Assembly, set-up and operation of a laboratory scale 1L photobioreactor

UNIVERSITAT POLITÈCNICA DE VALÈNCIA ESCOLA TÈCNICA SUPERIOR D !ENGINYERIA AGRONÒMICA I DEL MEDI NATURAL Assembly, set-up and operation of a laboratory scale 1L photobioreactor TRABAJO FIN DE GRADO EN BIOTECNOLOGÍA ALUMNA: María Cristina Loureiro Casalderrey TUTOR: Javier Fermín Urchueguía Schölzel DIRECTOR EXPERIMENTAL: David Fuente Herraiz Curso Académico: 2015/2016 VALENCIA, Julio de 2016  ! 2 Title: Assembly, set-up and operation of a laboratory scale 1L photobioreactor. Abstract: Currently, cyanobacteria are photosynthetic microorganisms that have been considered as potential candidate for the production of biofuels or pharmaceutical compounds among others. For their cultivation, there are specific systems called photobioreactors in which all their natural requirements (light, CO2 and nutrients) are provided. Moreover, the cultivation of these microbes involves a set of sensitive parameters, such as temperature, pH, mass transfer or mixing, that should be maintained under specific ranges to get the photobiological process stable. These parameters are controlled by a serie of associated devices that conform the photobioreactor system. The assembly and operation of a 1L lab-scale flat-bed photobioreactor and its associated devices using a computer program were performed in this work. Furthermore, a set of calibration procedures and characterization experiments were done to check the proper operation of the photobioreactor system and to collect relevant information for future cultivation processes. This investigation is set inside the European project CyanoFactory (FP7-Energy 308518). This project is carried out by a consortium composed of several European universities and research companies. It pursues the aim of generating photosynthetic microorganisms cultivated in photobioreactors with the capacity of producing biohydrogen. Keywords: Photobioreactor, cyanobacteria, process engineering, control of process variables. Author: María Cristina Loureiro Casalderrey! Tutor: Javier Fermín Urchueguía Schölzel Experimental director: David Fuente Herraiz City and date: Valencia, July of 2016! 3 4 Título: Montaje y operación de un fotobiorreactor de un litro a escala de laboratorio. Resumen: En la actualidad, las cianobacterias son microorganismos fotosintéticos que han sido considerados como fuentes prometedoras para la producción de biocombustibles o compuestos farmacéuticos entre otros. El cultivo de estas bacterias requiere de sistemas específicos denominados fotobiorreactores, los cuales proporcionan los requerimientos que necesita la cyanobacteria en condiciones naturales (luz, CO2 y nutrientes). Además, el cultivo de estos microorganismos lleva consigo la regulación de una serie de parámetros, como son la temperatura, el pH, la transferencia de calor o la homogeneización, para mantener el proceso fotobiológico estable dentro de unos rangos. Estos parámetros son controlados por una serie de dispositivos asociados que forman parte del sistema del fotobiorreactor. El ensamblaje y operación de un fotobiorreactor de tipo plano de 1L a escala laboratorio y de sus dispositivos asociados usando un programa informático fueron el objeto de este trabajo. Además, se realizaron una serie de calibraciones y experimentos de caracterización para comprobar el correcto funcionamiento del fotobiorreactor montado y para obtener información relevante que debe ser tenida en cuenta para futuros cultivos de cianobacterias. La presente investigación se enmarca dentro del proyecto de la Comisión Europea Cyanofactory (FP7-Energy 308518) conformado por un consorcio de universidades y empresas europeas que tiene el fin de crear microorganismos cultivados en fotobiorreactores que sean capaces de producir biohidrógeno. Palabras clave: Fotobiorreactor, cianobacterias, ingeniería de procesos, control de variables de proceso. Autora: María Cristina Loureiro Casalderrey! Tutor: Javier Fermín Urchueguía Schölzel Director experimental: David Fuente Herraiz Localidad y fecha: Valencia, Julio de 2016! 5 6 Table of contents 1. Introduction……………………………………………………………………… … 1 2. Objectives………………………………………………………………………… … 5 3. Materials and Methods……………………………………………………………. 6 3.1. Photobioreactor system parts characterization………………………………………. 6 3.2. Assembly of the photobioreactor system………………………………………….. 13 3.3. Computer programs’ performance…………………………………………………… 18 3.3.1. BacVisSingle……………………………………………………………………………………. 18 3.3.2. DinModule………………………………………………………………………………………. 19 3.3.3. Labview………………………………………………………………………………………….. 20 3.4. Device calibration procedure………………………………………………………… 25 3.4.1. Gas Flow Controllers (GFCs) and Gas Flow Meters (GFMs)…………………….……. 25 3.4.2. O2 sensor……………………………………………………………………………………….. 25 3.4.1. pH meter………………………………………………………………………………………… 25 3.4.1. LED panels……………………………………………………………………………………… 26 3.5. Characterization experiments……………………………………………………….. 27 3.5.1. Culture medium pump flow rate characterization………………………………………… 27 3.5.2. pH control………………………………….…………………………………………………… 27 3.5.3. Optics experiments. LED panels characterization………………………………………… 27 3.5.4. Warming process and heat transfer.……………………………….………………………… 28 4. Results and Discussion……………………………………………………….……. 29 4.1. Culture medium pump flow rate characterization.………………………………………….. 29 4.2. pH control.……………………………….……………………………….……………………… 31 4.3. Optics experiments. LED panels characterization.…………………………………………. 33 4.4. Warming process and heat transfer.……………………………….………………………….. 35 5. Future prospects…………………………………………………………………… 37 6. Conclusions………………………………………………………………………… 39 7. References………………………………………………………………………….. 40 7 I List of Tables List of Figures Table 1. Specific channels values for each device. AI is referred to Analog Input signal, AO to Analog output, DI to Digital input and DO to Digital output…………………………………………………….. 23 Figure 1. Steps carried out for the execution of this work.…………………………………………….. 4 Figure 2. Flat panel photobioreactor……………………………………………………………………. 6 Figure 3. Control Box opened.…………………………………….…………………………………… 6 Figure 4. LED panel.………………………………………………………………………………..….. 6 Figure 5. Computer.……………………………………………………….……………………………. 7 Figure 6. Air Mass Flow Meter (GFM).…………………………………………………………..……. 7 Figure 7. CO2 Mass Flow Controller (GFC).………………………………………………………..…. 7 Figure 8. O2 Sensor.………………………………………………………………………….…………. 8 Figure 9. pH meter with the cap.………………………………………………….……………………. 8 Figure 10. Piston air compressor.……………………………………………………..……………….. 9 Figure 11. Schott-Bottles.…………………………………………………………………………….… 9 Figure 12. Waste container.…………………………………………………………………..………… 9 Figure 13. Peristaltic pumps on the Control Box surface.……………………………………..……….. 10 Figure 14. Heat exchanger.………………………………..……………………………………………. 10 Figure 15. Air filter.………………………………………………….…………………………………. 11 Figure 16. Safe-Site valve on the left and check valve on the right.……………………………..…….. 11 Figure 17. Types of silicone tubes used. (1) 06/08 tube, (2) 04/06 tube, (3) 02/04 tube, (4) 01/03 tube……………………………………………………………………………………………………….. 11 Figure 18. Metal pipe………………………………………………………………………………….. 12 Figure 19. Scheme of both types of plugs……………………………………………………………… 12 Figure 20. Clamp in port 8…………………………………………………………………………….. 12 Figure 21. “Y” Connector………………………………………………………………………………. 12 Figure 22. Diagram of the PBR system that sums up the assembly process. It shows electrical connections (black channels), fluid piping system connections (color-coded conduits according to Spanish Regulation UNE 1063 (2000)) between the devices and where the accessory stuff has to be placed. Thick lines represent the main pipes needed for the biological process takes place, and thin lines are referred to secondary connections………………………………………..