BACKGROUND: Oleaginous filamentous fungi, such as Mucor circinelloides, are capable of accumulating high levels of single cell oil (SCO), making them attractive candidates for the production of biodiesel and other oleochemicals. Lignocellulosic feedstocks offer an abundant and cost-effective carbon source for SCO production due to their high polysaccharide content. However, most oleaginous microorganisms cannot directly utilize cellulose and hemicellulose polysaccharides, necessitating their conversion into monosaccharides. Lignocellulosic substrates can be saccharified either separately from fermentation (separate hydrolysis and fermentation; SHF) or simultaneously (simultaneous saccharification and fermentation; SSF). This study evaluated SSF using M. circinelloides, as well as SHF cultivations on two types of lignocellulosic hydrolysates, and two control fermentations, with process monitoring via four vibrational spectroscopy techniques: Fourier Transform Infrared (FTIR) spectrometer with fibre optic probe, FTIR microspectrometer, FTIR spectrometer with high throughput setting (HTS), and FT-Raman spectrometer with HTS. RESULTS: Quantitative estimation of glucose in the cultivation media and lipid content in the biomass was achieved using PLSR analysis of FT-Raman measurements from the cell suspension. FT-Raman spectroscopy demonstrated exceptional capability for online and at-line process monitoring among the tested techniques. It enabled direct and rapid analysis of raw cell suspensions (containing growth media, cellulose-rich pulp substrate, and fungal biomass) without the need for sample pre-treatment, purification, or modification. FT-Raman provided comprehensive biochemical profiles, effectively detecting key chemical changes in both the cellulose-rich pulp substrates and the fungal biomass, including lipid accumulation by the oleaginous fungi. FTIR with fiber optics is effective for monitoring glucose in SHF processes, but its accuracy is limited in SSF processes due to the very low glucose concentrations. The study demonstrates that FTIR microspectroscopy is a valuable tool for lab-scale fermentation process development, as well as for investigating the bioconversion of lignocellulosic biomass into fungal biomass and metabolites. CONCLUSIONS: FT-Raman spectroscopy is highlighted as a powerful process analytical technology (PAT) tool for real-time or near-real-time monitoring of SSF processes for intracellular SCO production. Its ability to provide rich chemical information rapidly and without extensive sample preparation holds significant promise for optimizing industrial SCO production from lignocellulosic feedstocks.

Faculty of Chemistry Biotechnology and Food Science Norwegian University of Life Sciences Postbox 5003 Ås 1432 Norway

Faculty of Chemistry Brno University of Technology Purkyňova 464 118 Brno 61200 Czech Republic

Faculty of Science and Technology Norwegian University of Life Sciences Postbox 5003 Ås 1432 Norway

SINTEF Industry Postbox 4760 TorgardenTrondheim 7465 Norway

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