Bioconversion of low-cost fat materials into high-value PUFA-Carotenoid-rich biomass

Due to an increasing focus on heathy diets and low-fat products, huge amounts of animal fat by-products are processed annually by fat processors and renderers. Most of the produced fat products are used for energy consumption. In order to add value to these low-value products, the LipoFungi project aims at upgrading Norwegian animal slaughter fat by-products to high-value fatty acids by fungal fermentation processes. Prior to the project, we showed in a pilot study that oleaginous fungi from the phylum Zygomycetes can convert saturated fatty acids from animal fat by-products containing triacylglycerols (SAT-TAGs), into polyunsaturated fatty acids containing triacylglycerols (PUFA-TAGs). The lion's share of the fat by-products that are produced in Norway and Europe today are either edible fats fitting for human consumption or so-called Category 3 fat that is suitable for animal feed. Therefore, depending on the type of fat by-product, high-value fatty acids produced from Category 3 fat rest materials or edible fat rest materials could be used for feed or for human consumption, respectively. The project addressed the most central biological and technological research challenges for developing a sustainable process for lipid upgrading from animal rest fat. It performed research for developing fermentation technology for submerged and solid-state fermentation, process optimization and upscaling. The project evaluated the final product and performed a preliminary economic evaluation of the process. The project developed fermentation media for a direct upgrading strategy for both submerged and solid-state fermentation and indirect upgrading by submerged fermentation. For the preparation of fermentation medium from animal fat for the direct fungal fermentation different types of emulsifiers were applied with Tween 80 showing the best emulsification. Different fat concentrations were tested for optimal fungal submerged fermentation and concentrations of 5% and 10% were found the most optimal. In addition to emulsion-based media, we tested media containing non-emulsified fat media. For the testing we used the oleaginous yeast Yarrowia lypolitica, which is able to release high amounts of lipases enzymes that degrade fat. We observed very good results on the fat degradation and utiliszation of fat by Yarrowia lypolitica. As an indirect upgrading strategy, several hydrolysis strategies were tested - acidic, base and enzymatic hydrolsysis. The best results with the highest glycerol yield were observed with enzymatic hydrolysis when commercial lipase enzyme was used. Acidic and base hydrolysis resulted in by-products that were inhibiting fungal growth. The project performed series of screening experiments for identifying the best fungal cell factories for bio-conversion of animal rest materials into high-value PUFA-carotenoid rich biomass. As a result, the following fungi have been suggested as the best converters of animal fat: (1) Mucor circinelloides for the production of biomass that is rich in GLA and carotenoid pigments; (2) Mortierella alpine for the production of biomass that is rich in GLA and ARA (3) red yeasts Rhodotorula, Sporobolomyces, Cystofilobasidium and Phaffia for the production of biomass that is rich in GLA and carotenoid pigments. With the aim to valorize animal waste fat by fungi during solid state fermentations (SSF), two fermentation strategies have been applied: SSF in plastic bags and SSF in rotary fermentation tank (30L). All fungal strains were able to grow on these substrates. Fungal hyphae not only covered the substrate surface but also penetrated into the substrate layer. The final fermented products were enriched with PUFA or pigments. The ability to utilize oil/fat material during solid state fermentation was also performed in a rotary fermentation tank. It is necessary to emphasize that conditions and regulation of SSF in a fermenter is different compared to plastic bag experiments. Therefore, we screened several substrates and optimized substrate/water ratios and substrate filling (amount) in the fermentation tank. The up-scale of the submerged fermentation was performed for fungi Mucor ciricinelloides and Mortierella alpine with 5% of fat in the media in 1,5L, 15L and 80L bioreactors at NORCE.

The main anticipated outcomes and impacts of the project are related to development of novel environmentally friendly Biorefinery processes for valorising low-value animal fat rest materials and producing high-value bioproducts enriched with essential oils and carotenoids. The project developed two alternative processes for the animal fat bioconversion: submerged fermentation and solid state fermentation by oleaginous yeast and filamentous fungi. The developed solid state fermentation processes utilize low amounts of water and increase thereby economic and environmental sustainability.

Due to an increasing focus on heathy diets and low-fat products, huge amounts of animal fat by-products are processed annually by fat processors and renderers. Most of the produced fat products are used for energy consumption. To add value to these products, the LipoFungi project aims at upgrading Norwegian animal slaughter fat by-products to high-value fatty acids by fungal fermentation processes. In a pilot study, we have shown that oleaginous fungi from the phylum Zygomycetes can convert saturated fatty acids from animal fat by-products containing triacylglycerols (SAT-TAGs), into polyunsaturated fatty acids containing triacylglycerols (PUFA-TAGs). Large parts of the produced fat by-products produced in Norway and Europe today are either edible fat, which fits for human consumption or so-called Category 3 fat, which is suitable for animal feed. Therefore, depending on the type of fat by-product, high-value fatty acids produced from Category 3 or edible fat rest materials can be used for either feed or for human consumption. The project addresses the most central biological and technological research challenges for developing a sustainable process for lipid upgrading from animal rest fat. It suggests research for developing fermentation technology for submerged and solid-state fermentation, process optimization and upscaling. The project will evaluate the final product and perform a preliminary economic evaluation of the process. The project will make use of the NordBioLab infrastructure facilities at NMBU and constitutes a multi-disciplinary and cross-institutional collaboration involving all competencies required for such a complex goal.