……………………. 14 8 II Figure 23. The inside of the Control Box with its components, transmitters and PCI cards………….. 15 Figure 24. Flat panel photobioreactor with the nine numbered ports and their usability………………. 15 Figure 25. Pictures that show the way in which some components have to be assembled. (A) Clipping of silicone tubes to ports 7, 8 and 9; (B) metal pipe placed at a GFM inlet; (C) silicone tubes hooked to their respective pumps; (D) Air filter inlet side; (E) heat exchanger insertion at port 3; (F) both valves put together with a male plug at the top…………………………………………………….. 16 Figure 26. BacVisSingle program. Window with a black frame shows the user graphic interface of the program. The green pathway on the right represents the subsequent windows that will display if Zero Adjustment is not checked. The blue pathway on the left represent the steps if Zero Adjustment is checked………………………………………………………………………………………………….. 18 Figure 27. DinModule user graphic interface………………………………………………………….. 19 Figure 28. DinModule configuration…………………………………………………………………… 20 Figure 29. Labview user graphic interface. (1) Warning window; (2) program information window; (3) Welcome card; (4) Setting register card; (5) Graphic register card; (6) Control buttons; (7) General operation unit…………………………………………………………………………………………….. 22 Figure 30. Labview user graphic interface. (1) Media flow rate and turbidity card; (2) Light intensity card; (3) Gas control card; (4) Gas measurement card; (5) pH, temperature and redox card; (6) Check filling levels and gas pressure card; (7) General AI/AO card; (8) Ports and Channels summary card; (9) Options/files card………………………………………………………………………………………… 21 Figure 31. (A) Water volume pumped per min at different flow rate setpoints (with slope 1 and intercept 0) and (B) at different slope and intercept values. Each point represent the resulting volume measured in the test-tube after 1 min.……………………………………………………………………. 30 Figure 32. Water volume pumped at different times in a 100 mL test-tube by acid and base pumps.It can be appreciate that flow rate of both pumps is approximately of 10 mL·min-1 and that base pumps is a 5% slower than acid pump…………………………………………………………………………….. 31 Figure 33. pH evolution during approximately 80 min. A value of 1 in Y pumps axis, means that the pump is on, while 0 value indicate that the pump is off. Moreover, blow the graphic, it is shown at which points pH value and tolerance ranges where changed……………………………………………. 32 Figure 34. Characteristic wavelength emission spectrum of LED panels light that corresponds to PAR radiation.…………………………………………………………………………………………………. 33 Figure 35. Changes in LED panel irradiance emitted when the radiometer sensor is moved away from the LED panel (at 2, 3 and 4 cm distance). Each dot represent the average of the three measurements taken in the three LED panel positions for each light intensity and distance (see Figure 36). ………………………………………………………………………………………………………. 34 Figure 36. LED panel positions where radiometer measurements were taken.………….…………….. 34 Figure 37. Temperature evolution in a 90 min time interval at three different LED panels distance from the PBR (1, 2 and 3 cm). The experiment was done flanking the PBR with both panel and using maximum LED light intensity.………….……………………………………………………………….. 36 Figure 38. Schott-bottle with the feed port specific for ISO glass jars and the air filter.……….. 38 9 III 2. Objectives The objective of this study is the assembly, operation and test of a 1L lab-scale flat-bed photobioreactor system and to compile all the information required for future users of the system. In order to accomplish this purpose, a set of sub-objectives were established: Characterization of all the parts, components and devices that make up the photobioreactor system. Assembly of the photobioreactor system. Determination and verification of the smooth running of the computer programs. Determination of the calibration procedures in devices which require it. Tests to check the proper assembly and functioning of the photobioreactor system. 5 3. Materials and Methods 3. 1. Photobioreactor system parts characterization As mentioned above, a PBR presents a set of components and devices that allow to maintain proper culture cyanobacteria conditions. In this way, our scale-lab 1L flat-bed photobioreactor is composed of the following parts: A. Flat panel photobioreactor (PBR). It is the recipient which contains the cyanobacteria culture and it is made of a biocompatible polymer. Its measurements are 240x290x40 mm, its capacity is 1.18 L, although its total capacity is 1.32 L, its empty weight is 3,5 kg and it also has a light capturing surface of 661 cm2. It presents two plastics surfaces on both sides which could take apart unscrewing white nuts to encourage disinfection and cleaning process. For further information see KSD Manual. B. Control Box. It is the key that allows communication between the computer program and the different measurement devices. It contains two PCI cards designed for digital/analog signal conversion and is connected with the following electronic transmitters and components: -pH-controller with temperature sensor. -Dose pump for HCl and NaCl (manual / automatic). -Dose pump to fill and for constant flow rate (manual / automatic). -Speed controller for dose pump. -Main switch ON / OFF. -Analog card - converter - for PC. -Controller and power supply for LED lights. -Gas controller and mixer for air and CO2. -PC C. LED panels. They provide the artificial light needed for photosynthesis process. The flat panel PBR is flanked on either site by a LED metal panel, two panels in total, encouraging the lighting of the complete culture medium surface in the PRB. Each panel has 84 LEDs in one of the slides, arranged in 4 rows of 21 LEDs. The measurements of the panels are 500x8x1 mm and they irradiate white light between 400-750 nm. The panel radiance intensity can be change but not the emission wavelength. For further information see KSD Manual. 6 Figure 2. Flat panel photobioreactor. Figure 3. Control Box opened. Figure 4. LED panel. D. Remote control system. It refers to the desktop computer with Windows 7 software. It has three computer programs related to the PBR: Labview, BacVisSingle and DinModule. Labview is the main photobioreactor program for data collection, data visualization and remote control of the photobiological process. This program allows to set up all the parameters of the system and saves all the measurements taken by the overall devices. The other both, BacVisSingle and DinModule, are used mainly for calibration procedure of O2 sensor and pH meter respectively. Controlling these devices and taking measurements are also possible with both programs. E. Mass Flow Meters (GFMs). GFMs are devices designed to measure and show the gas flow rate (mL·min-1) that passes through them. They do not operate with liquids, only with pure or filtered gases. There are two GFMs in our PBR system. One measures the air that comes out of the PBR. The other one indicates the total amount of CO2 which enters the PBR after air flow and a percentage of CO2 were mixed together. Since for photosynthesis and cell growth control, it is important to know how much of each gas is moving into and out of the PBR. For a proper operation, these devices should be collocated in vertical position and they need 30 minutes to warm-up since they were switched on. Related to their operation principles, only a small portion of the total