Developing microalga Nannochloropsis oceanica as a future source of protein, fatty acids, and vitamins

In years to come, the global demand for food, especially protein-rich foods, will increase as the world population climbs towards an estimated 10 billion in 2050. To be compatible with the planetary boundaries, future food production must be increasingly plant-based. However, some essential nutrients such as long-chain omega-3 fatty acids and vitamin D are scarce in plants. In addition, the global food system faces a lack of diversity, relying on only a handful of plant and animal species for most of our food supply. This calls for a need to explore and develop new, non-animal sources of protein, fatty acids, and vitamins. One alternative could be microalgae, such as the marine species Nannochloropsis oceanica. It is rich in protein and essential amino acids and can produce both long-chain omega-3 fatty acids and vitamin D. Production of N. oceanica could take place on non-arable land using salt water and would therefore not compete with traditional agriculture for fertile soil and freshwater resources. However, several technical challenges limit its potential. One crucial challenge is production cost, which is strongly linked to the efficiency of biomass production. Another major challenge is the low biomass digestibility. To fully unlock the potential of N. oceanica in future food production, these challenges must be addressed.

Considering these challenges, the MASSPROVIT research project was established, with the overall objective of facilitating Danish microalgae production as a new source of non-animal protein, omega-3 fatty acids and vitamins, focusing on Nannochloropsis oceanica. The project aimed at contributing to research that could improve biomass productivity and digestibility. Within the framework of the MASSPROVIT project, this PhD study aimed to explore the use of industrial side streams as a cost-friendly source of nutrients. Furthermore, the study aimed to investigate the use of cultivation conditions for optimizing growth and nutritional composition. In line with this, another objective of the study was to improve the production of vitamin D in N. oceanica through cultivation optimization and exploration of new UVB treatments. The study also aimed at investigating the use of cell disruption to increase the nutrient bioaccessibility of N. oceanica biomass. Finally, the study tested the above concepts on selected mutant strains, looking for potential improvements compared to wild type N. oceanica. These mutant strains were generated as part of a related MASSPROVIT PhD study with the aim of generating strains with improved traits, such as enhanced growth or improved biomass composition. To investigate all these aspects, several controlled cultivation experiments and biomass processing experiments were conducted, resulting in four separate studies and associated manuscripts, including additional results related to the mutant strains.

The cultivation studies carried out in the first study showed that side streams from industrial enzyme production could replace between 20-40% of a commercial nutrient substrate without negatively affecting growth and the content of limiting amino acids and unsaturated fatty acids. The cultivation studies of the second study showed that light intensity, temperature, and salinity, could be used to manipulate both biomass productivity and the level of protein, EPA, and MK-4. Using response surface methodology, biomass productivity and protein production was found to peak at 27 °C and 300–350 μmol·m⁻²·s⁻¹. In contrast, the production of both EPA and MK-4 were stimulated by the lower tested temperature of 19 °C. The experiments performed in the third study revealed that the cultivation temperature was also highly influential on the production of vitamin D3. When cultivated at 19 °C, vitamin D3 production was 2.7-fold higher than in biomass produced at 27 °C. This study further revealed that a short, post-cultivation treatment with UVB could successfully produce vitamin D3, achieving up to 0.260 μg D3·g^-1 DM. Finally, the processing experiments of the fourth study showed that bead milling could efficiently disrupt N. oceanica biomass, without compromising the concentration of amino acids, fatty acids, and vitamin K in the processed biomass. However, the bead milling treatment did not significantly increase the bioaccessibility of EPA and MK4, which remained in the range of 8-12%, similar to the non-disrupted biomass. The additional experiments performed on mutant strains, M34 and M181, revealed no major differences regarding growth performance and nutrient composition compared to wild type N. oceanica. The mutants also responded similarly to cell disruption by bead milling. However, exposure to post-cultivation UVB indicated a higher production of vitamin D3 in one mutant strain.

This PhD study has provided valuable results and information for the further development of N. oceanica as a potential source of protein, omega-3 fatty acids, and vitamins. The PhD study revealed that side streams from industrial enzyme production could serve as an alternative source of nutrients, and that abiotic cultivation factors, especially light intensity and temperature, could be used to optimize both biomass production and composition. The study further revealed that cell disruption, in this case bead milling, does not always guarantee improved bioaccessibility of intracellular nutrients. Furthermore, it documented that vitamin D3 could be efficiently produced post-cultivation. The study also investigated these concepts on mutant strains but found no major improvements compared to wild type N. oceanica, with exception of a potentially higher vitamin D3 production. In summary, the overall objective of MASSPROVIT in contributing to Danish microalgae production as a new source of non-animal protein, omega-3 fatty acids, and vitamins has been accomplished.

It is uncertain whether the use of food grade industrial side streams is a viable concept for production of N. oceanica. As the side streams did not improve the production/composition of the biomass, it is questionable whether the potential cost reductions are worth the inherent variability of the concept. It is also questionable whether one-stage cultivation is ideal for N. oceanica as there was a clear trade-off between biomass productivity and the accumulation of EPA and MK-4. Instead, two-stage cultivation with a stress-induction phase might be more efficient for production of these valuable lipids. The post-cultivation production of vitamin D3 showed great potential, and efforts should be aimed at developing more specialized UV exposure technology for production on a larger scale. Cell disruption should be combined with an oil extraction step to ensure proper bioaccessibility of the valuable lipids. This would also generate two fractions with a more specialized function. Finally, more effort should be put into generating mutant strains using more targeted screening methods. This could help fully unluck the genetic potential of Nannochloropsis oceanica.