QuEChERS: a simple extraction for monitoring quaternary ammonium biocide pollution in soils and antimicrobial resistance

Quaternary ammonium compounds (QACs) are broad-spectrum disinfectants used in a range of everyday materials. Their high usage rates, limited regulation and reporting has meant their environmental release is largely uncontrolled and impact unknown. With links to antimicrobial resistance (AMR) and adsorption to wastewater solids (that are recycled), there is a need for more controlled disposal measures and monitoring. These environmental matrices are highly complex requiring methods that are often laborious and costly to undertake. Using a robust quantitative reversed-phase LC-MS/MS method, we have shown that an ‘off the shelf’ QuEChERS product can reliably extract (<10% RSD) aromatic and aliphatic QACs anticipated within municipal, industrial and agricultural waste from water and soil, with reduced matrix effects of 95.7–104.4% for recoveries of up to 53% from soil when combined with extract dilution. Therefore, unlike current literature, this work has shown that, with minimal development, the QuEChERS product can provide a rapid, effective and low cost preparation for quantifying QAC pollution and monitoring AMR

BODIPY-labeled estrogens for fluorescence analysis of environmental microbial degradation

Biodegradation of estrogen hormone micropollutants is a well-established approach toward their remediation. Fluorescently labeled substrates are used extensively for rapid, near-real-time analysis of biological processes and are a potential tool for studying biodegradation processes faster and more efficiently than conventional approaches. However, it is important to understand how the fluorescently tagged surrogates compare with the natural substrate in terms of chemical analysis and the intended application. We derivatized three natural estrogens with BODIPY fluorophores by azide–alkyne cycloaddition click reaction and developed an analytical workflow based on simple liquid–liquid extraction and HPLC-PDA analysis. The developed methods allow for concurrent analysis of both fluorescent and natural estrogens with comparable recovery, accuracy, and precision. We then evaluated the use of BODIPY-labeled estrogens as surrogate substrates for studying biodegradation using a model bacterium for estrogen metabolism. The developed analytical methods were successfully employed to compare the biological transformation of 17β-estradiol (E2), with and without the BODIPY fluorescent tag. Through measuring the complete degradation of E2 and the transformation of BODIPY-estradiol to BODIPY-estrone in the presence of a co-substrate, we found that BODIPY-labeled estrogens are biologically viable surrogates for investigating biodegradation in environmental bacteria

BODIPY-labelled estrogens for fluorescence analysis of environmental microbial degradation

Biodegradation of estrogen hormone micropollutants is a well-established approach towards their remediation. Fluorescently labelled substrates are used extensively for rapid, near real-time analysis of biological processes and are a potential tool for studying biodegradation processes faster and more efficiently than conventional approaches. However, it is important to understand how the fluorescently tagged surrogates compare with the natural substrate in terms of chemical analysis and the intended application. We derivatized three natural estrogens with BODIPY fluorophores by azide-alkyne cycloaddition click reaction and developed an analytical workflow based on simple liquid-liquid extraction and HPLC-PDA analysis. The developed methods allow for concurrent analysis of both fluorescent and natural estrogens with comparable recovery, accuracy, and precision. We then evaluated the use of BODIPY-labelled estrogens as surrogate substrates for studying biodegradation using a model bacterium for estrogen metabolism. The developed analytical methods were successfully employed to compare the biological transformation of 17β-estradiol (E2), with and without BODIPY fluorescent tag. Through measuring the complete degradation of E2 and the transformation of BODIPY-estradiol to BODIPY-estrone in the presence of a co-substrate, we found that BODIPY-labelled estrogens are biologically viable surrogates for investigating biodegradation in environmental bacteria