gas stream that enters in the device is used for the measurement. This small portion is shunted through a capillary stainless steel sensor tube. There, the laminar gas flow is heated and carried from an upstream coil to a downstream coil windings. A temperature change is detected electronically and it is dependent of a resistance differential. The GFM provides an output signal that is a function of the amount of heat carried by the gas to indicate mass-molecular based flow rate. For further information see GFM Operating Manual Aalborg. F. Mass Flow Controllers (GFCs). GFCs are devices that control the gas flow rate (mL·min-1) that passes through them. They do not operate with liquids either, only with pure or filtered gases. There are also two GFCs in our PBR system, one regulates the air that enters the flat panel PBR and the other one, the CO2 input. These devices allow to control the mixing, aeration and the gas quantity that enters in the PBR. For a proper operation, they also should be collocated in vertical position and they need 30 minutes to warm-up since they were switched on. 7 Figure 7. CO2 Mass Flow Controller (GFC). Figure 6. Air Mass Flow Meter (GFM). Figure 5. Computer. They have the same operational components as the GFM plus a PIC and a solenoid valve. Related to their operation principles, they firstly make the same type of measurements as the GFMs, generating an output flow rate signal. Afterwards, this signal is detected by a closed loop control circuit (PIC) and compared continuously with the flow rate value selected in Labview program. Deviations are corrected by solenoid valve adjustments to maintain the constant setpoint. Thus, the valve allows to set the gas flow rate to any desired value within the range of 0-500 mL·min-1 in the Air GFC or 0-50 mL·min-1 in the CO2 GFC. This valve is closed when the Labview program is not running. For more information see GFC Operating Manual Aalborg. G. O2 sensor. This device measures de oxygen concentration that comes out of the PBR in a range over 0,1-25 % Vol. If the O2 % is know, the photosynthetic rate as well as the cell growth can be determined. It does not operate with gases which contain polymers, silicons, halogens (fluoride, chloride, bromide, etc.), SOx or H2S and it needs between 45 min and 1 hour to warm-up since it was switched on. Moreover, the relative humidity of the environment should be lower than 75 % for proper measurements. If the RH increases, the sensor has to be 12 h at 80 ºC in a hot air oven. If the red adaptor is unscrewed, there is a sealing ring to avoid possible leaks. There, under the sensor’s head, there are two air filters. The external filter can be replaced by another one but not the internal filter. In this case, contact custom service. Regarding its operational principles, it has a oxygen pump cell where gas O2 molecules are transferred since the cathode to the anode. The sensor correlates O2 molecules with electrons. Besides, the sensor must not be disconnected from the power supply when it is operating. For further information see the BluesSens BCP-O2 Manual. H. pH meter. This electrochemical device measures the pH of the culture inside the PBR. It is unscrewed in PBR port 5 (see Figure 24). Moreover, it has an integrated temperature sensor, which measures the temperature of the culture. These measurements allow to regulate both factors and keep them in a stable range. This electrolyte is filled of a 3M KCl solution. When this solution volume is low, it has to be refilled using the unscrewed black nut which is located on the side of the device. Furthermore, pH meter has a white-transparent cap with the same KCl solution. This cap should be placed at the tip of the pH meter when the device is not being used. For more information see the pH meter Manual. 8 Figure 8. O2 sensor. Figure 9. pH meter with the cap. I. Piston air compressor Nuair Vento OL 195. This device takes air from the environment and stores it in the tank until 8 Bar pressure is achieved. At that moment, the compressor stops automatically. Then, the compressed air is released at a specific pressure that can be regulated by the “Pressure regulator”. As the air is draining out, the tank depressurizes. When tank pressure reaches its lower limit, the device turns on automatically and stores air until re-pressurizing the tank. For a proper operation, the pressure should be maintained between 2-4 Bar using the “Pressure regulator”. The capacity of the air compressor is 6 L and has to be connected to the current supply using the power cable. Furthermore, the silicone tube is connected to the quick metal coupler. J. CO2 Cylinder. This bottles stores compressed CO2 which can be released using a valve. Since we have not started culture experiments, we have not bought it yet. Because of that, more specific information cannot be provided. This cylinder is required for the cyanobacteria culture due to the fact that these microorganisms need an extra percentage of this gas for photosynthesis process and thereby, for its growth. Synechocystis sp. PCC6803 needs air enriched with 3 % CO2 for its culture because the amount of CO2 that the compressed air has, is not enough. K. Schott-Bottles ISO GL45 1L. There are three 1L glass bottles in our PBR system. Each one stores a specific solution required for the PBR operation. One stores acid solution (HCl) and other, the base solution (NaCl). Both solutions permit the culture pH regulation. The last schott-bottle stores culture medium (BG11) for the culture dilution and renovation. The bottles’ volume is of 1L because it has been reported from other researched groups that for a continuous cultivation of the scale-lab 1L flat-bed PBR, 1L volume of this three solutions is enough. L. Waste container. It is the tank that stores the biological waste, which is the culture residues that overflowed of the PBR. The actual 3 L waste container is only for characterization experiments. For future culture experiments with cells, an adequate container fit for the biological waste collection will be required. 9 Figure 11. SchottBottles. Figure 12. Waste container. 1. On/off switch. 2. Comprised air outlet with quick metal coupler. 3. Operating pressure adjustment knob. 4. Air used pressure gauge. 5. Receiver condensation drainage tap. Figure 10. Piston air compressor. 1 2 3 4 5 M. Pumps. There are three pumps incorporated into the Control Box but located in the external surface of it. Theses pumps are peristaltic pumps. They have a rotor with a number of “rollers” around the external circumference. Specific flexible silicone tubes are placed inside the pumps, in contact with the “rollers”. The silicone tubes are the carriers whereby the solutions pass through from the Schott-Bottles into the PBR. When the rotor turns, the silicone tubes are compressed and closed by the “rollers”and the liquid between two rollers is forced to move through the tube. This type of peristaltic pumps run at ambient pressure and usually have a range of revolutions to deliver different amounts of fluid. Each peristaltic pump moves a different solution through the tube. The pump on the bottom, is the main pump. It controls the input culture medium flow rate (mL·min-1). According to the experimental results, its revolution range is 1-5 mL·min-1 (see section 4). The middle pump regulates the input base solution and the last one pumps the acid solution. These two pumps help in pH regulation. They do not have a range of revolutions. On the contrary, they only work at a specific rate revolution (10 mL·min-1) (see section 4) and they can only be switched on and off. In addition, for a correct