Physiological Characterisation of Yarrowia lipolytica Cultures Grown on Alternative Carbon Sources to Develop Microbial Platforms for Waste Cooking Oils Valorisation

With the economic development, the non-renewable resources consumption and the energy crisis, microbial-based platforms represent a promising alternative route for the production of valuable chemicals. Yarrowia lipolytica is a non-conventional oleaginous yeast, which is able to grow on hydrophobic substrates, such as triglycerides, as carbon source. Its growth is associated with the production of different high-value bio-products, such as enzymes, organic acids, and lipids. In recent years, both basic and applied research have been carried out for improving its genetic manipulation and industrial use. The thorough knowledge of Y. lipolytica biolipid conversion and production pathways makes it a good candidate for its use as a cell factory for the design of alternative bioprocesses based on renewable substrates. Waste cooking oils (WCOs) are vegetable oils and animal fats that are discarded after food processing. Globally, high amounts of this waste product are produced every year. Due to their composition in triglycerides, WCO can be used as feedstock for microbial growth to create novel routes for the so-called “industrial symbiosis” following the circular economy approach, to upgrade a waste as a renewable feedstock for the bio-based industry. In particular, the goal of this study was the creation of new value chains from waste cooking oil (WCO) for the sustainable production of added-value compounds to valorize this type of waste and to reduce its incorrect disposal. Specifically, Y. lipolytica was investigated to evaluate its ability to grow in the presence of different concentrations of WCO compared to glucose as carbon source and determine its response both in terms of industrially-relevant compounds production and cell robustness. Flow cytometry analysis was performed to investigate the response of Y. lipolytica at increasing WCO concentrations in terms of intracellular lipids quantification and cellular viability. growth inhibition above. The results showed that biomass production was improved to OD 600 0.7 in YP supplemented with WCO, showing that Y. lipolytica W29 more efficiently WCO than LA. The growth curve on WCO was comparable to the curve obtained in YP with 20 g/L glucose, while in YNB yeast growth with WCO was slightly lower. However, the Sunrise Tecan™ system showed a low performance due to interferences of WCO when it was used as substrate. This problem was probably caused by low lipid emulsification in the small volume of each well, despite TWEEN ® 80 was added in the nutrient media as surfactant. In order to optimize growth conditions on WCO in the Sunrise Tecan™ system, media dilutions, higher initial OD 600 and lower well volume were investigated. When Y. lipolytica W29 is inoculated at initial OD 600 0.15 in a final volume of 200 µL of tenfold diluted medium, the growth curves did not show any interference (Figure 2a). This better performance could mainly be ascribed to the dilution, which included both nutrient medium and substrate. The highest biomass in the rich medium reached a final OD 600 of 0.7, instead it was only 0.3 in YNB medium. Similar results were obtained when an initial OD 600 of 0.3 was used (data not shown). Due to higher growth in YP medium compared to YNB medium, YP medium was chosen for further experiments. The effect of different WCO concentrations (10, 20 and 40 g/L) were evaluated, using tenfold-diluted YP medium. As shown in Figure 2b, by increasing the initial WCO concentration from 10 to 40 g/L, the lag phase decreased from 5-6 h to 0 h, highlighting Y. lipolytica W29 ability to efficiently use higher amounts of WCO. To date, most research studies on transforming WCO into high-value products identified optimal initial concentration values for the substrate in the range from 10