Fungal Production of Fumaric Acid From Crude Glycerol: Different Approaches to the Process Including Intermediate Accumulation of Microalgal Biomass

Background: Interest in processing high-tonnage biodiesel production waste (crude glycerol and microalgal biomass after lipid extraction) into commercial products is steadily growing. Moreover, it is attractive to utilize these wastes for the microbiological production of fumaric acid (FA) using immobilized microorganisms, as it combines chemical and biological processes. Methods: The authors developed original approaches to FA production, which immobilize microalgal Chlorella vulgaris cells and the mycelium of the fungus Rhyzopus oryzae. Traditional techniques were employed for cultivating microorganisms, extracting lipids from accumulated microalgal biomass, and producing biomass hydrolysates through thermolysis under acidic conditions. The biochemical composition of C. vulgaris biomass obtained under different growth conditions (autotrophic, heterotrophic, and mixotrophic) was analyzed using standard methods. Results: The maximum accumulation of microalgal biomass was achieved under mixotrophic conditions using an inoculum immobilized in polyvinyl alcohol (PVA) cryogel in Tamiya medium supplemented with crude glycerol and bubbled with air containing 1% vol. CO2. A total of 4.8 ± 0.2 g DW/L of free C. vulgaris biomass was accumulated in five batch cycles. Two types of C. vulgaris hydrolysates were obtained: hydrolysates from whole cell C. vulgaris biomass (ChVH1) and hydrolysates from C. vulgaris biomass waste after lipid extraction (ChVH2), respectively. In combination with crude glycerol (10–50 g/L), microalgal hydrolysates were transformed to FA using fungi R. oryzae immobilized in PVA cryogel. The highest concentration and yield of FA from the converted substrate were obtained when 20 g/L of crude glycerol, in combination with C. vulgaris hydrolysates, was administered alongside the fungi for 40 h. Multiple uses (five cycles, 200 h total) of immobilized fungi added to the medium with 20 g/L of crude glycerol and ChVH2 produced 186 g of FA. The average FA yield was 309 ± 12 mg/g substrate. Conclusion: A combined approach to producing FA by bioconversion of crude glycerol and hydrolysates of lipid-free microalgae biomass waste is of practical importance. These results expand the potential of bioscience in utilizing microorganisms in an immobilized form, contributing to the integration of environmental biotechnology into bioenergy production.