Fungal biorefineries are important players in the emerging global bioeconomy and contribute to the transition from the traditional fossil-based production to a renewable, sustainable and environment-friendly bio-production. In such biorefineries, fermentation utilizing fungi as cell factories is a central process. Development of sustainable fungal biorefineries involves optimization of fungal fermentation for efficient feedstock utilization and high product yields. Since lignocellulosic biomass is sustainable and of high abundance, lignocellulose hydrolysates are considered as key carbon sources for large scale fungal fermentation. Mucoromycota filamentous fungi are powerful cell factories able to valorize hydrolyzed lignocellulose materials into a range of marketable products, such as lipids, biopolymers, pigments, proteins, enzymes and organic acids. Currently, the use of Mucoromycota for industrial production of fungal lipids for food, feed and biofuels applications is not economically viable. Therefore, a co-production concept has been proposed where several valuable bio-products can be produced in a single fungal fermentation process. This PhD work has focused on optimizing co-production of several metabolites in oleaginous Mucoromycota fungi by manipulation of growth media composition, with the ultimate goal of improving the economic sustainability of fungal biorefineries. The following media parameters were manipulated-type of nitrogen source, concentration of phosphorus substrate, and concentration of different metal ions. The optimization was performed using a high-throughput micro-cultivation system (Duetz- microtiter plate system) combined with different analytical techniques including vibrational spectroscopy. Total fungal lipid content was estimated either by gravimetry or gas chromatography (GC), while lipid profile was characterized by GC and nuclear magnetic resonance spectroscopy (NMR). NMR was also used for the characterization of phosphates in Mucoromycota biomass, in addition to estimation of total phosphorus by assay-based UV-visible (UV-VIS) spectroscopy. Fourier transform infrared spectroscopy (FTIR) and Fourier transform Raman spectroscopy (FT-Raman) were utilized for monitoring fungal fermentation in Duetz-MTPS and for biochemical fingerprinting of fungal biomass in order to measure the co-production of intracellular metabolites. The influence of two nitrogen sources (yeast extract and ammonium sulphate) and different amounts of phosphate substrate on the co-production of lipids, chitin/chitosan and polyphosphate, and on the lipid accumulation, in nine oleaginous Mucoromycota fungi was studied in Paper I and Paper II, respectively. To verify co-production, high-throughput FTIR spectroscopy was used as a main analytical method in Paper I. In Paper II, gas chromatography was used for analyzing the fatty acid profile and total lipid content was estimated gravimetrically. Strains with co-production potential and media components affecting the co-production and lipid accumulation were identified. In Paper III, the role of the metal ions calcium, copper, cobalt, iron, magnesium, manganese and zinc for growth of Mucor circinelloides was assessed. This strain was used since it is one of the most promising strains for the co-production of lipids, chitin/chitosan and polyphosphate. It was observed that calcium ions have a significant effect on the lipid accumulation in Mucor circinelloides. In order to investigate whether the effect of calcium ions is generally valid for other oleaginous Mucoromycota fungi, a study where six Mucoromycota fungi were grown in the presence and absence of calcium ions was performed in …