Simultaneous Saccharification and Fermentation with Ustilago maydis and Analytical Methods for Cell and Product Quantification.

Ustilago maydis is an emerging microbial host for the sustainable production of itaconic acid, an important platform chemical. Its robustness toward impurities and compatibility with renewable resources make it an attractive alternative to the industrial producer Aspergillus terreus. Here, a detailed protocol for converting brewer's spent grains (BSG), an exemplary lignocellulosic feedstock, into itaconic acid using U. maydis is presented. The workflow describes an extended valorization route, integrating pretreatment, fermentation, and analysis. The solid fraction of BSG undergoes organosolv pretreatment to enhance enzymatic accessibility, while the liquid fraction from pretreatment is recovered and detoxified for potential fermentation use. Nitrogen availability is assessed to account for its key role in triggering secondary metabolism. Simultaneous saccharification and fermentation (SSF) is then applied, enabling continuous sugar release and conversion into itaconic acid. The protocol further details analytical methods, including HPLC for substrates, products, and inhibitors, as well as fluorescence microscopy-based cell counting for biomass monitoring.