Fig. 3

Experimental setup for comparison between CBP and SSF. T. reesei RUT-C30 (RFP1) cultures were grown for 1 week in co-culture medium buffered with 33 g/L CaCO₃ to produce cellulases (shaded yellow). Thereafter, the cultures were pooled and phosphate and ammonium content of the culture was measured. Residual NH₄⁺ was equivalent to 1.2 g/L NH₄Cl and was not necessary to supplement before the inoculation of U. maydis since the NH₄Cl concentration in itaconic acid production medium is only 0.8 g/L. Residual KH₂PO₄ was 0.18 g/L and was supplemented to a final concentration of 0.5 g/L to prevent preliminary phosphate limitation. For the SSF experiment, half of the pooled T. reesei culture broth was sterile filtrated and the filtrate distributed into three replicate Erlenmeyer flasks (25 mL each). For the CBP experiment, the pooled T. reesei culture broth was directly distributed to three replicate Erlenmeyer flasks (25 mL each). All cultures were supplemented with 120 g/L α-cellulose and 33 g/L CaCO₃ and finally inoculated to OD 0.7 with U. maydis Δcyp3 ΔP_ria1::P_etef Δfuz7 P_etef mttA. While the SSF culture simutanously hydrolyses cellulose (shaded green) and produces itaconic acid (shaded blue) using the enzymes from the T. reesei pre-culture, the CBP culture is able to continue cellulase enzyme production in addition due to the presence of living T. reesei cells