operation of pumps, the Control Box must be placed in vertical position. N. Heat exchanger. It is a glass tube to control culture media temperature inside the PBR. It is located in port 3 (see Figure 24) unscrewing the black nut. Water at a particular temperature passes through it, warming up or cooling down the culture’s temperature. The water enters on the top, passes through a thin tube with an open bottom end that is inside the exchanger, flows in the outer layer and finally, exits by the side output. Heat is transferred through the glass wall of the exchanger to the culture. Photosynthetic processes usually require more cooling down because LED panels as well as cell metabolism produce heat, warming up the culture and having negative effects on cell growth and bioprocess development. O. Thermal circulator. It is a tank that stores water and it can also regulate its temperature. Moreover, it has a pump that forces the water to move from the tank to the heat exchanger and come back. So, it works in a closed system. However, since we do not have this device, more detailed information cannot be given. In addition, plastic tubes will be bought to connect the thermal circulator with the heat exchanger. P. Other items: 10 Figure 13. Peristaltic pumps on the Control Box surface. Figure 14. Heat exchanger. Air filters. They remove solid particles from the air such as bacteria, mould, dust, etc. thanks to their 0.2 µm Millipore membrane. They avoid the introduction of any potential flow impediment in GFCs, GFMs and O2 sensor, guaranteeing the good functioning of the devices, and they keep sterile culture conditions inside the PBR (cyanobacteria are very sensitive to this). The filter positions in the PBR system are displayed in Figure 22. Moreover they have to be set in a specific position when they are clipped to the silicone tubes. Its location depends on the fluid flow direction. Their blue blind plug side (see Figure 25(D)) is the inlet side whereby the flow enters, and the other one is the outlet side. These filters can be reused 10-15 times, but if an air filter gets wet, it should be replaced by another one immediately. Actually, not all the filters have been placed in their specific positions due to the fact that it is not necessary for the characterization experiments. But for culture experiments, all the air filters have to be located suitably. Valves. Two types of valves are used in the PBR system: check valves (green color in Figure 16) and Safe-Site injector valves (lilac color in Figure 16). The first one is an anti-reflux valve, i.e. a mechanical valve, whereby liquids and gases flow in one direction only, preventing process flow from reversing. The Safe-Site valve injects the fluid in a specific direction. These valves have to be located in specific positions in the system, primarily in PBR ports. The precise positions are shown in Figure 22. In some cases, both valves should be put together but in others, only one of them has to be placed. In this last case, the color of the valve to use is also specified in Figure 21. As well as air filters, valves have not been placed. They have to be set for culture experiments once the PBR is sterilized and all the other material autoclaved. Moreover, if a valve is stuck, it should be replaced by another one. Silicone tubes. They are the carriers that allow the movement of liquid and gases over the PBR system. There are four types of silicone tubes depending on their diameter and use. They can be seen in Figure 17. According to the sequential numbering of the right-hand figure: (1) 06/08 tube (6 mm of inner diameter and 8 mm of outer diameter) is used for gas flow, (2) 04/06 tube used for PBR output liquids, (3) 02/04 tube to pump culture medium and (4) 01/03 tube for acid/base solutions. As can be appreciated in this figure, tubes 3 and 4 have a thick wall because they have to be very resistant to pump’s friction. If the part of the tube that is in contact with the pump is worn, it should be covered with masking tape. 11 Figure 15. Air filter. Figure 16. Safe-Site vale on the left and check valve on the right. Figure 17. Types of silicone tubes used. (1) 06/08 tube, (2) 04/06 tube, (3) 02/04 tube, (4) 01/03 tube. 1 2 3 4 With the actual PBR system configuration, there are some tubes that are excessively long, but they have not been cut because the PBR will be moved to another laboratory at some point, and by then, its configuration could change and long silicone tubes might be necessary. Metal pipes. They are hollow cylinders made of steel. They have a 1/4’’ diameter and a 5 cm length. There are eight metal pipes in our system, two for each gas device (except for the O2 sensor). They are placed in the inlet and outlet of each GFC and GFM to propel gas passage and tube clipping, minimizing gas leaks. Luer locks. They are plastic plugs that facilitate silicone tubes’s clipping. There are two types: male plugs and female plugs. Male plugs have an external thread while female plugs have an internal thread. The specific position where each type of plug have to be placed is shown in Figure 22. Clamp. It is used to block liquid passage through a silicone tube. It is placed in the PBR drainage spot to avoid the emptying of it. The clamp should only be removed when an experiment finishes so as to drain the PBR. “Y” connector. It is a plastic piece with a “Y” shape that is used to mix or split gases or liquids that move through the silicone tubes. 12 Figure 18. Metal pipe. Figure 19. Scheme of both types of plugs. Figure 20. Clamp in port 8. Figure 21. “Y” connector. 3. 2. Assembly of the photobioreactor system The parts described above were assembled as it is shown in Figure 22. Assembly process was done in a set of steps. Firstly, electric connections were made and after, the fluid piping system. The electric channels are symbolized as black conduits in Figure 22, while the others are represented using a color code. Concerning electric connections, primarily all the devices with a direct connection to the Control Box were plugged into it following the circuit diagram that can be found in KSD PBR Manual. GFMs and GFCs were connected using usual 9-pol cables. CO2 GFM connections are not displayed in KSD PBR Manual. In this case, red and orange cables were plugged into pin 62 (+) and pin 28 (-) respectively in PCI-1710UL Multifunction card. LED panels were connected to the transmitter 1G2 shown in Figure 23, red cables to pin 5 and black cables to pin 6. The pH meter has a shielded cable composed of a set of color-coded wires. The connection of each wire to the pH transmitter MV 3010 (see also Figure 23) is the following: A2-black, A4-red, A5-green, A7-white and A8-yellow. Pins A1 to A4 are related with the pH sensor while pins A5 to A8 are matched up with the integrated temperature meter. These six devices are powered and controlled by the Control Box. Likewise, both PCI cards of the Control Box were plugged directly into the computer using two 68-pol cables, so these six electronic devices can be controlled using the Labview program. The Control Box was represented in Figure 22 with a colored box which has a PIC controller (Programmable Interface Controller) inside. This controller is an electronic circuit that can be programmed to carry out a vast range of tasks. However, the O2 sensor, the thermal circulator and the air compressor were not connected to the Control Box. They are powered directly by the power supply. The O2 sensor was also connected to the computer by a serial USB cable. So it can be controlled by Labview and BacVisSingle too. Nevertheless, the termal circulator and the air compressor have