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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

Abstract

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

Introduction

Global warming is one of the most important environmental issues that the world faces today1. Studies suggest that the major cause is the increase in greenhouse gas (GHG) emissions, mainly CO2, in the atmosphere due to human activities2,3,4,5,6,7. In the U.S., the largest density of CO2 emissions originates mainly from fossil fuel combustion in the energy sector, specifically electric power generation plants3,7,8,9. Thus, carbon capture and utilization (CCU) technologies have emerged as one of the major strategies to reduce GHG emissions2,7,10. These include biological systems that utilize sunlight to convert CO2 and water via photosynthesis, in the presence of nutrients, to biomass. The use of microalgae has been proposed due to the fast growth rate, high CO2 fixation ability, and high production capacity. Additionally, microalgae have broad bioenergy potential because the biomass can be converted into products of interest, such as biofuels that can replace fossil fuels7,9,10,11,12.

Microalgae can grow and achieve biological conversion in a variety of cultivation systems or reactors, including open raceway ponds and closed photobioreactors13,14,15,16,17,18,19. Researchers have studied the advantages and limitations that determine the success of the bioprocess in both cultivation systems, under either indoor or outdoor conditions5,6,16,20,21,22,23,24,25. Open raceway ponds are the most common cultivation systems for carbon capture and utilization in situations where flue gas can be distributed directly from the stack. This type of cultivation system is relatively inexpensive, is easy to scale up, has low energy costs, and has low energy requirements for mixing. Additionally, these systems can easily be co-located with the power plant to make the CCU process more efficient. However, there are some drawbacks that need to be considered, such as the limitation in CO2 gas/liquid mass transfer. Although there are limitations, open raceway ponds have been proposed as the most suitable system for outdoor microalgal biofuel production5,9,11,16,20.

In this article, we detail a method for microalgae cultivation in open raceway ponds that combines carbon capture from the flue gas of a natural gas power plant. The method consists of a semi-automated system that controls flue gas injection based on the culture pH; the system monitors and records the Chlorella sorokiniana culture status in real time using optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperature sensors. Algal biomass and flue gas injection data are collected by a data logger every 10 min at the Tucson Electric Power facility. Algae strain maintenance, scale up, quality control measurements, and biomass characterization (e.g., correlation between optical density, g/L, and lipid content) are performed in a laboratory setting at the University of Arizona. A previous protocol outlined a method for optimizing flue gas settings to promote microalgae growth in photobioreactors via computer simulation26. The protocol presented here is unique in that it utilizes open raceway ponds and is designed to be implemented on-site at a natural gas power plant in order to make direct use of the flue gas produced. Additionally, real time optical density measurements are part of the protocol. The system as described is optimized for a hot semiarid climate (Köppen BSh), which exhibits low precipitation, significant variability in precipitation from year to year, low relative humidity, high evaporation rates, clear skies, and intense solar radiation27.

Protocol

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, OD750nm ≥ 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 (OD750nm).
  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 OD750nm > 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.

Results

Prior experimental results from our lab indicate that microalgae cultivation using a semi-automated open raceway pond can be coupled with carbon capture processes. To better understand the synergy between these two processes (Figure 2), we developed a protocol and tailored it for cultivating the green algal species Chlorella sorokiniana under outdoor conditions in a hot semiarid climate. Natural gas flue gas was obtained from an industrial power generation station. This protocol use...

Discussion

In this study, we demonstrate that synergistically coupling flue gas carbon capture and microalgae cultivation is possible in a hot semi-arid climate. The experimental protocol for the semi-automated raceway pond system integrates state-of-the-art technology to monitor relevant parameters in real time that correlate to algal growth when using flue gas as a carbon source. The proposed protocol is intended to reduce uncertainty in algal cultivation, which is one of the main drawbacks of raceway ponds20

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported through the Regional Algal Feedstock Testbed project, U.S. Department of Energy DE-EE0006269. We also thank Esteban Jimenez, Jessica Peebles, Francisco Acedo, Jose Cisneros, RAFT Team, Mark Mansfield, UA power plant staff, and TEP power plant staff for all their help.

