# Integrated Carbonate-Based CO\({}_{2}\) Capture--Biofixation through Cyanobacteria
## 4 Discussion  
Based on the previous considerations, further biofixation experiments with constant light PPFD (260 \(\upmu\)mol/s\(\cdot\)m\({}^{2}\)) and higher bicarbonate concentrations (0.45 M and 0.56 M) were set up. Unfortunately, these tests revealed a decreased productivity (n. 1-4, Table 2). This decline can be attributed to abiotic stresses due to high ionic strength, a known growth inhibitor for most microalgal strains [36]. The tolerance of high salinity appears to be enhanced at higher temperatures (n. 4, 5 vs. 1, 2), a behavior also observed for Spirulina platensis [37]. Anyway, productivity was never higher than that obtained with 0.36 M aqueous bicarbonate (n. 5 of Table 1, reported as n. 3 in Table 2 for better comparison). Not much more was obtained at the concentration of 0.45 mol/L when part of the NaHCO\({}_{3}\) was changed with the respective potassium salt (n. 6, 7 Table 2). This suggests that the limitations in strain proliferation depend on the overall ionic strength rather than a cation-specific one [38]. In order to favor strain adaptation and possibly reduce the initial lag phase observed for Spr02 (Figure 3b), a new inoculum featuring a higher NaHCO\({}_{3}\) concentration (0.36 M instead of 0.09 M) was prepared and subjected to three consecutive renewals, one every 14 days. When employed to start a new Spr02 culture under the best-known conditions (n. 8, Table 2), a shorter lag time was observed. The OD680 of the biomass suspension reached 2.59 after 48 h, an improved value compared to before (Figure 3b). Unfortunately, the OD680 at 72 h stopped at 3.25, corresponding to a productivity limited to 620 mg\({}_{\text{biomass}}\)/L\(\cdot\)d.  
Dealing now with the CO\({}_{2}\) utilization efficiency (U, Equation (7)), its calculation reveals a maximum value of 46% (right column of Table 2). This means that, in the best- conditions (n. 3), more than half of the inorganic carbon moles were lost during the biofixation process. Moreover, the final pH value of 12.2 indicates that no residual bicarbonate was present. This is a clear confirmation of the occurrence of CO\({}_{2}\) outgassing, which is probably more pronounced during the initial growth phase when the pH is lower. We thought that the continuous air bubbling used to keep the cultures in agitation may cause the removal of the dissolved CO\({}_{2}\), leading to a continuous equilibrium shift towards the loss of other inorganic carbon species. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**n.**} & \multirow{2}{*}{**T (\({}^{\circ}\)C)**} & **NaHCO\({}_{3}\)** & **KHCO\({}_{3}\)** & **P** & **R** & **U** \\  & & **(mol/L)** & **(mol/L)** & **(mg\({}_{\text{biomass}}\)/L\(\cdot\)d)** & **(mg\({}_{\text{CO2}}\)/L\(\cdot\)d)** & **(mol\%)** \\ \hline
1 & 29 & 0.45 & 0 & 52 \(\pm\) 2 & 83 \(\pm\) 3 & 2 \\
2 & 29 & 0.54 & 0 & - & - & - \\
3 & 35 & 0.36 & 0 & 760 \(\pm\) 26 & 1323 \(\pm\) 45 & 46 \\
4 & 35 & 0.45 & 0 & 550 \(\pm\) 19 & 958 \(\pm\) 33 & 29 \\
5 & 35 & 0.54 & 0 & 350 \(\pm\) 12 & 610 \(\pm\) 21 & 15 \\
6 & 35 & 0.27 & 0.18 & 600 \(\pm\) 21 & 1045 \(\pm\) 36 & 32 \\
7 & 35 & 0.36 & 0.09 & 590 \(\pm\) 20 & 1027 \(\pm\) 36 & 31 \\
8 \({}^{\text{b}}\) & 35 & 0.36 & 0 & 620 \(\pm\) 19 & 1110 \(\pm\) 36 & 40 \\ \hline \hline \end{tabular}  
* PPFD = 260 \(\upmu\)mol/s\(\cdot\)m\({}^{2}\), T = 35 \({}^{\circ}\)C, t = 72 h; 0.09 M NaHCO\({}_{3}\) inoculum; \({}^{\text{b}}\) PPFD = 260 \(\upmu\)mol/s\(\cdot\)m\({}^{2}\), T = 35 \({}^{\circ}\)C, t = 72 h; 0.36 M NaHCO\({}_{3}\) inoculum.  
\end{table} 
Table 2: Higher bicarbonate concentration experiments \({}^{\text{a}}\). A possible solution to this issue could involve recirculating the gas used for mixing the biomass culture, thereby achieving a sealed PBR design. In our small-scale setting, this was easily accomplished using a peristaltic pump (depicted in Figure 6), with the added simplification of removing the gas humidification stage (see Section 2.3).