an autonomous operation, which means that they cannot be driven by any computer program. They only can be regulated by hand. 13 14 Acid solution Base solution Culture medium Air compressor PBR Waste container Thermal circulator Heat exchanger Valve Valve Flowmeter Flowmeter CO2 GFM Air GFM pH O2 sensor Air GFC CO2 cylinder Computer LED panel LED panel CO2 GFC Control Box CO2 Air Air CO2Air Air + % CO2 Air Air Air Water Water Acid Acid Base Base Culture media Culture media Acid + base Biological waste Biological waste serial - USB cable adapter 9-pol cable 9-pol cable 9-pol cable 9-pol cable Red-black cables Red-black cables Shielded cable Power supply 1 2 3 4 5 6 789 Air filter Air filter Air filter Air filter Safe-Site valve Check valve T Clamp Acid pump Base pump Media pump CA CA CA Power supply Power supply P I C P I C P I C “Y” p iece “Y” piece “Y” piece 06/08 tube 06/08 tube 06/08 tube 06/08 tube 06/08 tube 06/08 tube 06/08 tube 04/06 tube 04/06 tube 02/04 tube 01/03 tube 01/03 tube 02/04 tube 01/03 tube 01/03 tube LLM LLM LLM LLW LLW 68-pol cable (2) Figure 22. Diagram of the PBR system that sums up the assembly process. It shows electrical connections (black channels), fluid piping system connections (color-coded conduits according to Spanish Regulation UNE 1063 (2000)) between the devices and where the accessory stuff has to be placed. Thick lines represent the main pipes needed for the biological process takes place, and thin lines are referred to secondary connections. parameter value evolution during the bioprocess can be visualized; (6) it has four buttons: Start makes Labview run once all parameters have been set, Pause stops program running which can continue if Pause is unclicked, Stop finishes the running but the program remains opened and End finishes program running and besides Labview is closed; (7) it is the general operation unit and provides an overview of the most important measured values. In addition, once Stop or End are clicked, the program takes a while to carry out the action. If Stop is clicked, the parameter values will have to be set again. The program must be closed always by pressing End button, do not close it by pressing File —> Exit, since it will not end at all. Besides, BacVisSingle and DinModule cannot be opened and used while Labview is running, this will lead to an error and Labview will close. If Settings card is clicked, there are nine sub-register cards to set the parameter values of Labview controlled devices. Figure 30 shows the nine cards with the data that has to be inserted. The first card is Media Flow Rate and Turbidity for the medium pump configuration (Figure 30(1)). There, Media pump button allows to switch the pump on or off. A Flow rate set point value has to be inserted as well as the Channel media, the Slope and the Intercept to adjust the analog output signal to the used pump. The flow rate setpoint refers to the rate at which the medium culture is pumped since the Schott-bottle to PBR port 2. The specified Channel media is shown in Table 1. The Slope and Intercept values and the Flow rate set point range values were determined according to the experiment results (see Section 4). Moreover, the pump can be switched on and off while Labview is running clicking Mode arrows, not the Media pump button. The growth rate of the culture is calculated by: (Time media pump is on)*(Pump rate). The time span can be chosen to calculate the growth rate (average). The second register card is Light intensity (Figure 30(2)). Two types of light can be configured. In our PBR system we only have LED panels, so Light intensity control (fluorescent lamps) section should be avoided. Regarding LED panels, LED panel A and B buttons have to be clicked to On mode. All A and B Channels have to be filled out with number 0. The operation mode has to be chosen in Source and Manually [µE] or [V]/[mA] are recommended. Depending on the mode clicked, both A and B LED panels 21 1 2 3 4 5 6 7 Figure 29. Labview user graphic interface. (1) Warning window; (2) program information window; (3) Welcome card; (4) Setting register card; (5) Graphic register card; (6) Control buttons; (7) General operation unit. 22 Figure 30. Labview user graphic interface. (1) Media flow rate and turbidity card; (2) Light intensity card; (3) Gas control card; (4) Gas measurement card; (5) pH, temperature and redox card; (6) Check filling levels and gas pressure card; (7) General AI/AO card; (8) Ports and Channels summary card; (9) Options/files card. 1 2 3 4 5 6 7 8 9 boxes have to be filled out in Manually section, which refers to radiation intensity ([µE] or [V]/[mA]). If a value of 0µE or 0 [V]/[mA] is set in this last section for both panels, they will be switched off. It does no work for only one panel. Finally, Slope and Intercept values for both panels allow the radiation intensity regulation. Gas Control card is related to the Gas Flow Controllers (GFCs) (Figure 30(3)). Our devices are Aalborg brand, so only Gas mass flow control (Aalborg) section should be configured. Firstly, the On button should be pressed. As our PBR system has two GFCs (Air GFC and CO2 GFC), two connected controllers have to be selected with their respective names, flow rate setpoints, maximal flow rates and channels. The Air GFC has a flow rate range between 0-500 mL·min-1 and a maximal rate of 500 mL·min-1. However, the CO2 GFC has a range of 0-50 mL·min-1 and hence, a 50 mL·min-1 maximal rate. Their respective AO and AI channels values are in Table 1. They can be switched off, changing the flow rate setpoint to 0. Besides, GFCs may overheat if the gas flow rate is too low while the controller’s valves are turned open. Flow rate safety shutter is a feature that stops the controllers from overheating and closes the valve if the measured flow rate is smaller than half of the flow rate setpoint. Therefore, the controllers are turned off and eventually they should not be restarted. Also, it is recommended that Flow rate safety shutter is turned off for a short time and that the gas flow rate is controlled manually and regularly to ensure the safety of the controllers. The next card, Gas measurement, is related to O2 sensor and Air GFM (Figure 30(4)). In the first section, Gas content (Bluesens), the On button should be pushed. Only one controller should be connected since we only have one O2 sensor in the system. Moreover, the type of connection Bluesens sensor has should be specified, since as it was mentioned above, the sensor is connected to the computer by a serial USB cable adapter to COM5. Thus, serial/USB port and COM5 should be chosen by clicking in the arrows. Next, in Total mass flow (gas out) section the Air GFM configuration should be set. The main button should be turned on and the AI channel box should be filled out with its specific value (see Table 1). AI AO DI DO Media pump 3 LED pannels 0 Air GFC signal/setpoint 6 1 CO2 GFC signal/setpoint 8 2 Air GFM signal 10 pH signal 0 Temperature signal 2 Pump small lower (BASE) 0 Pump small upper (ACID) 1 CO2 GFM signal 12 23 Table 1. Specific channels values for each device. AI is referred to Analog Input signal, AO to Analog output, DI to Digital input and DO to Digital output. Here, the CO2 GFM configuration cannot be set since the program came with this configured interface. It will be checked in a following card. Finally, CO2 in liquid section should be avoided for our PBR system. pH, temperature and redox card is used to configure the pH and temperature sensors (Figure 30(5)). The last Redox sensor section should not be filled out since no redox sensor is in our system. Regarding the pH meter, it should