*3.1. Carbon Source Assessment*

With the aim of identifying potential raw materials for the production of itaconic acid, initial experiments were carried out to evaluate the performance of *A. terreus* when cultivated in di fferent carbon sources. As can be seen in Table 2, hexoses, especially glucose, were the preferred carbon sources used by the strain to produce itaconic acid. Glucose has also been reported as being the preferred carbon source for other filamentous fungi, and this could be explained by the fact that this hexose enables the most direct conversion pathway, eliminating the need for additional biochemical steps [18]. In the present study, the production of itaconic acid from other carbon sources including xylose, sucrose, mannose, and glycerol were clearly lower that that observed from glucose (Table 2). The relevance of glucose as compared with the other carbon sources is also highlighted by the values of itaconic acid yield per substrate consumed, which was of 0.61 g/g of glucose consumed, more than three times higher than that observed for mannose, which was the second best carbon source evaluated in our study.


**Table 2.** Itaconic acid concentration and yield and biomass yield for the fermentations with *A. terreus* using di fferent carbon sources.

> *YP*/*S*, itaconic acid yield per sugar consumed; *YX*/*S*, biomass yield per sugar consumed.

It is also interesting to note that the strain presented a very good ability to consume all the carbon sources, including C3 (glycerol), C5 (xylose), and C12 (sucrose), being a potential candidate for use in bioprocesses. However, unlike glucose, the other carbon sources were mainly used for biomass growth instead of itaconic acid formation, which is evidenced by the higher values of biomass yield ( *YX*/*S*) obtained (Table 2). The biomass formation in glucose was the lowest as compared with the other carbon sources, which was due to the main use of this carbon source for product formation. These results play a crucial role in selecting novel lignocellulosic biomass sources for use on the production of itaconic acid, opening up good possibilities for integrating the production of this acid in a biorefinery.

### *3.2. Cellulose Pulp as Feedstock for Itaconic Acid Production*

Since glucose was the best carbon source for itaconic acid production by *A. terreus*, a cellulose-rich material can be considered to be the ideal candidate for use in this bioprocess as an alternative to replace pure glucose as the carbon source. During recent years, studies have reported the use of cellulose pulp as a substrate for the production of di fferent compounds by fermentation, including ethanol and hydrogen [19,20]. However, there are no studies reporting the use of bleached cellulose pulp for the production of itaconic acid. This study confirms that bleached cellulose pulp can be a feasible feedstock for use in the production of itaconic acid since this material is produced in high amounts in the pulp and paper industries and has attracted grea<sup>t</sup> interest for use in the production of valuable compounds (rather than for use in the production of paper only). In addition to its grea<sup>t</sup> availability, other important advantages of using bleached cellulose pulp for itaconic acid production include its high content of cellulose (which can be converted into glucose) and the possibility of applying an enzymatic hydrolysis directly, without previous pretreatment, saving time and energy, which can positively impact on the final costs of the itaconic acid production process, making it more economical.

The cellulose pulp used in this study was produced from Eucalyptus wood. The chemical composition of this material was cellulose 89.7% and hemicellulose 10.3% (dry weight). Lignin was present in trace amount. Enzymatic hydrolysis of this material under the conditions used in this study yielded a hydrolysate containing 72.3 g/<sup>L</sup> of glucose and 14.8 g/<sup>L</sup> of xylose, representing a rich carbon source for use as fermentation medium by *A. terreus*. Fermentation results from this medium are discussed in the next sections.
