# Integrated Carbonate-Based CO\({}_{2}\) Capture--Biofixation through Cyanobacteria
## 3 Results  
Some preliminary growth tests were first conducted in the PBR (Figure 2) with both Spr01 and Spr02 strains to define the most significant operating variables and their ranges. Three independent parameters were chosen: culture temperature, light PPFD, and bicarbonate concentration. Biomass culture temperature is one of the most significant operating parameters [15] as variations can lead to significant shifts in metabolic pathways. Additionally, certain microalgae or cyanobacterium strains may have an optimal growth temperature [31]. The operating temperature is also linked to the energy input of the process. Light intensity strongly affects the growth and metabolism of photosynthetic organisms, including different Spirulina species [32; 33]. Increased light irradiation can result in enhanced growth rates [34]. Moreover, bicarbonate concentration is a crucial parameter, not as a growth enhancer, but because it is linked to the inorganic carbon density in the solution. Working at high bicarbonate concentration means having a lower chemofixation volume for CO\({}_{2}\), with substantial advantages in both PBR dimensions and the CO\({}_{2}\) adsorption column. These parameters were included in a multivariate statistical DoE evaluation using the following ranges: from 29 to 35 \({}^{\circ}\)C for cultivation temperature, from 260 to 390 \(\mathrm{\SIUnitSymbolMicro mol/s\cdot m^{2}}\) for light PPFD, and from 0.09 to 0.36 mol/L (7.6-30.2 g/L) for bicarbonate concentration. As suggested by the DoE full factorial model, eleven experiments for each strain were performed, and the obtained outputs, in terms of P (Equation (5)) and R (Equation (6)), are reported in Table 1.  
A significant productivity difference was observed between the two strains: while Spr01 reaches a productivity of 249 mg/L\(\cdot\)d at its best (n. 3, Table 1), Spr02 achieves the excellent value of 760 mg/L\(\cdot\)d (n. 5). Big differences between the two strains can also be observed in the corresponding daily growth rates (Figure 3). The lag, exponential, and stationary growth phases of Spr01 are difficult to define (Figure 3a), while Spr02 clearly shows a 36 h long lag phase, followed by a steeper exponential phase and then a short stationary phase (Figure 3b). OD680 values higher than 2.0 were never observed in the case of the Spr01 strain, while values exceeding 4.0 were observed in some instances of the Spr02 strain. Accurate pH measurements of the best experiment (n. 5, Table 1), verified by titration with HCl 0.1 M, allowed us to assess a pH shift from 8.1 (at the start) to 12.2 (at the end), corresponding to the complete conversion of bicarbonate (LCC+) to carbonate (LCC-, Figure 1). This also indicates that, after 72 h of growth, no more biofixable carbon is present for the Spr02 strain. Therefore, extending the growth experiments beyond 72 h was judged to be of little interest. From the statistical assessment of the correlation between (single or a combination of) independent parameters and the output variable, it came out that none of the studied parameters gave a significant correlation to P, in the case of the Spr01 strain (Figure S3a). On the other hand, the bicarbonate concentration and the interaction between bicarbonate concentration and temperature (AC, Figure S3b) give a significant correlation with productivity for the Spr02 strain. Temperature significance, when considered alone, was uncertain, i.e., showing a borderline correlation with P, according to the statistical model. Based on these data and considering the easier separation due to favorable morphology (Figure S1b), our attention from hereon was focused on the Spr02 strain only. The strong dependence of P on bicarbonate concentration suggests a favorable match between carbon capture on carbonate and biofixation on Spirulina. Moreover, the statistical non-correlation between light intensity and productivity suggests that light saturation was reached.