also be turned on and the analog input channel has to be set according to Table 1. Slope and intercept values were adjusted based on calibration results (see section 4). Moreover, to control the pH automatically, Control pH button should be pressed and pH setpoint, upper tolerance and lower tolerance values should be chosen depending on each culture experiment. Upper and lower tolerance are referred to the tolerable pH deviation from pH setpoint without acid/base pumps activation. Thus, these pumps will turn on if the pH value is higher or lower than setpoint plus/minus the tolerance. Furthermore, acid and base channels have to be filled out based on Table 1 and output type must be changed to digital. Pump acid and Pump base buttons allow pump activation despite the fact that pH value is within the proper range. Both pumps can be turned off unclicking Control pH button, even if Labview is running. Concerning Temperature sensor section, the same steps as pH meter should be performed. Nevertheless, Check filling levels and gas pressure and Ports and channels summary cards (Figures 30(6) and 30(8)) should not be filled out with any configuration data and in General AI/AO card (Figure 30(7)), only CO2 GFM information has to be set. In this card, both PCI all analog input and PCI all analog output sections have to be turned on and only their first row will be completed with CO2 GFM name and its AI/AO channel value displayed in Table 1. Lastly, in Options/files card (Figure 30(9)), the above configuration could be saved in a new file or conversely, a configuration file could be loaded instead of configuring all card parameters. Save data as… is to create a new file in which all photobiological process data will be saved once Labview is running. Other option is to save the data in a file that already exists by checking the Add data to file name checkbox. The time interval at which the data is saved, could also be chosen. Once all parameters were set and files were saved, the Start button can be pressed and Labview will begin running. While the program is running, all the above mentioned parameter values can be regulated and changed to other adequate values. The signal channels shown in Table 1 are referred to the pin connection each device has with the Control Box. Hence, this values are important for the communication and activation of PBR system devices. In most of the cards there are three repeated features, Signal type (Differential, Single-Ended), Output (Voltage, Current) and Output type (Analog and Digital). These properties must not be changed because they specified the type of electrical connection between the devices and the Control Box . Finally, in the Graphic register card there are four graphics that could give an overview of the raw parameter data evolution during the photobiological process. The graphic time scale can be changed by clicking in the arrows. Each graphic can register until eight different plots of different parameters. Graphic data and graphic representation can be saved by clicking on the right mouse button. The graphic representation is saved by clicking on Export Simplified Image, while the data can be exported to an Excel document or copied to Clipboard and pasted in a text file (i.e. Microsoft Word). 24 3. 4. Device calibration procedure Once the PBR is assembled and the computer programs performance is known, the next step is the calibration of some devices that require it. These devices are both GFCs, both GFMs, the O2 sensor, the pH meter and LED panels. 3.4.1. Gas Flow Controllers (GFCs) and Gas Flow Meters (GFMs) GFCs and GFMs could not be calibrated because currently we do not have access to a specific type of equipment that includes a flow calibration standard, a certified high sensitive multimeter (which together have a collective accuracy of ±0.25% or better), a flow regulator installed upstream from the device and a pressure regulated source of dry filtered reference gas. Furthermore, a bottle of a reference gas is need but it could not be bought so far because of funding shortage. Once the calibration material is available, the calibration procedure provided at each device manual should be followed. 3.4.2. O2 sensor O2 sensor calibration corresponds to BacVisSingle Zero Adjustment. To get started with the calibration procedure, the following steps must be taken: 1. Connect the sensor to the power and wait about 45min-1hour until the sensor is warm. 2. Turn on the air flow (room air) and open the BacVisSingle program. 3. Firstly, perform a measurement (no Zero Adjustment) (review section 3.3) and wait until O2% value is stable (usually 30 min and small changes are tolerable). 4. Once the measurement is ended, start Zero Adjustment, which will take other 30 min. 5. Finally, close BacVisSingle. LabView can now be started. 3.4.3. pH meter The pH meter can be calibrated in two ways: using the DinModule or the three pH transmitter buttons. In both cases, you will need distilled water and the two pH standard solution whose pH must be matched to the pre-defined standards (see cal. key1 and key 2 in Figure 28). Via DinModule is recommended since there are more calibration options and the configuration can be modified. Here, calibration using DinModule is only detailed, for calibration using pH transmitter buttons see pH meter Manual. If DinModule is used, the following steps must be taken: 1. Connect the pH transmitter to the computer using the USB cable with a Phone connector. 2. Open DinModule program and configure the COM-Port in Setup and the Device Address. 3. Review Calibration parameters in Calibration button. If NBS standards are used, fix temperature checkbox must be checked. 25 4. Press Calibration button to start the menu-guided calibration. 5. Rinse the electrode with distilled water and dry it with paper. 6. Immerse the electrode in the first pH standard solution (cal. key1 in Figure 27) and confirm the immersion with OK in the first pop-up window. 7. Stir the solution and wait until the pH value is stabilized (pay attention to the necessary response time and temperature equilibria) and then press OK. 8. Enter the pH value of the used standard (at the actual temperature) or select the nominal value of the buffer solution at 25 °C of stored buffer sets. 9. Take the electrode out of the first standard solution, rinse it with distilled water, dry it with paper and immerse it in the second pH standard solution. Confirm it with OK. 10. Repeat the steps 7, 8 and 9 with the second standard solution. 11. Once the calibration procedure finishes, a new dialog window appears with the new calculated characteristic sensor data (calibration data). If Save button is pressed, these new data will be saved in Configuration information, otherwise if Exit is pressed, data will not be saved. 3.4.4. LED panels Finally, LED panels can also be calibrated. For this procedure, a radiometer is needed. A radiometer is a device that measures the radiant flux (light energy) of electromagnetic radiation. The calibration procedure requieres of 100µE of light intensity and a 2 cm distance between LED panels and the PBR, since this is the proper distance for cultivation process. Both panels have to be calibrated separately. The following steps should be taken for the calibration: 1. Set one LED panel at a 2 cm distance from the PBR. 2. Start Labview, configure 100 µE of light intensity and run it. 3. Place the radiometer sensor over the PBR plastic surface and take the measurements diagonally, from an upper corner to a bottom corner. Measurements have to be taken en each LED line and between the lines. 