*Article* **Kinetic and Stoichiometric Modeling-Based Analysis of Docosahexaenoic Acid (DHA) Production Potential by** *Crypthecodinium cohnii* **from Glycerol, Glucose and Ethanol**

**Kristaps Berzins 1, Reinis Muiznieks 1, Matiss R. Baumanis 1, Inese Strazdina 1, Karlis Shvirksts 1, Santa Prikule 1, Vytautas Galvanauskas 2,3, Daniel Pleissner 4,5, Agris Pentjuss 1, Mara Grube 1, Uldis Kalnenieks <sup>1</sup> and Egils Stalidzans 1,2,\***


**Abstract:** Docosahexaenoic acid (DHA) is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), with numerous health benefits. *Crypthecodinium cohnii*, a marine heterotrophic dinoflagellate, is successfully used for the industrial production of DHA because it can accumulate DHA at high concentrations within the cells. Glycerol is an interesting renewable substrate for DHA production since it is a by-product of biodiesel production and other industries, and is globally generated in large quantities. The DHA production potential from glycerol, ethanol and glucose is compared by combining fermentation experiments with the pathway-scale kinetic modeling and constraint-based stoichiometric modeling of *C. cohnii* metabolism. Glycerol has the slowest biomass growth rate among the tested substrates. This is partially compensated by the highest PUFAs fraction, where DHA is dominant. Mathematical modeling reveals that glycerol has the best experimentally observed carbon transformation rate into biomass, reaching the closest values to the theoretical upper limit. In addition to our observations, the published experimental evidence indicates that crude glycerol is readily consumed by *C. cohnii,* making glycerol an attractive substrate for DHA production.

**Keywords:** Krebs cycle; central metabolism; kinetic model; constraint-based model; FTIR spectroscopy
