**Production of Itaconic Acid from Cellulose Pulp: Feedstock Feasibility and Process Strategies for an E** ffi**cient Microbial Performance**

### **Abraham A. J. Kerssemakers, Pablo Doménech, Marco Cassano, Celina K. Yamakawa, Giuliano Dragone and Solange I. Mussatto \***

Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, Building 220, 2800 Kongens Lyngby, Denmark; aajker@biosustain.dtu.dk (A.A.J.K.); pablo.domenech@ciemat.es (P.D.); marcocassano08@gmail.com (M.C.); celinayamakawa@biosustain.dtu.dk (C.K.Y.); giudra@biosustain.dtu.dk (G.D.) **\***Correspondence:smussatto@biosustain.dtu.dkorsolangemussatto@hotmail.com;Tel.:+45-93-511-891

Received: 27 February 2020; Accepted: 31 March 2020; Published: 2 April 2020

**Abstract:** This study assessed the feasibility of using bleached cellulose pulp from Eucalyptus wood as a feedstock for the production of itaconic acid by fermentation. Additionally, di fferent process strategies were tested with the aim of selecting suitable conditions for an e fficient production of itaconic acid by the fungus *Aspergillus terreus*. The feasibility of using cellulose pulp was demonstrated through assays that revealed the preference of the strain in using glucose as carbon source instead of xylose, mannose, sucrose or glycerol. Additionally, the cellulose pulp was easily digested by enzymes without requiring a previous step of pretreatment, producing a glucose-rich hydrolysate with a very low level of inhibitor compounds, suitable for use as a fermentation medium. Fermentation assays revealed that the technique used for sterilization of the hydrolysate (membrane filtration or autoclaving) had an important e ffect in its composition, especially on the nitrogen content, consequently a ffecting the fermentation performance. The carbon-to-nitrogen ratio (C:N ratio), initial glucose concentration and oxygen availability, were also important variables a ffecting the performance of the strain to produce itaconic acid from cellulose pulp hydrolysate. By selecting appropriate process conditions (sterilization by membrane filtration, medium supplementation with 3 g/<sup>L</sup> (NH4)2SO4, 60 g/<sup>L</sup> of initial glucose concentration, and oxygen availability of 7.33 (volume of air/volume of medium)), the production of itaconic acid was maximized resulting in a yield of 0.62 g/g glucose consumed, and productivity of 0.52 g/L·h.

**Keywords:** lignocellulosic biomass; cellulose pulp; hydrolysis; oxygen availability; C:N ratio; fermentation; biorefinery; itaconic acid; *Aspergillus terreus*