4. With light measurements and the voltage, the calibration graphic can be plotted with the specific slope and intercept of the linear regression line. 5. Repeat the process for the other LED panel. 26 3. 5. Characterization experiments 3.5.1. Culture medium pump flow rate characterization For the media pump flow rate characterization (mL·min-1), it was used a full water Schott-bottle, testtubes (250 and 100 mL) and a stopwatch. The pumped deionized water volume from the Schott-bottle to the test-tube at 2 min and at different flow rate setpoints was measured (0.1, 0.5, 0.8, 1, 2, 3, 4, 5, 10, 20, 50, 100, 200 and 500 mL·min-1). Two minutes was an adequate time for pump warming and detecting the right pumped water volume. Moreover, different flow rate setpoints were also detected by changing the Labview slope and intercept of the media pump (see Figure 30(1)). 3.5.2. pH control For acid and base pumps characterization, it was used a full water Schott-bottle, a test-tube (100 mL) and a stopwatch. In this case, pumped deionized water volume from the Schott-bottle to the test-tube was measured at different times (1, 2, 3, 4 and 5 min), but at the same flow rate setpoint. Remember that these pumps can only be switched on and off, so they only have a specific flow rate revolution. To characterize pH meter operation and Labview functioning, an experiment in which the pH was changing in a water solution over time, was performed. This experiment allowed to see whether the pH meter measured properly, whether the program could detect pH changes and how it respond to these changes activating acid or base pump, increasing or decreasing the pH value. Besides, the pH tolerance setpoint was also studied in order to see if it had any influence in achieving an specific pH setpoint. Prior to the experiment, the PBR was filled up with deionized water, air bubbling was activated and acid (pH 3) and base (pH 11) solutions were prepared. It was waited until CO2 saturation in water and the pH was measured at zero time, being around 5.73 —although it varied due to CO2 solubility in water. It was adjusted at pH 6.5, as initial pH value. During the experiment, Labview pH setpoints were changed to 7, 6.5 and 7.5, in that order. The tolerance was also changed and interested values were 0.01, 0.05, 0.1 and 0.2. 3.5.3. Optics experiments. LED panels characterization For optics experiments, a radiometer was used. This instrument measures light radiation intensity in the electromagnetic spectrum (200-1200 nm). Thus, the radiation intensity emitted by one LED panel was measured at three different LED radiation powers (maximum, medium and minimum), at three panel locations (upper right side, middle central side and bottom left side) and at three different distances from the LED panel (2, 3 and 4 cm). Besides, one specific measurement was also taken in the dark — turning off the artificial light sources and blocking sunlight pass through the window — to see whether these other two light sources were having influence in LED light measured values. In addition, PBR plastic surface transmittance was measured. For this, LED radiation intensity at minimum power, on the middle central side and at 3 cm distance was measured and compared with the previous result without the PBR surface. 27 3.5.4. Warming process and heat transfer The last experiment consisted on studying the warming process, specifically, the heat transfer from LED panels to the PBR culture at different LED panels distances (1, 2 and 3 cm). Prior to the experiment, the PBR was filled up with deionized water, air bubbling was activated and the temperature was measured at time zero. Afterwards, temperature measurements were taken each 5 min, during 90 min. Furthermore, in order to maximize the heat transfer and basing on optics experiments’ results, the radiation intensity was set at its maximum value and the thermometer was placed in the middle central side of the PBR. 28 4. Results and Discussion The aim of this work is the assembly, set-up and operation of our 1L scale-lab flat-bed photobioreactor system. For that, a set of steps were carried out (see Figure 1). All these steps require a biological knowledge of the cyanobacteria, the interdependence of the parameters and its effects on cultivation and on the photobiological process. First, all the parts were characterized in order to know how they operate and with which other parts they have to be connected. Once all features were understood, the assembly process was performed as it can be visualized in Figure 22. Each device position and each connection was selected taking into account the cyanobacteria requirements and their cultivation process. Afterwards, it was checked whether everything was properly connected using the already described computer programs. Corresponding program manuals were read for this purpose and their main characteristics are shown in this work (see section 3.3). Subsequently, device manuals were also read to know their operational mechanisms and how they should be calibrated. With that information, calibration procedures were performed following the steps described in section 3.4. GFCs and GFMs could not be calibrated due to lack of equipment. However, the rest of devices were correctly calibrated according the specifications described before. It should be remarked that LED panels radiation can only be regulated by changing slope and intercept values in the corresponding software menu, and not directly changing their intensity value in Manually section (see Figure 30). Finally, a set of experiments were performed to characterize the operation ranges of all PBR system components. Moreover, these experiments were also useful to ensure that the assembly process was done correctly and to have a better knowledge on how the computer programs have to be used. The characterization experimental results gave relevant information of how the system should operate during real cultivation process. For that, knowledge about the cyanobacteria cultivation procedure, i.e. the devices operation ranges should be previously known. 6.1. Culture medium pump flow rate characterization It is known that the maximal reached growth rate of Synechocystis sp. PCC6803 is 0.135 h-1 (Zavřel et al., 2015). However, in normal conditions, growth rates are lower, 0.02-0.03 h-1 (Kwon, Rögner and Rexroth, 2012), since there is not enough light and nutrients supply in typical PBR operation conditions. Part of the light that cyanobacteria receive, specially for the case of cells next to the surface, is dissipated as heat or fluorescence, and so light does not reach cells in the internal side of the PBR volume. But, a light intensity increment will cause photoinhibition phenomenon. Thus, it is very difficult to optimize the light. Moreover, the culture medium is not suitable for high culture densities, so it is very difficult to reach optimum growth rates in a photobioreactor (Lopo et al., 2012; Shuler and Kargi, 2002). Consequently, as growth rates are lower, the culture dilution rate in this type of reactors should also be lower, in order not to wash out the culture. Furthermore, for the maximal growth rate of 0.135 h-1, its respective culture dilution rate can be calculated and it is 2.25 mL·min-1. So a 3 mL·min-1 dilution rate will be enough for cyanobacteria cultivation at high growth rates. 