Fig. 2.

SSF-based itaconic acid production using U. maydis Δcyp3 ΔP_ria1::P_etef Δfuz7 P_etef mttA with different cellulose substrates and sources of cellulases. a Shows the itaconic acid production during SSF using T. reesei (TR) enzymes (2.2 FPU/mL), b Shows the itaconic acid production during SSF using P. verruculosum (PV) enzymes (0.6 FPU/mL). c, d Show the corresponding pH profiles of the SSF cultures containing T. reesei or P. verruculosum enzymes, respectively. e Shows a comparison of the achieved itaconic acid production yields based on the consumed amount of glucose equivalents (1 g cellulose can yield 1.1 g glucose). Nitrogen-free itaconic acid production medium for U. maydis was supplemented with sterile filtered culture supernatants of T. reesei RUT-C30 (RFP1) or P. verruculosum M28-10. The residual NH₄⁺ concentration in the culture supernatant was determined and the supernatants were diluted accordingly (1/5), so that the NH₄⁺ transferred from the cellulase-containing supernatants is equivalent to the 0.8 g/L NH₄Cl which is usually added to the medium as nitrogen source. Both culture supernatants were combined with either 120 g/L Sigmacell, 120 g/L α-cellulose or 50 g/L of glucose as a reference. The medium was buffered to pH 6.5 using 100 mM MES buffer. The cultures were inoculated to a final OD₆₀₀ of 0.5 using a pre-culture of U. maydis Δcyp3 ΔP_ria1::P_etef Δfuz7 P_etefmttA with an OD₆₀₀ of 18.2. The culture was performed with 25 mL filling volume in 250 mL Erlemeyer flasks at 200 rpm, 50 mm shaking diameter and 30 °C. Values shown are means of biological duplicates, error bars show deviation from the mean. The 100 h time point only shows single measurments from one replicate. The dotted line indicates the additional feeding of 60 g/L α-cellulose