## 3 Results and Discussion
### Stability of CA-Cu and CO\({}_{2}\) Capture Ability  
We compared the relative activity of both the free and immobilised enzyme. The relative enzyme activity is the ratio of the retained activity of an immobilised CA or a free enzyme and was investigated over 60 days. The results in Figure 5 showed that the immobilised enzyme efficiency was 95%. Previous studies presented similar findings for the immobilised CA [46; 47]. In our study, the free CA enzyme lost 34% of its initial activity within 7 days when incubated in PBS (pH 7.4) at room temperature. Conversely, CA-Cu hybrid carriers maintained 94% of their initial activity under the same conditions even after one month (Figure 5a). Thus, the immobilisation strategy lowers enzyme deactivation so that enzymes can be used for extended periods, as previously reported [48]. The increased stability of the CA-Cu is crucial as it is an essential factor in biocementation. Figure 4: (**a–c**) SEM images of Cu-CA hybrid nanoflower at different magnifications (coloured photograph courtesy of public resources in the network), (**d**) a “Dahlia pinnata” flower. The pH stability of the free CA and immobilized CA-Cu was assessed after incubating at specified pH values. pH is a critical factor in bioccentation, as it controls the precipitation and dissolution of biominerals; the enzyme is therefore required to be stable in the alkaline range where calcite bioprecipitation occurs [19]. Figure 6a shows the relative activity of the CA enzyme (free or immobilized) compared to the initial activity of the enzyme, under different pH values. From Figure 6a, it can be seen that when the incubation pH increased, the free enzyme lost its activity steadily but still preserved about 85% of its stability at pH > 9. On the other hand, the immobilized CA was even more stable retaining about 92% of its original activity at pH > 9. Similar results from the literature also showed that the CA enzyme remained stable and maintained its secondary structure for pH values of 7.0-11.5 [49]. The activity of the free and immobilized CA enzyme was also measured under different temperatures; the results are presented in Figure 6b in terms of the relative activity of the enzyme compared to its initial activity. The free enzyme lost approximately 30% of its activity at temperatures higher than 50 \({}^{\circ}\)C. The activity loss of the CA-Cu at the same temperature range was only 10%. Overall, the activity of the immobilized CA was minimally affected for a wide temperature range until around 40 \({}^{\circ}\)C, when a more notable activity loss started. Previous studies also found that immobilised CA enzyme had a higher thermal stability/activity than the free CA enzyme and that the optimal temperature for CA immobilized within polyurethane foam was 40 \({}^{\circ}\)C [50]. Other studies of thermostable CA found it to be active in the temperature range 0-100 \({}^{\circ}\)C, with an optimal temperature of approximately 80 \({}^{\circ}\)C [51]. Despite the difference in optimal temperature observed in the two papers, it can be concluded that CA enzymes can operate at wide temperature ranges. This finding is important, as in bioccentation it is critical that the enzyme remains stable at different soil temperatures. In our study, we investigated temperature ranges from 5 to 60 \({}^{\circ}\)C; we found the immobilised enzyme to lose only up to 10% of its initial  
Figure 5: (**a**) Stability of CA and CA-Cu hybrid over time, (**b**) electrical conductivity changes over time, (**c**) pH changes over time, (**d**) change curve of calcium concentration. activity within this temperature range; moreover, only a 1-2% activity loss was observed for temperatures up to 35 \({}^{\circ}\)C. The foundation soil temperature commonly ranges between 10 and 24 \({}^{\circ}\)C. Therefore, the CA and CA-Cu enzymes would suit ground improvement applications in most world regions, while contributing to climate change mitigation by converting CO2 into calcium carbonate.