Real-time monitoring of Komagataeibacter xylinus biofilm dynamics using a microfluidic SERS platform integrated with gold nanoparticles

Komagataeibacter xylinus is an established model organism for bacterial cellulose (BC) production attracting considerable interest in the fields of biomaterials, plant-associated biofilms, and cell–cell signaling. Rapid, non-destructive monitoring of subtle biochemical variations during biofilm formation is critical for optimizing BC production. The objective of this work is to demonstrate a proof-of-concept platform for time-dependent monitoring of chemicals released during K. xylinus biofilm production using surface-enhanced Raman spectroscopy (SERS). SERS is a versatile technique known for its exceptional sensitivity to ultra-low concentrations and its ability to provide molecular-level information in real time with minimal sample volume. This work represents one of the first demonstrations of a biologically mediated SERS platform for monitoring BC production. This study compares chemically and biologically synthesized plasmonic enhancers for integration into the SERS platform. Gold nanoparticles (AuNPs) were synthesized using the classical Turkevich citrate reduction method and an in-situ biologically mediated approach. The Turkevich method was selected for its reproducibility and consistency in size control. Polydimethylsiloxane (PDMS) was laser-etched at 405 nm to create a microfluidic bioreactor channel integrated with SERS-active AuNPs components to facilitate continuous, spatially resolved, and low volume (5 μL) monitoring. Multivariate analyses were applied to the SERS dataset to track subtle biochemical changes over time using principal component analysis (PCA). PCA effectively reduced data dimensionality and revealed temporal biochemical shifts corresponding to evolving biofilm composition. Overall, this study demonstrates the feasibility of combining chemically and biologically synthesized AuNPs, a microfluidic device, SERS, and PCA as an integrated platform for real-time monitoring of BC production and dynamics. This platform holds promise for broader applications in monitoring metabolite exchange and optimizing industrial bioprocesses involving BC.