BACKGROUND: Non-invasive spectroscopic methods are increasingly valued in life sciences, where preserving the native state of biomolecules is essential for accurate interpretation. Traditional analyses of microbial compounds typically involve solvent-based extraction and chromatographic separation processes, which are time consuming, damaging to samples, and can alter biomolecular structures of complexes. To overcome these limitations, we developed a novel spectroscopic workflow for direct metabolite monitoring in microbial cells. RESULTS: In this study, we established a combined spectroscopic methodology that allows direct pigment and polyhydroxyalkanoates (PHAs) analysis in complex biological samples without requiring chemical extraction procedures. The UV-Vis spectroscopy technique using an integrating sphere enables direct monitoring of pigments even in turbid whole cell suspensions, providing detailed fingerprints of bacteriochlorophyll a and carotenoids in their natural environment. Together, these techniques provide consistent information about cellular composition. Using the photosynthetic bacterium Rhodospirillum rubrum as a model organism, we demonstrate that our combined spectroscopic approach can resolve pigment states, reveal intracellular PHA content and crystallinity, and measure carotenoids and bacteriochlorophylls directly in native whole cell suspensions. Furthermore, advanced data processing provided an improved interpretation of pigment and PHA states in different cellular forms. CONCLUSIONS: This innovative combination of spectroscopic techniques reduces sample manipulation, preserves cellular integrity and provides rapid, precise, and environmentally friendly analysis of microbial metabolites in their natural physiological conditions. The demonstrated workflow is broadly applicable to biological samples where maintaining biomolecular integrity is crucial, and it has strong potential for applications in process analytical technology and industrial biotechnology.

Faculty of Chemistry Brno University of Technology Purkynova 464 118 612 00 Brno Czech Republic

Institute of Chemistry NAWI Graz University of Graz Heinrichstrasse 28 4 8010 Graz Austria

Institute of Life Sciences University of Applied Sciences and Arts Western Switzerland Valais Wallis Sion Switzerland

Institute of Scientific Instruments v v i Czech Academy of Sciences Kralovopolska 147 612 64 Brno Czech Republic

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