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.

1. Growth system: outdoor open raceway pond settings
<ol>
	<li>Set up the open raceway ponds close to the flue gas source (containing 8&#8211;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).</li>
	<li>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).</li>
	<li>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&#8210;13.8% water28. In addition, the condenser and pipeline cool the flue gas16.</li>
	<li>Connect the following sensors to a datalogger to monitor algal growth: (1) a real time optical density sensor29, which measures absorbance at two wavelengths&#8212;650 and 750 nm&#8212;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.</li>
	<li>Ensure that all components of the algal growth system are calibrated and properly working before inoculation.</li>
</ol>
2. pH control system
<ol>
	<li>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).</li>
	<li>Use a tube to direct the flue gas from the control valve to the bottom of the raceway pond through a stone diffuser.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Maintain cultures at room temperature (25 &#176;C) using a 12 h/12 h light/dark cycle.</li>
	<li>Keep light intensity at 200 &#181;M/m2/s for culture maintenance grown on plates&#160;and in small liquid cultures (50 mL to 500 mL).</li>
	<li>Keep light intensity for scale up grown in liquid cultures 50 mL to 500 mL at 400 &#181;M/m2/s, and liquid cultures 5 L to 20 L at 600&#8210;800 &#181;M/m2/s.</li>
</ol>
4. Scale up and quality control
<ol>
	<li>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 &#176;C.</li>
	<li>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 &#181;L from a re-suspended frozen algal stock culture and add Ampicillin (100 &#181;g/mL); incubate the algal plates in a shaker table (120 rpm) for 1 to 2 weeks.</li>
	<li>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.</li>
	<li>Transfer 50 mL of algae culture (linear growth phase, OD750nm &#8805; 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 &#181;m air sterilization filters. Let the culture grow for one to two weeks. Monitor cell density using a spectrophotometer (OD750nm).</li>
	<li>Place the 500 mL liquid culture&#160;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.</li>
	<li>Monitor stock plate and liquid cultures (in steps 4.2&#8210;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.</li>
</ol>
5. Concentrated medium preparation for open pond cultivation
<ol>
	<li>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&#160;and fill the flask to the 1 L volume mark.</li>
	<li>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&#8217;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&#8217;s final volume. Fill the glass bottle to 1 L.</li>
</ol>
6. Outdoor open raceway pond inoculation
<ol>
	<li>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.</li>
	<li>Calibrate all the sensors before algae inoculation according to their corresponding calibration procedure.</li>
	<li>Dilute the concentrated media (in step 5) using the water source by filling the raceway pond up to 80%.</li>
	<li>Inoculate the reactor using a 10 L carboy filled with algae (linear growth phase OD750nm &#62; 2) and bring it to its&#160;final volume.</li>
	<li>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:&#160;This period will also provide time for the microalgae to&#160;adapt to the stress caused by the direct injection of flue gas.</li>
</ol>
7. Batch growth experiment at the generating station
<ol>
	<li>Inspect and record any day-to-day variations including water evaporation, paddlewheel motor, sensor functionality, and anything out of the ordinary.</li>
	<li>Drain and inspect the compressor and water trap every day to remove any excess&#160;water to minimize corrosion since flue gas is highly corrosive34.</li>
	<li>Configure the data logger to scan each sensor measurement&#160;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.</li>
</ol>
8. Discrete sampling and monitoring
<ol>
	<li>Make sure water level remains constant at the reactor&#8217;s final volume otherwise the optical density measurement will be affected.</li>
	<li>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.</li>
	<li>Take a 500 mL sample three times per week for microscope observations and biomass concentration based on ash-free dry weight (AFDW).
	<ol>
		<li>Perform microscope observations with 10x and 40x objective lenses. Additionally, these microscope magnifications are used&#160;as part of the algal quality control described in step 4.6.</li>
		<li>Use 400 mL of the sample in step 8.3 for AFDW
		<ol>
			<li>Set each 0.7 &#181;m 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 &#176;C.</li>
			<li>Label each aluminum foil tray using a #2 pencil, record its weight (A), and place it in the vacuum filter apparatus.</li>
			<li>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.&#160;&#160;&#160;&#160;</li>
			<li>Place each filter containing the algae sample in its foil tray in the oven at 105 &#176;C for at least 12 h.</li>
			<li>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).</li>
			<li>Place the foil tray/filter in the 540 &#176;C muffle furnace for 4 h.</li>
			<li>Turn off the muffle furnace, cool down foil trays/filters, place them into the desiccator, and record each foil tray/filter weight (C).</li>
			<li>Calculate AFDW using gravimetrical analysis: 
			% AFDW= C &#8211; A x&#160;100 / B</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Hold 2 L of algae before harvesting for microwave-assisted extraction (MAE) lipid extraction analysis using solvents.
	<ol>
		<li>Centrifuge the algae sample at a relative centrifugal force (RFC) of 4,400 x&#160;g for 15 min. Take the algae pellet and dry it using an oven at 80 &#176;C for at least 24 h.</li>
		<li>Grind the algae sample and weigh the algal powder (recommended biomass ranges from 0.3 g to 0.5 g).</li>
		<li>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.</li>
		<li>Place the vessels into the MARS machine using the solvent sensor for 60 min at 70 &#176;C and 800 W of power.</li>
		<li>Take vessels out of the MARS and let them cool down under the hood.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Calculate lipid content (wt. %) using gravimetric analysis: 
		Lipid content (wt. %) = Dry biomass of lipids x&#160;100/ Dry Algal mass</li>
	</ol>
	</li>
</ol>
9. Algal harvesting and crop rotation
<ol>
	<li>Harvest 75% of the total algae culture volume when the culture is close to reaching the stationary phase. Take 2&#8210;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.</li>
	<li>Re-grow the open raceway pond by using the 25% algae remaining as inoculum. Add water up to 80% of the total reactor&#8217;s volume, add the concentrated media, and then finish filling up to the reactor&#8217;s final volume if necessary.</li>
	<li>Cultivate the appropriate algae strain according to the season, based on temperature and light intensity conditions.</li>
</ol>
10. Data management
<ol>
	<li>Record data in the data logger and collect for analysis as in step 7.3.</li>
	<li>Consider saving raw and analyzed data in the Regional Algal Feedstock Testbed (RAFT) share drive. RAFT project collaborators contribute&#160;their data to simulate and model algal productivity and validate outdoor cultivation.</li>
</ol>

