Microstructured Polymer Surfaces for Growth of Probiotic Marine Bacteria

Marine probiotic bacteria of the Phaeobacter species can produce the antibacterial compound tropodithietic acid (TDA), which effectively inhibits pathogenic microorganisms. Since TDA is biosynthesized and has not been chemically synthesized, culturing live TDA-producing bacteria offers continuous antibacterial effect. Biofilms, which are aggregates of bacteria embedded in an extracellular matrix. This study aims to investigate surface topographies and properties that support biofilm formation by Phaeobacter piscinae S26, with the potential of enhancing their antibacterial functionality in fish farming applications.

Engineered surface topography can significantly influence bacterial behavior. In this study, hexagonal pit and pillar microstructures were fabricated on silicon (Si) wafers in the cleanroom, serving as molds for polymer replication. The height and gap distance of these microstructures were varied to explore the correlation between topographical features and bacterial attachment. 
The fabrication process was optimized to produce microstructures with critical dimensions and smooth sidewalls. Maskless lithography was employed, and the exposure parameters were systematically studied to ensure accurate feature sizes. During dry etching, sidewall roughness and angles were tuned to achieve optimal conditions for polymer demold. Additionally, thermal oxidation followed by oxide removal was applied to further improve surface smoothness and enhance demolding performance. 

Polymers are promising candidates for large-scale surface production due to their cost-effectiveness and ease of scaling fabrication. Surface properties of polymers, such as surface energy, can influence bacterial attachment. In this study, flat polymer surfaces made of polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), polypropylene (PP), and polydimethylsiloxane (PDMS) were selected to investigate the correlation between bacterial attachment and surface energy. These polymers span a wide range of surface energies, from 11.3 mN/m to 47.1 mN/m, allowing for comparative analysis. The most hydrophobic surface, PDMS, exhibited higher bacterial attachment after 6 hours of incubation as an early phase. However, after long-term culture of 24 hours and 96 hours, bacterial attachment and TDA production were comparable across all polymer surfaces. Therefore, no clear correlation was found between surface energy and bacterial behavior during long-term culture, suggesting that material selection can be guided by practical considerations such as production efficiency and scalability.

Various polymer replication techniques were explored. Nanoimprint was primarily used to prototype microstructures on the polymer sheets, specifically using COC and PP. Imprinting and demolding parameters were optimized, with imprinting and demolding temperatures identified as the critical factors for achieving complete pattern transfer and minimizing damage. Effective demolding techniques were essential to prevent defects in the replicated structures during demolding. As a result, high-quality microstructures were successfully fabricated in polymer to support bacterial growth. Additionally, injection molding was employed to produce polymer chips designed for use in a dynamic flow cell platform for bacterial culture. 

Hierarchical structures were further developed to enable the integration of features across nano-, micro-, and macro-scales within a single device. While the replicated microstructures typically have smooth surfaces derived from the mold, the addition of polystyrene (PS) particles during nanoimprint introduced random nanoscale indentations. This fabrication method allowed investigation of bacterial behavior in response to surface roughness on microstructures. In the other method, the 3D printer first fabricated the macroscale mold frame. The printing process was then paused to integrate customized nano- and microstructures, transferred from a silicon mold, at designated layers and positions. Printing subsequently resumed to complete the overall structure. This flexible approach enables the incorporation of any desired testing structures into a single device. Both methods facilitate comprehensive studies of bacterial interactions with engineered surfaces.

A static carrier test platform and a dynamic flow cell platform were established to evaluate bacterial growth on the polymer surfaces. Bacterial attachment was visualized using scanning electron microscopy (SEM) after bacteria fixation, dehydration, and drying. This enabled close observation of bacterial behavior on different surfaces. SEM analysis provided insights into bacterial coverage and interactions with various polymer materials and microstructured topographies, supporting further investigation into surface- dependent biofilm formation.

In conclusion, this study explored and evaluated the factors that influence bacterial behavior, and various fabrication techniques were applied to develop substrates and platforms to support bacterial growth. These approaches offer strong potential for the large-scale production of functional surfaces tailored for bacterial studies.