*2.3. Hydrolysate Sterilization*

Three di fferent methods (membrane filtration, autoclave at 112 ◦C for 15 min, and autoclave at 121 ◦C for 20 min) were tested for sterilization of the hydrolysate prior its use as fermentation medium. The autoclave assays were carried out in an autoclave MultiControl 2 (CertoClav, Austria); while for the membrane method, Nalgene RapidFlow ™ PES-membrane filters with a pore size of 0.2 μm (Thermo Fisher Scientific, USA) were used.

### *2.4. Fermentation Media and Conditions*

Initially, di fferent synthetic media were tested for the production of itaconic acid by *A. terreus,* which contained only one type of carbon source (glucose, xylose, sucrose, mannose, or glycerol) at a concentration of 50 g/L. Later, the cellulose pulp hydrolysate was used as fermentation medium, which contained around 53 g/<sup>L</sup> of glucose as carbon source. For all the experiments, the initial pH of the media was adjusted to 3.0.

All the fermentation media, synthetic and hydrolysate, were supplemented with the following nutrients (in g/L): KH2PO4 (0.2), (NH4)2SO4 (3.0), MgSO4·7H2O (3.0), CaCl2·1H2O (0.2), ZnSO4·7H2O (0.15), FeSO4·7H2O (0.16), and CuSO4·5H2O (0.015). To assess the e ffect of nitrogen concentration on itaconic acid production from cellulosic hydrolysate, the following three di fferent concentrations of (NH4)2SO4 were evaluated: 1, 3, and 5 g/L. For comparison, hydrolysate without any nutrient supplementation was also used as fermentation medium.

Fermentation experiments were carried out in 250-mL Erlenmeyer flasks at 35 ◦C and 200 rpm for 3 to 5 days (72 h to 120 h). A working volume of 50 mL was used in the experiments with pure carbon sources. Experiments performed to assess the e ffect of aeration on itaconic acid production from cellulosic hydrolysate were carried out with di fferent working volumes varying from 20 to 50 mL in order to result in di fferent air-to-liquid ratios (Vair/V m) as shown in Table 1. All other fermentations from cellulosic hydrolysate medium were performed using a working volume of 30 mL. All experiments were carried out in duplicate.


**Table 1.** Di fferent air-to-liquid ratios used for the fermentation experiments.

Vf, volume of the flask; Vm, volume of medium; Vair, volume of air.

### *2.5. Analytical Methods and Statistical Analysis*

Nitrogen content in the hydrolysates was determined by using an elemental analyzer Vario MACRO cube (Elementar Analysensysteme GmbH, Germany), following the Dumas method.

Cell mass concentration during the fermentations was estimated by dry weight measurement. The fermentation broth was centrifuged at 10,000 rpm for 10 min and the biomass pellet was rinsed two times with deionized water and dried at 60 ◦C for 48 h. The supernatant of centrifuged samples was used for pH measurement and determination of sugars, itaconic acid, and potential by-products.

The concentrations of glucose, cellobiose, xylose, sucrose, mannose, glycerol, organic acids (itaconic, acetic and formic), 5-hydroxymethylfurfural (5-HMF) and furfural were determined by high-performance liquid chromatography (HPLC) using a Dionex Ultimate 3000 HPLC equipment (Thermo Scientific, USA) coupled with a Biorad Aminex ® HPX-87H column (300×7.8 mm). For analysis, the column was maintained at 65 ◦C and a 5 mM H2SO4 solution was used as mobile phase at a flow rate of 0.5 mL/min. Sugars, glycerol, and organic acids were detected using a Shodex RI-101 refractive index detector, whereas 5-HMF and furfural were detected using an ultraviolet detector at 254 nm.

Statistical analysis including graphs and quantitative information such as mean and standard deviation was performed using the software OriginPro 9.1.0 (OriginLab Corporation, USA).
