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