*2.1. Microorganism and Inoculum Preparation*

The filamentous fungus *Aspergillus terreus* NRRL 1960 was used in the experiments. The strain was obtained from the ARS Culture Collection (Peoria, IL, USA) and preserved in the form of spores in 20% (*v*/*v*) glycerol stock solution at −80 ◦C.

For inoculum preparation, the stock culture was activated on 2.4% ( *w*/*v*) potato dextrose broth (PDB) medium at 35 ◦C for 3 days, and subsequently on 3.9% ( *w*/*v*) potato dextrose agar (PDA) plates at 35 ◦C for 7 days. Then, spores were collected from plates by using a sterilized solution of 4% ( *w*/*v*) Tween 80. The spore suspension was diluted with sterile MilliQ water in order to obtain a concentration of 10<sup>6</sup> spores/mL at the beginning of the fermentation.

### *2.2. Cellulose Pulp Characterization and Hydrolysis*

Bleached cellulose pulp from Suzano S/A (Brazil) was used as raw material for the production of itaconic acid. The cellulosic material, which was produced from Eucalyptus wood and had a moisture content of approximately 5% (w/w), was ground to particle size ≤ 1.0 mm by means of a mill Polymix PX-MFC 90D (Kinematica, Switzerland) and its composition was determined by following standard methods [14–16].

Enzymatic hydrolysis of the cellulose pulp was carried out using the enzyme concentrate Cellic ® CTec2, kindly supplied by Novozymes (Bagsværd, Denmark). The cellulase activity of the concentrate, which was measured according to standard protocol [17] and expressed in filter paper units (FPU), was 217.5 FPU/mL. One unit of FPU was defined as the amount of enzyme required to liberate 1 μmol of glucose from Whatman no.1 filter paper per minute at 50 ◦C.

For the experiments, an enzyme load of 10 FPU/g cellulose was added to 0.1 M sodium citrate bu ffer (pH 4.7), and then mixed with the cellulose pulp in a concentration of 12% ( *w*/*v*). The reactions were carried out in 2-L Duran laboratory bottles with vertical ba ffles containing 0.6 L of working volume. The bottles were accommodated horizontally in a Bottle/Tube Roller system (Thermo Scientific, USA) placed inside an incubator, and kept at 50 ◦C and 20 rpm for 96 h. Afterwards, the hydrolysate was separated by centrifugation (10,000 rpm, 5 ◦C, 20 min).
