# Vibrational spectroscopy methodology for profiling of mixed carbon metabolism in carotenogenic and oleaginous Mucor circinelloides

## TL;DR

This study uses advanced spectroscopy to track how a fungus uses mixed carbon sources to produce lipids and carotenoids, helping improve sustainable bioproduction.

## Contribution

A novel multi-modal spectroscopy method with stable isotope labelling is introduced to trace carbon metabolism in mixed substrate fermentations.

## Key findings

- Glucose is primarily responsible for lipid accumulation in Mucor circinelloides.
- Polyphosphates serve as important energy reserves alongside triglycerides during fermentation.
- Glycerol contributes to carotenoid synthesis but not significantly to lipid accumulation.

## Abstract

Mixed carbon substrate fermentation is gaining interest in industrial biotechnology for effectively utilizing sustainable resources like waste glycerol and lignocellulose to produce food, feed, biofuels, and platform chemicals. However, challenges arise from the inefficient co-utilization of carbon sources as most microorganisms exhibit a preference for one substrate. This study evaluated the use of stable isotope labelling with infrared and Raman spectroscopies and microspectroscopies to investigate carbon utilization in fermentations with mixed carbon substrates. As a model system, the carotenogenic and oleaginous filamentous fungus Mucor circinelloides was grown on varying ratios of glucose and glycerol under nitrogen-limited conditions to induce lipid accumulation during the stationary growth phase.

The multi-modal spectroscopic approach successfully identified the flux of carbon from mixed substrates (glucose and glycerol) into specific metabolites, providing a detailed timeline of metabolite production. In the early phase of fermentation (first 8 h), biomass was rich in proteins and carbohydrates, primarily resulting from yeast extract utilization and some glucose consumption. Between 8 and 14 h, the production of polyphosphates, lipids, and carotenoids began. In media with abundant glycerol, carotenoids were assembled from both glucose and glycerol, with potential contributions from yeast extract. The lipid accumulation, primarily in the form of triglycerides (TAGs), is largely attributed to the utilization of glucose. Beyond 14 h, the biomass continues to accumulate polyphosphates, while high TAG levels were only observed when glucose was plentiful. In contrast, media with excess glycerol or glycerol as the sole carbon source resulted in only modest or negligible TAG accumulation, respectively. Polyphosphates were identified as important additional energy reserves alongside TAGs.

This study demonstrates that stable isotope labelling coupled with multi-modal infrared and Raman spectroscopies is a powerful approach for tracking carbon flow in mixed substrate fermentations. The findings highlight the pivotal roles of glucose in lipid accumulation and polyphosphates as alternative energy reserves in Mucor circinelloides. The combined use of multiple infrared and Raman techniques revealed complementary spectral features, improving data reliability and providing a comprehensive chemical insight into the fermentation process. This approach can contribute to the development of more efficient bioprocesses for sustainable production in industrial biotechnology.

The online version contains supplementary material available at 10.1186/s12934-026-02963-6.