During this master’s thesis work an enzymatic process for the industrial production of 2,5-diformylfuran (also known as DFF) was developed at Bio Base Europe Pilot Plant in the port of Ghent, Belgium, in the frame of the SMARTBOX project. The project aims to create value added chemicals from lignocellulosic waste. Therefore, the developed process contributes to the sustainability of the biorefineries while decreases the need for fossil-based chemicals thus actively combats today’s environmental crisis. DFF can serve as a building block for various materials such as bioplastics, antifungal agents, Schiff-base polymers, resins, fluorescens agents and macrocyclic ligands. The upstream process was developed via 26 individual bioconversion runs utilizing a 4x2L parallel bioreactor system. Parameters were optimized through design of experiments approach. An effective product purification method was developed as well, with response surface methodology as a key tool for optimization.