Materials

NameCompanyCatalog NumberComments
Adjustable speed motor (paddle wheel system)Leeson174307Lesson 174307.00, type: SCR Voltage; Amps:10
Aluminum weight boatsFisher Scientific08-732-102Fisherbrand Aluminum Weighing Dishes
Ammonium Iron (III) (NH?)?[Fe(C?H?O?)?]Fisher Scientific1185 - 57 - 5Medium preparation. Ammonium iron(III) citrate
Ammonium PhosphateSigma-Aldrich7722-76-1This chemical is used for the optimized medium
Ampicillin sodium saltSigma AldrichA9518-5GThis chemical is used for avoiding algae contamination
AutoclaveAmerex Instrument IncHirayama HA300MII
Bacto agarFisher ScientificBP1423500Fisher BioReagents Granulated Agar
BleachCloroxGermicidal Bleach, concentrated clorox
Boric Acid (H3BO3)Fisher Scientific10043-35-3Trace Elelements: Boric acid
Calcium chloride dihydrate (CaCl2*2H2O)Sigma-Aldrich10035-04-8Medium preparation. Calcium chloride dihydrate
Carboys (20 L)Nalgene - Thermo Fisher Scientific2250-0050PKPolypropylene Carboy w/Handles
CentrifugeBeckman Coulter, IncJ2-21
ChloroformSigma-Aldrich67-66-3This chemical is used for lipid extraction
Citraplex 20% IronLoveland ProductsSDS No. 1000595582 -17-LPIhttps://www.fbn.com/direct/product/Citraplex-20-Iron#product_info
Cobalt (II) nitrate hexahydrate (Co(NO3)2*6H2O)Sigma-Aldrich10026-22-9Trace Elements: Cobalt (II) nitrate hexahydrate
CompressorMakitaMAC700This equipment is used for the injection CO2 system
Control ValveSierra InstrumentsSmartTrak 100This item needs to be customized for your application. In our case, it was used a 5% CO2 and 95% air mixture.
Copper (II) Sulfate Pentahydrate (CuSO4*5H2O)Sigma-Aldrich7758-99-8Trace Elements: Copper (II) Sulfate Pentahydrate
Data Logger: Campbell unit CR3000Scientific CampbellCR3000This equipment is used for controlling all the system, motoring and recording data
Dissolvde Oxygen SolutionCampbell Scientific14055Dissolved oxygen electrolyte solution DO6002 - Lot No. 211085
Dissolved Oxygen probeSensorex?DO6400/T Dissolved Oxygen Sensor with Digital Communication
Electroconductivity calibration solutionRicca Chemical Company2245 - 32 ( R2245000-1A )Conductivity Standard, 5000 uS/cm at 25C (2620 ppm TDS as NaCl)
Electroconductivity probe sensorHanna InstrumentsHI3003/DFlow-thru Conductivity Probe - NTC Sensor, DIN Connector, 3m Cable
Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA*2H2O)Sigma-Aldrich6381-92-6Medium Preparation: Ethylenediaminetetraacetic acid disodium salt dihydrate
FiltersFisher Scientific09-874-48Whatman Binder-Free Glass Microfiber Filters
FlasksFisher scientific09-552-40Pyrex Fernbach Flasks
FurnaceHogentoglerModel: F6020C-80Thermo Sicentific Thermolyne F6020C - 80 Muffle Furnace
Glass dessicatorVWR International LLC75871-430Type 150, 140 mm of diameter
Glass funnelFisher ScientificFB6005865Fisherbrand Reusable Glass Long-Stem Funnels
Laminar flow hoodFisher Hamilton SafeairFisher Hamilton Stainless Safeair hume hood
Magnesium sulfate heptahydrate (MgSO4*7H2O)Fisher Scientific10034 - 99 - 8Medium Preparation: Magnesium sulfate heptahydrate
MethanolSigma-Aldrich67-56-1Lipid extraction solvent
Micro bubble DiffuserPentair Aquatic Eco-Systems1PMBD075This equipment is used for the injection CO2 system
Microalgae: Chlorella SorokinianaNAABBDOE 1412
MicrooscopeCarl Zeiss 4291097
