Nanocultures as an Assessment Tool for Microbial Dynamics

<p dir="ltr">This dissertation presents the systematic design, engineering, and application of microbial nanocultures as a versatile platform for studying microbial dynamics under controlled and physiologically relevant conditions. By integrating polymer science, microfluidics, and microbiology, this work addresses a central challenge in the field: the lack of scalable, tunable, and high-resolution tools to interrogate microbial behavior in diverse environments. A baseline nanoculture system was developed using poly(dimethylsiloxane) (PDMS) membranes, rigorously characterized for mechanical robustness, osmotic stability, and compatibility with real-time imaging. Chemical functionalization with spacer molecules enabled tunable control over hydrophobicity, swelling, and stretchability, while maintaining cytocompatibility and selective permeability. Embedding magnetic nanoparticles produced responsive nanocultures that could be retrieved, mobilized, and manipulated without compromising growth, expanding their use to environmental sampling and targeted interrogation. The platform was applied to explore microbial interactions, with Pseudomonas aeruginosa–Staphylococcus aureus co-cultures revealing how stressors and secreted metabolites drive spatial organization, collapse, and resilience within confined microenvironments. A complementary computational framework was developed to classify motility trajectories of pathogens such as P. aeruginosa and E. coli, demonstrating motility as a phenotypic signature of microbial physiology and interaction. Together, these advances establish microbial nanocultures as an integrated experimental–computational toolkit for investigating growth, motility, and interactions at single-cell and community levels. The framework retains compatibility with high-throughput imaging and downstream analysis, positioning nanocultures as a powerful approach for antibiotic discovery, microbiota-directed therapeutics, and mechanistic studies of microbial ecology in engineered complex environments.</p>