29 Therefore, the operational flow rate range of our pump was studied, with a special emphasis in lower rates from 1 to 5 mL·min-1. For this first experiment, we assumed that the culture medium pump was well calibrated, so we worked with a Labview slope value of 1 and an intercept of 0. As it can be appreciated in Figure 30(A), at flow rate values lower than one, the pump does not work. In fact, it does not operate either with decimalized values (data not shown). Moreover, we found that at ranges of interest (1-5 mL·min-1), larger volumes than expected were pumped. This fact indicates that the pump should be adjusted changing slope and intercept values in software entry. In addition, at 5 mL·min-1 flow rate, the pump reaches its maximum rate, pumping around 200 mL·min-1. At higher rates, the peristaltic pump moves the same water volume (Figure 31(A)). This indicates that the inner diameter of the silicone tube is crucial for the flow rate. At higher inner diameter of the tube, larger volumes can be pumped. This maximum rate could be used to filling up the PBR with the culture medium in the initial experimental stages. As the first obtained results were not adequate for our purpose, we tried to calibrate the pump adjusting Labview slope and intercept values. As a 2.25 mL·min-1 dilution rate is needed for the maximal growth rate. We wanted to see if a 3 mL·min-1 rate is pumped with our pump, since this rate would largely cover high growth rates. Henceforth, we tried to adjust a 3 mL·min-1 rate with our pump. To this end, first the intercept was set to zero but the slope value was increased in one unit each time. It was found that at slope 4, the pump does not work and that at slope 2, a volume of 8 mL·min-1 was pumped; however, the pumped volume at slope 3 was closer to the objective. Fixing this last slope value and modifying the intercept, it was found that 3 mL·min-1 were pumped at slope 3 and intercept (-0,5) (see Figure 31(B)). Henceforth, a 3 mL·min-1 rate can be fixed in our culture medium pump. This rate allows to dilute the culture, set a desirable growth rate by matching dilution rate to growth rate and maintain an exponential growth and stable experimental conditions. Thus, our pump is suitable to largely cover cyanobacteria cultivation. 30 Figure 31. (A) Water volume pumped per min at different flow rate setpoints (with slope 1 and intercept 0) and (B) at different slope and intercept values. Each point represent the resulting volume measured in the test-tube after 1 min. Pumped volume in 1 min (mL) 0 20 40 60 80 100 Flow rate setpoint of the media pump (mL/min) 0 1 10 100 1.000 0 0 0 3 28 52 79 96 95 98 96 99 98 98 Final volume in 250mL tube-test. Volume pumped in 1 min (mL) 0 1,2 2,4 3,6 4,8 6 Slope + intercept values 3 + 1 3 + 0 3 + (-0,5) 3 + (-1) 0 1 3 6 Final volume in 100mL tube-test. 5. Future prospects Some further actions have to be taken if cell cultures are to be grown. Thus, regarding the assembly process, some components have to be placed to carry out culture experiments. Air filters as well as check and Safe-Site valves should be placed in accordance to Figure 21. Moreover, a feed port specific for ISO glass jars should be put in each Schott-bottle stopper (see Figure 38). This piece has two ports, one is used for liquid output and the other one allows air inlet, preventing vacuum generation inside the Schott-Bottle. An air filter has to be placed in the gas inlet to avoid liquid contamination. Moreover, the thermal circulator and the CO2 cylinder have to be bought and also the 10/12 silicone tubes for water movement between the thermal circulator and the heat exchanger. The waste container should be replaced by a container suitable for biological waste. If it is wanted to carry out more accurate light experiments, some guide-rails can be bought to fix LED panels and control its distance to the PBR. A scaffolding or a block structure for gas devices (GFCs, GFMs and O2 sensor) could also be built. Hence, gas devices can be fixed to it, being protected from impacts and making its visualization easier. Besides, a gas-liquid phase separator could also be bought. When liquid overflows through port 6, some gas can flow out with it too. So, the phase separator will separate both fluids and the outlet gas could be recirculated and mixed with port 5 outlet gas to get more accurate gas measurements by Air GFM and O2 sensor. However, these recommendations are not strictly necessary since the PBR can also operate without them. Furthermore, if the PBR system is moved to another laboratory, the next aspects should be taken into consideration: the laboratory has to be adapted to have a CO2 cylinder inside and must have specific equipment for the photobiological process. In addition, the air compressor has to be placed in a different bench, apart from the other PBR system components, since the compressor vibrates when it is operating and these vibrations can affect the other devices and even, the photobiological process. Further characterization experiments could be done before cultivation. The first one is a mass transfer characterization. For that, turbulence should be measured inside the PBR at different gas flow rates. Each gas flow rate produces a specific inlet gas pressure, resulting in a particular bubble size. Lower bubble sizes facilitate mass transfer, since the surface area per volume unit increases and the ascent rate is lower, remaining the bubble more time in contact with the culture. However, higher bubble sizes increase culture turbulence and mixing process but cause greater cell damage which could be detrimental for the bioprocess. Thus, finding a trade-off between the turbulence and the cell damage is important for the culture. The second experiment is about heat transfer characterization. It could be interesting to know the water flow be pumped by the thermal circulator, the water temperature for culture cooling and the time it does take to reduce the culture temperature one degree Celsius. 37 Figure 38. Schott-bottle with the feed port specific for ISO glass jars and the air filter. Furthermore, it would be interesting to carry out a few more optics experiments for LED panels light characterization. It could be studied whether there are significant differences between dark and light conditions, to check at which specific wavelengths the PBR plastic surface absorbs and also verify whether the culture medium BG11 absorbs light due to its mineral composition. Finally, a sterilization process for most of the PBR components should be performed before cultivation. This is the most important step before cultivation, since if it is not properly done, there will be biological contamination, cyanobacteria will grow suboptimally and the overall photobiological performance will be reduced. A chemical sterilization protocol by incubation with 5mM peroxyacetic acid solution for 1 h can be found in PBR Manual. 38 6. Conclusions All the proposed objectives have been achieved and can be summarized by the following results: 1. The assembly and start-up of a 1L lab-scale flat-bed photobioreactor system was performed. For that, all the parts, components and devices were first characterized to know their specifications and operational principles. 2. Thanks to the data collected during the characterization process and taking into account previous biological knowledge in cyanobacteria cultivation and photobiological processes, all components could be interconnected and assembled making up the photobioreactor system. 3. According to software manuals specifications and additional information supplied by the producer company, the correct program functioning could be assessed. Besides, computer programs were used to ensure that the assembly and set-up were performed properly. 4. Calibration procedures were determined and correctly done thanks to the information provided by their respective manuals. 5. 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