Microwave assistant extractionMARS, CEM CorportationCEM Mars 5 Xtraction 230/60 Microwave Accelerated Reaction System. Model: 907601
MnCl2*4H2OSigma-Aldrich13446-34-9Manganese(II) chloride tetrahydrate
MortarsFisher ScientificFB961BFisherbrand porcelein mortars
Nitrogen evaporatorOrganomationN-EVAP 112 Nitrogen Evaporatpr (OA-SYS Heating System)
OvenVWR International LLC89511-410Forced Air Oven
Paddle Wheel8-blade horizontal axis propeller. This usually comes as part of the paddlewheel reactor.
Paddle wheel motorLeesonM1135042.00Leeson, Model: CM34025Nz10C; 1/4 HP; Volts 90; FR 34; 62 RPM.
PestlesFisher ScientificFB961MFisherbrand porcelein pestles
pH and EC TransmitterHanna InstrumentsHI98143Hanna Instruments HI98143-04 pH and EC Transmitter with Galvanic isolated 0-4V.
pH calibration solutionsFisher Scientific13-643-003Thermo Scientific Orion pH Buffer Bottles
pH probe sensorHanna InstrumentsHI1006-2005Hanna Instruments HI1006-2005 Teflon pH Electrode with matching pin 5m.
Pippete tipsFisher Scientific1111-28211000 ul TipOne graduated blue tip in racks
PippetterFisher Scientific13-690-032Eppendorf Reserch plus Variable Adjustable Volume Pipettes: Single-channel
Plastic cuvettesFisher scientific14377017BrandTech BRAND Plastic Cuvettes
PlatesFisher scientific08-757-100DCorning Falcon Bacteriological Petri Dishes with Lid
PotashThis chemical is used for the optimazed medium preparation. It was bought in a fertilizer local company
Potassium phosphate dibasic (K2HPO4)Sigma-Aldrich7758 -11 - 4Medium Preparation: Potassium phosphate dibasic
Pyrex reusable Media Storage BottlesFisher scientific06-414-2A1 L and 2 L bottels - PYREX GL45 Screw Caps with Plug Seals
Raceway PondSimilar equipment can be bought at https://microbioengineering.com/products
Real Time Optical Density SensorUniversity of ArizonaThis equipment was design and build by a member of the group
RS232 CableSabrentSabrent USB 2.0 to Serial (9-Pin) DB-9 RS-232 Converter Cable, Prolific Chipset, Hexnuts, [Windows 10/8.1/8/7/VISTA/XP, Mac OS X 10.6 and Above] 2.5 Feet (CB-DB9P)
Shaker TableAlgae agitation 150 rpm
Sodium Carbonate (Na2CO3)Sigma-Aldrich497-19-8Sodium carbonate
Sodium molybdate dihydrate (Na2MoO4*2H2O)Sigma-Aldrich10102-40-6Medium Preparation: Sodium molybdate dihydrate
Sodium nitrate (NaNO3)Sigma-Aldrich7631-99-4Medium Preparation: Sodium nitrate
SpectophotometerFisher Scientific Company14-385-400Thermo Fisher Scientific - 10S UV-Vis GENESTYS Spectrophotometer cylindrical Longpath cell holder; internal reference dectector, Xenon flash lamp; dual silicon photodiode; 240V, 50 to 60Hz selected automatically.
Test tubesFisher Scientific14-961-27Fisherbrand Disposable Borosilicate Glass Tubes with Plain End (10 ml)
Thermocouples type KOmegaKMQXL-125G-6
UreaSigma-Aldrich2067-80-3Urea
Vacuum filtration systemFisher ScientificXX1514700MilliporeSigma Glass Vacuum Filter Holder, 47 mm. The system includes: Ground glass flask attachment, coarse-frit glass filter support, and flask
Vacuum pumpGraingerMarathon Electric AC Motor Thermally protected G588DX - MOD 5KH36KNA510X. HP 1/4. RPM 1725/1425
Zinc sulfate heptahydrate (ZnSO4*7H2O)Sigma-Aldrich7446-20-0Zinc sulfate heptahydrate

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