This chapter provides a comprehensive review on the development of A. gossypii bioprocesses for the biosynthesis of metabolites of biotechnological interest. The first subsection focuses on bioprocess development, mostly focusing on riboflavin production, the main staple in Ashbya biotechnology. It covers the evolution of A. gossypii bioprocesses in what regards to the establishment and impact of nutritional requirements and media formulation, definition of process parameters (pH, temperature, inoculum), process configuration (e.g. batch and fed-batch), scaling up and downstream processing. The second subsection focuses on the development of biosustainable strategies mainly translated by the usage of low-cost, renewable and sustainable feedstocks, such as agro-industrial by-products and wastes. In this section, alternative nitrogen and carbon sources are explored, with particular emphasis on emerging substrates such as oil wastes, lignocellulosic residues and crude glycerol and their application to produce riboflavin as well as emergent value-added metabolites such as microbial oils, terpenes and orotic acid.