Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

1. Growth system: outdoor open raceway pond settings

1. Set up the open raceway ponds close to the flue gas source (containing 8–10% CO₂). Ensure water and electricity are available at the pond reactor location and that the reactor is not in the shade the majority of the day (Figure 1).
2. Capture flue gas during the post-combustion process using a 0.95 cm fuel hose, a few meters before the flue gas enters the stack to be discharged into the atmosphere (Figure 2).
3. Remove water from the flue gas using a 20 L water trap and a condenser (coil length ~12 m) between the stack and the compressor (Figure 2).
   NOTE: Flue gas typically contains approximately 9‒13.8% water²⁸. In addition, the condenser and pipeline cool the flue gas¹⁶.
4. Connect the following sensors to a datalogger to monitor algal growth: (1) a real time optical density sensor²⁹, which measures absorbance at two wavelengths—650 and 750 nm—and can detect a maximum algal cell concentration of 1.05 g/L; (2) a DO sensor; (3) air and pond thermocouples; (4) a pH sensor; and (5) an EC sensor.
   NOTE: Additionally, the pH and EC sensors are connected to a transmitter. The data logger unit configuration is shown in Figure 3.
5. Ensure that all components of the algal growth system are calibrated and properly working before inoculation.

2. pH control system

1. Manage flue gas injection by using a compressor, a control valve system, and the data logger program, as shown in Figure 2 and Figure 3 (Supplementary material A).
2. Use a tube to direct the flue gas from the control valve to the bottom of the raceway pond through a stone diffuser.
3. Inject the flue gas into the growth system based on pH. When the pH value is greater than 8.05, the system will inject flue gas, whereas when the pH is less than 8.00, the system will stop the flue gas injection in periods of no growth. The flow rate is measured in standard liters per minute (SLPM).
   NOTE: In the control valve, the inlet flue gas pressure is limited to a maximum of 50 psi.

3. Algae selection and strain maintenance (light and temperature)

NOTE: The green algae Chlorella sorokiniana DOE 1412 was isolated by Juergen Polle (Brooklyn College)^30,31 and selected by the National Alliance for Advanced Biofuels and Bioproducts (NAABB); its selection was based on the previous strain characterization studies performed by Huesemann et al.^32,33 . Their research regarding algal screening, biomass productivity, and climate-simulated culturing (e.g., temperature and light) in the Southwest region when using outdoor open raceway ponds informed the method used in this project.

1. Maintain cultures at room temperature (25 °C) using a 12 h/12 h light/dark cycle.
2. Keep light intensity at 200 µM/m²/s for culture maintenance grown on plates and in small liquid cultures (50 mL to 500 mL).
3. Keep light intensity for scale up grown in liquid cultures 50 mL to 500 mL at 400 µM/m²/s, and liquid cultures 5 L to 20 L at 600‒800 µM/m²/s.

4. Scale up and quality control

1. Prepare the BG11 culture medium using deionized water and the following salts, for macronutrients, in g/L: 1.5 NaNO₃, 0.04 K₂HPO₄, 0.075 MgSO₄*H₂O, 0.036 CaCl₂*H₂O, 0.006 (NH₄)₅Fe(C₆H₄O₇)₂, 0.006 Na₂EDTA*2H₂O, 0.02 Na₂CO₃; add 1 mL/L of trace element solution, which contains the following micronutrients in g/L: 2.86 H₃BO₃, 1.81 MnCl₂*4H₂O, 0.22 ZnSO₄*7H₂O, 0.39 Na₂MoO₄*2H₂O, 0.079 CuSO₄*5H₂O, 0.0494 Co(NO₃)₂*6H₂O.
   NOTE: For plate inoculation and/or long-term storage, add 7.5 g/L of Bacto agar; for culture inoculation, no addition of agar is needed. Sterilize culture medium in the autoclave for 21 min at 121 °C.
2. Pour the BG11 medium with agar into Petri dishes in a sterile laminar flow hood or biosafety cabinet. Once plates are firm and cool, pipette 500 µL from a re-suspended frozen algal stock culture and add Ampicillin (100 µg/mL); incubate the algal plates in a shaker table (120 rpm) for 1 to 2 weeks.
3. Use a sterile loop to select a single algal colony from a culture plate and inoculate it in a 50 mL tube containing sterile growth medium in a clean biosafety cabinet. Grow the small liquid culture on a shaker table (120 rpm) for one week.
4. Transfer 50 mL of algae culture (linear growth phase, OD_750nm ≥ 1) into a 1 L flask with 500 mL liquid medium. Fit each flask with a rubber stopper and stainless-steel tubing to provide aeration. Filter the air using 0.2 µm air sterilization filters. Let the culture grow for one to two weeks. Monitor cell density using a spectrophotometer (OD_750nm).
5. Place the 500 mL liquid culture into a 10 L carboy containing 8 L of non-sterile culture medium and inject a mixture of 5% CO₂ and 95% air. Then, cultivate algae under the same conditions as in step 4.4.
6. Monitor stock plate and liquid cultures (in steps 4.2‒4.5) once a week. Take an aliquot and observe it under the microscope at 10x and 40x magnification to ensure the growth of the desired strain. Kept cultures until they have been compromised or used for experiments. Discard contaminated cultures.

