# Integrated Carbonate-Based CO\({}_{2}\) Capture--Biofixation through Cyanobacteria
## Materials and Methods
### PBR Design and Operation  
The photobioreactor (PBR) employed for this study was custom-built and consisted of a thermostatic recirculated water glass tank, constructed by bonding together five glass plates (each 5 mm thick) using silicon glue. Within the tank, eight biological glass tubes were positioned as culture slots, with each tube having an inner diameter of 3.0 cm, a height of 20 cm, and a working volume of approximately 70 mL. These culture tubes were arranged in two rows, and placed in the two longer sides of the tank. For illumination purposes, two LED panels are employed, with one panel positioned on each side of the PBR. Each LED panel comprised four LED strips, and each strip was aligned with the corresponding culture tube. The LED elements offer a lighting capacity of 18 W/m, producing 6300 lumens of cold white light with a color temperature of 4000 K. Power to the LED is supplied by a 24 V DC power supply unit.  
To ensure stability and proper alignment of the system, two 3D-printed holders in PET polymer were designed: one external and one internal. The internal support comprises two parts: an upper component with eight holes and a bottom component with eight concave sockets, perfectly aligned with four M4 stainless steel threaded bars. These components work together to securely hold the culture tubes in place. Additionally, a central hole on the top part is included to accommodate a thermometer. On the other hand, the external support is designed with a central zone to hold the glass tank and three slots on each side to accommodate the LED panels. Using these three slots, the LED panel can be positioned at various distances from the tank, enabling the adjustment of illuminance levels.  
The PBR is illustrated in Figure 2, which includes both a schematic representation and a photograph of the system.  
Figure 2: Scheme and real prototype of the custom-built PBR: (**a**) side view, (**b**) top view.  
Lux measurements were taken using an M1 professional portable lux-meter from OxyTech, adhering to reference standards EN 13032, with a photocell measuring 34 \(\times\) 21 mm in dimensions. The recorded lux values for the three distances were as follows: 14,000 \(\pm\) 600 lux for the farthest slot, 18,000 \(\pm\) 800 lux for the middle slot, and 27,000 \(\pm\) 1000 lux for the slot nearest to the tank. These measurements were conducted in the air, with the lux-meter placed at the same distances from the LED panels as the outer surface of the culture tubes. The values, averaging five measurements, were gathered at different heights of the LED strips, demonstrating excellent illumination uniformity. Photosynthetic Photon Flux Density (PPFD) values were derived through numerical conversion, accounting for the emission spectra of the light source [30]. The resulting PPFD values were as follows: 200 \(\pm\) 9 \(\mu\)mol/s\(\cdot\)m\({}^{2}\) for the farthest slot, 260 \(\pm\) 11 \(\mu\)mol/s\(\cdot\)m\({}^{2}\) for the middle slot, and 390 \(\pm\) 14 \(\mu\)mol/s\(\cdot\)m\({}^{2}\) for the slot closest to the tank.  
Temperature control was achieved by utilizing an external thermostatic fluid circulator using a mixture of water and propylene glycol. The local temperature inside the glass tank was monitored using a standard mercury thermometer. To ensure gentle mixing of the cultures, an air flux was introduced through 2 mm inner diameter glass tubes, one tube inserted in each culture tube. The air was supplied at a controlled rate of 0.8 cm\({}^{3}\)/s by a pump, which has proven to ensure proper mixing, even in the later stages of growth where the highest viscosity occurs.  
Before entering the culture tubes, the air passed through a 2 M NaOH aqueous solution. This step serves two purposes: first, to remove naturally occurring CO\({}_{2}\), and second, to achieve proper humidification.