# Effect of hydrogen peroxide and carbon-to-nitrogen ratio on growth and biochemical profile in oleaginous mucoromycota

## TL;DR

This study explores how hydrogen peroxide and carbon-to-nitrogen ratios affect the growth and biochemical profile of oleaginous Mucoromycota fungi for biofuel production.

## Contribution

The study introduces new insights into the compatibility of hydrogen peroxide with Mucoromycota in lignocellulose-based SSF for biofuels.

## Key findings

- Mucoromycota strains tolerate higher H2O2 concentrations than typically used in saccharification.
- Changes in C/N ratios significantly affect fungal biochemical composition and carbon allocation strategies.
- H2O2 exposure slightly reduced biomass production near minimal inhibitory concentrations in some strains.

## Abstract

Hydrogen peroxide (H2O2) has gained attention as cofactor of lytic polysaccharide monooxygenases (LPMOs) during lignocellulose saccharification. The action of these enzymes has been shown to significantly enhance saccharification efficiency. However, in simultaneous saccharification and fermentation (SSF) processes, H2O2 can have deleterious effects on the fermenting microorganism. In addition to oxidative stress, at certain concentration ranges, H2O2 can play a crucial role in redox biology mediating metabolic crosstalk. Indeed, some works have explored the influence of H2O2 and other stress molecules in lipid accumulation. In this study, nine strains from eight different species of Mucoromycota were grown at different sublethal concentrations of H2O2 and two carbon-nitrogen (C/N) ratios. The aim of this study was to investigate whether H2O2 could enhance lignocellulose-based SSF with oleaginous Mucoromycota fungi to produce second-generation biofuels. Therefore, effects of H2O2 concentration, beneficial or deleterious, were identified under different C/N conditions.

In general, all the strains tolerated H2O2 at much higher concentrations than those commonly used to improve enzymatic saccharification (1-19 mM vs 1-240 µM). Vibrational spectroscopy (mid-infrared and Raman) was used to analyze the biochemical composition of the fungi. The exposure to sublethal H2O2 doses did not increase any metabolite in particular but slightly reduced biomass production at concentrations near the minimal inhibitory concentration (MIC) in some cases. For Lichtheimia corymbifera grown in standard C/N medium, an accumulation of intracellular proteins with oxidative damage was positively correlated to the H2O2 concentration. This was not observed for other strains. The biggest changes in the biochemical composition of the fungal biomass were linked to changes in medium C/N ratios. This included different carbon allocation strategies among the tested species, such as accumulation of lipids and polyphosphates, lipids and saccharides, etc.

Our results suggest that the Mucoromycota strains used in this study are compatible with H2O2 feeding in lignocellulose-based SSF to enhance efficiency while sustaining minimal risk of oxidative damage.

The online version contains supplementary material available at 10.1186/s12934-025-02863-1.