• Droumpali, Ariadni

Abstract

Global production of fish and other aquatic organisms from aquacultures will continue to increase ensuring high quality protein for the growing world population. Infectious diseases represent a key challenge to aquaculture and to reduce or avoid using antibiotics, new solutions for disease control are required. One sustainable solution is to introduce marine probiotic bacteria of the Roseobacter group, such that the TDA-producing Phaeobacter species. By producing the antibacterial compound tropodithietic acid (TDA), these bacteria can antagonize other microorganisms, including fish pathogens. For some TDA-producing bacteria, their antagonism is most pronounced when growing in biofilms. The integration of these beneficial bacterial biofilms on surfaces, such as tank walls in rearing units would be a way of supplying the probiotic bacteria.<br/>Bacterial attachment and biofilm formation depends on the physical and chemical properties of the surface; thus, the goal of this project was to fabricate micro-structured surfaces facilitating the formation of biofilms by beneficial bacteria. In addition, the present work aimed to reach an understanding of which topographies and length scales influence the contact between the cells and the surface material. Silicon surfaces with different periodicities, diameters and depths were fabricated by steps of UV-lithography, followed by deep reactive ion etching (DRIE) and then replicated into an electroplated nickel mold. The mold consisted of arrays of honeycomb pits and pillars with a range of dimensions and was further used for injection moulding of polymer surfaces. The overall process was based on DEEMO (Dry Etching, Electroplating and MOulding). Different challenges occurred during the optimization of polymer surfaces such as stretching of structures and demolding effects. The optimized micro-patterned surfaces were used for studying the biofilm attachment of <i>Phaeobacter inhibens</i>. <br/>Two experimental studies were carried out to explore the bacterial attachment in micro-patterned surfaces. A first study, using fluorescent staining and confocal laser scanning microscopy of bacterial biofilms on silicon surfaces, demonstrated that surface morphology may influence the spatial organization of the bacterial biofilm. In a second study, bacterial biofilm formation in a microfluidic flow system was investigated using polymeric planar, pit and pillar surfaces. The micro-patterned surfaces influenced the bacterial biofilm development, and the honeycomb pit surfaces resulted in significantly higher bacterial biomass than the planar or pillars surfaces. Liquid Chromatography-Mass Spectrometry (LC-MS) analyses of the effluent from the flow cells demonstrated that the honeycomb pillars facilitated the production of the antimicrobial compound TDA even though biofilm biomass was not increased.<br/>Overall, a full fabrication cycle for honeycomb micro-patterned surfaces from a silicon master to nickel shim and polymer replication was successfully established. Those surfaces were effectively used for marine bacterial attachment experiments. The present method is suitable for scaling up of the process to large areas allowing further investigations in the field, and for the successful application in several industrial environments, like aquaculture systems.

Topics

• impedance spectroscopy
• morphology
• surface
• compound
• polymer
• nickel
• experiment
• Silicon
• spectrometry
• lithography
• microscopy
• liquid chromatography
• plasma etching
• dry etching
• liquid chromatography-mass spectrometry