By 2030, the EU aims to increase the share of renewable energy in transport to at least 14%, including a minimum share of 3.5% of advanced biofuels (1). It is anticipated that advanced biofuels, mainly 2^nd generation biofuels, will boost EU economy by creating 1.2 million jobs and a revenue of 400 billion euros by 2030. As such, feedstock costs need to remain competitive, and sustainability criteria need to be met.

Enzymes are one of the biggest cost in producing fermentable sugars, accounting up to 20% of the cost for traditional 2^nd generation bioethanol production (2). Enzyme immobilization could reduce significantly the use of cellulase to achieve a total cost reduction of 10% (3). When applying immobilized enzymes within heterogeneous processes where the substrate is initially insoluble, the efficient enzyme separation and recovery is crucial for enzyme reuse. Enzyme immobilization offers many advantages over enzymes in solution, including economic convenience, higher stability, and the possibility to be easily removed from the reaction mixture leading to pure product isolation.

Aim of this work is the preparation, characterization and application of Cross Linked Enzyme Aggregates (CLEAs) based on commercial multi-enzymatic bends with cellulase activity, Cellic® Ctec2 and Cellic® CTec3 HS (Novozymes A/S, Bagsværd, Denmark). CLEA preparation includes two distinct steps: a precipitation step, where the enzyme is aggregated with the addition of a saturated salt solution or organic solvent and a crosslinking step, where the aggregates are stabilized by a crosslinker e.g. glutaraldehyde.

Each step was optimized investigating the effect of different parameters on the recovery of cellulase activity (filter paper activity, endoglucanase activity, exoglucanase activity and β-glucosidase activity). The parameters investigated in the precipitation step were the type of precipitant, concentration of precipitant and time of precipitation, while in the cross-linking step the parameters investigated were the glutaraldehyde concentration, enzyme load, time and temperature for crosslinking. Moreover, the magnetic CLEA (m-CLEAs) formation using magnetic nanoparticles (4) was investigated. The prepared CLEAs and m-CLEAs were characterized in terms of optimal temperature performance, temperature stability and were compared to the free enzyme. Then, they were applied in the hydrolysis of two renewable lignocellulosic feedstocks, a cellulose fraction obtained from organosolv pretreated beechwood and an organic fraction of municipal waste. The substrate concentration, enzyme load and hydrolysis time were optimized. The formed CLEA and m-CLEAs showed good performance when compared to the free enzyme and their reusability potential was investigated for >5 cycles.