Influence of Surface Heterogeneity on Biofilm Attachment and Stability on Patterned 2D hBN-Cu Systems

This study explores the preferential attachment and biofilm development of Citrobacter

freundii on a chemically heterogeneous surface composed of hexagonal boron nitride (hBN)

patterns embedded in a copper (Cu) layer deposited on a silicon (Si) substrate. The heterogeneity

arises from the contrasting chemical reactivity of inert hBN and reactive Cu regions. The

objective is to assess whether such patterned 2D material systems can influence microbial

colonization behavior.

Biofilm formation was first evaluated under static conditions using Scanning Electron

Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). These techniques

quantified bacterial coverage, surface roughness, and biofilm thickness across hBN and Cu

domains. Results revealed substrate-specific differences in biofilm morphology and density,

suggesting that surface chemistry governs microbial attachment dynamics.

To examine environmental resilience, a dynamic exposure experiment was conducted using a

custom-built setup that simulated hydrodynamic shear stresses and introduced environmental

constituents such as ions, organic matter, and native microorganisms. The dynamic study

assessed biofilm adhesion strength, mechanical stability, and redistribution under flow

conditions.

These combined investigations—static and dynamic—offer insights into microbial behavior on

chemically heterogeneous surfaces, with implications for designing antifouling coatings and

biofunctional materials for real-world applications.