Non-enzymatic Raman sensing of lactic acid in microbial cultures: applications in resolving protonation states

Lactic acid is a key microbial metabolite with widespread industrial applications in food preservation, cosmetics, pharmaceuticals, polymer industries, and others. The detection of lactic acid directly in complex microbial culture remains a challenge. Enzymatic assays and high-performance liquid chromatography are traditional methods for lactic acid detection which require extensive sample preparation. In this study, we report a non-enzymatic Raman sensing framework for the direct detection and mapping of lactic acid in microbial culture. We used Lactobacillus plantarum and Lactobacillus acidophilus as representative lactic acid producing microbes, and the characteristic Raman signal of lactic acid was detected from microbial culture supernatants. An additional multiplex advantage of the approach is that both protonated and deprotonated forms of lactic acid were detected under varying culture pH conditions across species. Raman imaging was also performed directly on microbial cultures, making it possible to visualise the spatial distribution of lactic acid. Raman images revealed a diffuse distribution of lactic acid within the culture matrix, consistent with metabolic secretion by actively growing cells. This work pipeline highlights the analytical utility of Raman spectroscopy for a reagent-free, non-invasive, and minimal signal processing sensing platform for lactic detection and spatially resolved analysis for the future integration for in-line monitoring in bioreactor systems.