*2.4. Model-Based Determination of DHA Production Potential*

The effectivity of carbon conversion into biomass can be analyzed in several ways. We looked at the biomass production rate and the efficiency of substrate carbon transformation into biomass (Table 3). The experimentally observed biomass production rate μ is the highest in the case of glucose and the lowest in the case of glycerol. However, glycerol shows the highest efficiency of substrate transformation into biomass (57.4 mmoLC1·gDW−1), while glucose is the least efficient (76.5 mmoL mmoLC1·gDW−1). The optimization of the stoichiometric model, without taking into account the fluxes simulated by the kinetic model, reveals that any substrate of interest can be transformed into biomass with a ratio of about 42 mmoLC1·gDW<sup>−</sup>1. This means that the experimentally observed transformation rate of glycerol is the closest to the theoretical value using 35% more carbon than predicted by the model in an optimal case. In the case of glucose and ethanol, that is 80% and 45%, respectively.

The DHA production potential was determined by the stoichiometric model without taking into account the kinetic model fluxes for different biomass production intensities (Figure 6). The calculations were carried out by the stoichiometric model at experimentally observed substrate uptake rates of glucose, glycerol and ethanol. The maximal specific growth rate (μmax) was determined by maximizing biomass function, assuming that all substrates will be targeted at biomass production with DHA as a part of the biomass. Knowing that the DHA fraction in the experimentally produced biomass was variable (Figure 3), we introduced a DHA production reaction to simulate DHA overproduction, which increases in cases when 80% or 40% of the maximal biomass produced. Taking into account equal substrate transformation ratios into biomass, calculated numbers are equal for all substrates.

The stoichiometric model simulations indicate that DHA production potential increases when biomass production decreases. In the case of the maximal biomass production, the percentage of substrate carbon that forms DHA grows from 27% at the maximal biomass production rate up to 70% in the case of 40% of maximal biomass production rate. The percentage of DHA in total fatty acids (TFA) increases from 39% to 81%, respectively. These calculations are based on the assumption that all metabolic resources that do not form biomass are directed by the available metabolic reactions towards the production of DHA.


**Table 3.** The efficiency of substrate transformation into biomass for experimentally observed and optimized data.

**Figure 6.** Estimation of DHA production potential by the constraint-based stoichiometric model at different biomass production intensities: 100% (=μmax), 80% and 40%.
