Insights into Spectroscopic Signatures of Substrate-Driven EPS Modulation in Diatom Biofilms: Implications for Biofilm Functionality

Diatom biofilms represent a promising platform for industrial applications with extracellular polymeric substances (EPSs) playing a pivotal role in adhesion, cohesion, and interfacial interactions with substrates. This study employs an integrated spectroscopic strategy to unravel substrate-induced EPS remodeling in two benthic diatoms, Amphora coffeiformis and Nitzschia microcephala, cultivated on hydrophilic glass and hydrophobic polyethylene. ATR–FTIR analysis revealed a functional group distribution shift in polysaccharides, proteins, and lipids, while X-ray photoelectron spectroscopy (XPS) provided insights on elemental bonding states and chemical-state distributions of carbon, oxygen, and sulfur. 1 H NMR further captured a solution-state biochemical shift in EPS. The EPS of A. coffeiformis biofilm on the PE substrate is highly dominated by the strong –CH 2 stretching and aliphatic carbon C–C/C–H (55.9%), indicating hydrophobic adaptation. Conversely, the N. microcephala biofilm on glass is dominated by hydroxyl and amide groups, reflecting carbohydrate- and protein-enriched EPS that promote hydrogen bonding with the polar substrates. XPS supported this biochemical shift by exhibiting balanced aliphatic (C–C/C–H) and oxygenated (C–O/C–H) functionalities with enriched carbonyl groups (C═O). 1 H NMR analysis corroborated these findings, with A. coffeiformis on PE displaying strong aliphatic proton signals, whereas N. microcephala on glass revealed strong glycosidic and sugar-ring resonances consistent with the polar adhesion. Together, all these results provide integrated spectroscopic evidence that diatom EPS is not static but actively modulated in response to substrate properties. These mechanistic insights advance the molecular understanding of biofilm–substrate interactions and establish a baseline framework for optimizing biofilm systems for industrial applications in bioprocessing, bioproduct recovery, and bioremediation approaches.