Exploring the potential of Chlorella sorokiniana as a source of vitamin B12

Details

Supervisors

Larondelle, Yvan

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

The food system emits 23-42% of the overall anthropogenic greenhouse gas emissions and provides powerful mitigation opportunities concerning the climate change. (IPCC, 2022). The present master’s thesis focuses on the intake of vitamin B12, also called cobalamin, which is reduced in vegetarian diets. The major sources of cobalamin are indeed meat and animal-derived products, which acquire vitamin B12 through symbiotic associations with bacteria in their digestive tract. The long-term purpose of the project in which the present master’s thesis contributes to establish a symbiotic relationship between a microalga, specifically Chlorella sorokiniana, and a bacterium producing bioactive cobalamin. The microalgae would supply the bacteria with fixed carbon in return of vitamin B12. As far as possible, the carbon dioxide needed for the growth of the microalgae would come from local breweries, wine industries and cider mills. In that framework, the first specific objective consisted in determining a liquid chromatography-mass spectrometry analytical procedure allowing to distinguish different molecular forms of cobalamin, namely cyanocobalamin and hydroxycobalamin. The second specific objective was to observe the commercially available Chlorella-based products, specifically their cell walls, which are indigestibles for mammals, to evaluate whether cobalamin could be made available. The third specific objective was to set up the cultivation of C. sorokiniana and to compare its growth with and without cobalamin. Finally, the fourth specific objective was to evaluate cell wall disruption methods through microscopy observations and measurements of cobalamin concentration in the cells and in the media, through an enzyme-linked immunosorbent assay (ELISA). Concerning the injection of cyanocobalamin and hydroxycobalamin in a mass spectrometer, the parameters applied on the device led to distinguished clear spikes. Regarding the evaluation of market products, most cells were spherically shaped, while some presented unsculptured outer wall. In regard to the implementation of C. sorokiniana cultures, cell growth was not significantly different with and without cobalamin supplementation. The use of the ELISA kit was successful since it detected significant cobalamin concentrations in supplemented samples, and low concentrations in non-supplemented cells. Except for some unidentified fragments, freezing at -20°C, autoclaving and freeze-drying of C. sorokiniana did not have any disrupting effect and lead to intact globular cells. Freezing at -80°C led to many empty shells around intact cells. Grinding the cells after -20°C freezing did not have any significant effect, while grinding after -80°C showed destructed cell walls. Cobalamin concentrations increased in the positive control medium, which indicated a possible bacterial contamination. Future research is needed to investigate unanswered questions. First, bead-milling after liquid nitrogen freezing, enzymes, high pressure processing and microwaves are disruption methods to be explored. Secondly, further research on pseudo-cobalamin is warranted to ensure it does not end in the final product. Once these steps accomplished, co-culture of Chlorella sorokiniana and the bacterium can be tested to observe the growth of both organisms together.