5. Concentrated medium preparation for open pond cultivation

1. To prepare trace elements solution partially fill a 1 L volumetric flask with distilled water (DW). Insert a magnetic stir bar and add the chemicals shown in Table 1 sequentially. Ensure that each ingredient dissolves before the addition of the next constituent. Remove the magnet and fill the flask to the 1 L volume mark.
2. Partially fill a 1 L glass bottle with DW and insert the magnetic stir bar. Place the container on the top of a magnetic stirrer plate and add the chemicals for the reactor’s final volume, adding them sequentially, ensuring each fully dissolves. Table 2 lists the chemicals to prepare 1 L of medium, so multiply all the values by the reactor’s final volume. Fill the glass bottle to 1 L.

6. Outdoor open raceway pond inoculation

1. Thoroughly clean the reactor using 30% bleach before each inoculation and after harvesting. It is recommended to leave the bleach overnight. Rinse the reactor well to remove all bleach.
2. Calibrate all the sensors before algae inoculation according to their corresponding calibration procedure.
3. Dilute the concentrated media (in step 5) using the water source by filling the raceway pond up to 80%.
4. Inoculate the reactor using a 10 L carboy filled with algae (linear growth phase OD_750nm > 2) and bring it to its final volume.
5. Acclimate microalgae by partially shading the raceway pond with wooden pallets for ~ 3 days (Figure 4), once the exponential phase has passed, as an adaptation strategy to avoid photoinhibition.
   NOTE: This period will also provide time for the microalgae to adapt to the stress caused by the direct injection of flue gas.

7. Batch growth experiment at the generating station

1. Inspect and record any day-to-day variations including water evaporation, paddlewheel motor, sensor functionality, and anything out of the ordinary.
2. Drain and inspect the compressor and water trap every day to remove any excess water to minimize corrosion since flue gas is highly corrosive³⁴.
3. Configure the data logger to scan each sensor measurement every 10 s and to store the average data every 10 min. These include DO, pH, EC, real time optical density as well as air and reactor temperature.

8. Discrete sampling and monitoring

1. Make sure water level remains constant at the reactor’s final volume otherwise the optical density measurement will be affected.
2. After replenishing water in the reactor, take a 5 mL sample for cell mass measurements by optical density (540, 680, and 750 nm) using an ultraviolet-visible spectrophotometer. Repeat the process daily.
3. Take a 500 mL sample three times per week for microscope observations and biomass concentration based on ash-free dry weight (AFDW).
   1. Perform microscope observations with 10x and 40x objective lenses. Additionally, these microscope magnifications are used as part of the algal quality control described in step 4.6.
   2. Use 400 mL of the sample in step 8.3 for AFDW
      1. Set each 0.7 µm pore size glass microfiber filter in an aluminum foil tray and pre-treat each aluminum foil tray/filter using a furnace for 4 h at 540 °C.
      2. Label each aluminum foil tray using a #2 pencil, record its weight (A), and place it in the vacuum filter apparatus.
      3. Stir the algae sample vigorously before measuring out a volume to be filtered. Filter enough algae sample to give a pre/post ash weight difference of between 8 and 16 mg. Pick a weight difference to use throughout the course of the experiment and keep this value constant.    
      4. Place each filter containing the algae sample in its foil tray in the oven at 105 °C for at least 12 h.
      5. Remove the foil tray/filter from the drying oven and place it in a glass desiccator to prevent water uptake. Record each foil tray/filter weight (B).
      6. Place the foil tray/filter in the 540 °C muffle furnace for 4 h.
      7. Turn off the muffle furnace, cool down foil trays/filters, place them into the desiccator, and record each foil tray/filter weight (C).
      8. Calculate AFDW using gravimetrical analysis:
         % AFDW= C – A x 100 / B
4. Hold 2 L of algae before harvesting for microwave-assisted extraction (MAE) lipid extraction analysis using solvents.
   1. Centrifuge the algae sample at a relative centrifugal force (RFC) of 4,400 x g for 15 min. Take the algae pellet and dry it using an oven at 80 °C for at least 24 h.
   2. Grind the algae sample and weigh the algal powder (recommended biomass ranges from 0.3 g to 0.5 g).
   3. Add the algae powder (dry algal biomass) into the microwave accelerated reaction system (MARS) Xpress vessels, add 10 mL of chloroform:methanol (2:1, v/v) solvent solution under the hood, close the vessels, and let stand overnight.
   4. Place the vessels into the MARS machine using the solvent sensor for 60 min at 70 °C and 800 W of power.
   5. Take vessels out of the MARS and let them cool down under the hood.
   6. Use a funnel and glass wool to separate the liquid part which contains chloroform, methanol, and lipids by transferring each liquid sample to a pre-weighed glass test tube and keep the solids (biomass free of lipids) for other analyses.
   7. Take the test tubes containing the lipids to the nitrogen evaporator, remove them once the liquid has been evaporated, and then leave the tubes overnight under the hood to ensure complete dryness.
   8. Calculate lipid content (wt. %) using gravimetric analysis:
      Lipid content (wt. %) = Dry biomass of lipids x 100/ Dry Algal mass

9. Algal harvesting and crop rotation

1. Harvest 75% of the total algae culture volume when the culture is close to reaching the stationary phase. Take 2‒5 L of culture to perform biomass productivity analyses in the laboratory. Process and convert the rest of the algae into the desired algal products.
2. Re-grow the open raceway pond by using the 25% algae remaining as inoculum. Add water up to 80% of the total reactor’s volume, add the concentrated media, and then finish filling up to the reactor’s final volume if necessary.
3. Cultivate the appropriate algae strain according to the season, based on temperature and light intensity conditions.

10. Data management

1. Record data in the data logger and collect for analysis as in step 7.3.
2. Consider saving raw and analyzed data in the Regional Algal Feedstock Testbed (RAFT) share drive. RAFT project collaborators contribute their data to simulate and model algal productivity and validate outdoor cultivation.