Investigation of mycolic acids and biosurfactants from wastewater-foaming-associated bacteria

Foaming in activated sludge wastewater treatment plants represents a persistent operational challenge that can compromise treatment performance and increase operational costs. Activated sludge foaming can cause the release of inadequately treated wastewater to receiving waters systems, having detrimental consequences for ecosystems and living organisms, and posing risks to environmental and public health. This thesis aimed to investigate lipidomic profiles, biosurfactant production, and microbial community dynamics in activated sludge in order to improve understanding of the biochemical and microbial processes associated with foaming events. Initially, robust experimental and analytical methodologies were developed to enable lipidomic investigation of foaming-associated bacteria, such as Gordonia amarae. Reproducible growth conditions for Gordonia amarae were established in media that supported foaming formation. In parallel, a sensitive and reliable liquid chromatography–mass spectrometry (LC–MS) method was developed for mycolic acid analysis, and cyclic ion mobility spectrometry (cIMS) parameters were optimised to achieve high-resolution separation of lipid isomers. Application of cIMS revealed substantial isomeric complexity within mycolic acids from both Gordonia amarae and other mycobacteria species (Mycobacterium tuberculosis), with multiple conformations detected within single mass spectral peaks. Species-specific patterns of isomer separation and fragmentation were observed, highlighting the value of isomer-resolved lipid analysis for improved structural characterisation. Biosurfactant production by Gordonia amarae was shown to be strongly induced under foaming-related conditions, particularly in the presence of hydrophobic carbon sources, while being negligible in nutrient-rich media. Lipidomic analyses revealed reproducible shifts in mycolic acid composition under these conditions, including increased unsaturation and changes in oxidation state, indicating adaptive lipid remodelling in foaming conditions. Finally, investigation of wastewater mixed liquor suspended solids using 16S rRNA gene sequencing demonstrated that both foaming-related conditions and the presence of Gordonia amarae significantly influence microbial community structure and foaming potential. Distinct microbial interactions were observed, underscoring the importance of community-level processes in foaming behaviour. Overall, this thesis demonstrates that foaming in activated sludge systems arises from the combined effects of lipid adaptations, biosurfactant production, and dynamic microbial interactions. The integrated analytical framework developed here provides a foundation for identifying lipid- and community-based biomarkers to improve prediction and management of foaming events in wastewater treatment plants.