Microbial conversion of biodiesel waste for carotenoid production

Enhanced production of isoprenoid compounds, including carotenoids, is needed to meet the growing demand in the food, cosmetic, pharmaceutical, and biotechnology sectors. Haloferax volcanii represents a promising microbial platform for sustainable isoprenoid production, as this halophilic archaeon is well suited for metabolic engineering, thrives under harsh conditions (e.g., UV irradiation, high temperatures, and metal-induced stress) compatible with bioprocessing, and naturally synthesizes carotenoids including the high-value C₅₀ bacterioruberin.

In this study, we optimized carotenoid yield in H. volcanii using a chemically defined medium supplemented with cost-effective, industrially favorable feedstocks of crude glycerin and urea as the sole carbon and nitrogen sources, respectively. Following optimization by reuse of medium and supplementation with additional glycerin, carotenoid production was evaluated. Transcript abundance of the carotenoid 3,4-desaturase gene (crtD, HVO_2528) was measured, and β-galactosidase (bgaH) reporter activity was used to assess crtD promoter activity.

H. volcanii was found to display comparable growth rates on crude glycerin to laboratory grade glycerol. Following optimization by reuse of medium and supplementation with additional glycerin, an 8-fold increase in carotenoid yield was observed when urea (84.1 ± 8.4 mg⋅L^-1) served as the nitrogen source compared to cultures only grown with crude glycerin and NH₄Cl (10.0 ± 2.4 mg⋅L^-1) when normalized. The higher carotenoid yield on urea vs. NH₄Cl was found to be correlated with a 3- to 4-fold increase in transcript abundance of the carotenoid 3,4-desaturase gene (crtD, HVO_2528) that was regulated at the level of transcription. The crtD promoter was therefore identified as a strong candidate based on β-galactosidase (bgaH) reporter activity for use in metabolic engineering.

Urea offers higher nitrogen content, reduced acidification potential, and greater scalability than NH₄Cl for bioprocessing applications. Together, our findings support the development of a sustainable, circular approach for repurposing industrial glycerin waste streams to support carotenoid production using urea as a nitrogen source and H. volcanii as a microbial biocatalyst for renewable biomanufacturing.