The authors have nothing to disclose.

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</s...

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 (K&#246;ppen 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1767px;" width="1766">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Adjustable speed motor (paddle wheel system)</td>
			<td style="width:204px;">Leeson</td>
			<td style="width:161px;">174307</td>
			<td style="width:1116px;">Lesson 174307.00, type: SCR Voltage; Amps:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Aluminum weight boats</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td style="width:161px;">08-732-102</td>
			<td>Fisherbrand Aluminum Weighing Dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Iron (III) (NH&#8324;)&#8325;[Fe(C&#8326;H&#8324;O&#8327;)&#8322;]</td>
			<td>Fisher Scientific</td>
			<td>1185 - 57 - 5</td>
			<td>Medium preparation. Ammonium iron(III) citrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Ammonium Phosphate</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>7722-76-1</td>
			<td>This chemical is used for the optimized medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Ampicillin sodium salt</td>
			<td style="width:204px;">Sigma Aldrich</td>
			<td>A9518-5G</td>
			<td>This chemical is used for avoiding algae contamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoclave</td>
			<td>Amerex Instrument Inc</td>
			<td></td>
			<td>Hirayama HA300MII</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td>BP1423500</td>
			<td>Fisher BioReagents Granulated Agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Bleach</td>
			<td style="width:204px;">Clorox</td>
			<td style="width:161px;"></td>
			<td>Germicidal Bleach, concentrated clorox</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Boric Acid (H3BO3)</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td>10043-35-3</td>
			<td>Trace Elelements: Boric acid</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:285px;">Calcium chloride dihydrate (CaCl2*2H2O)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>Medium preparation. Calcium chloride dihydrate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Carboys (20 L)</td>
			<td style="width:204px;">Nalgene - Thermo Fisher Scientific</td>
			<td>2250-0050PK</td>
			<td>Polypropylene Carboy w/Handles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Centrifuge</td>
			<td style="width:204px;">Beckman Coulter, Inc</td>
			<td style="width:161px;"></td>
			<td>J2-21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Chloroform</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>67-66-3</td>
			<td>This chemical is used for lipid extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Citraplex 20% Iron</td>
			<td style="width:204px;">Loveland Products</td>
			<td>SDS No. 1000595582 -17-LPI</td>
			<td>https://www.fbn.com/direct/product/Citraplex-20-Iron#product_info</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Cobalt (II) nitrate hexahydrate (Co(NO3)2*6H2O)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:161px;">10026-22-9</td>
			<td>Trace Elements: Cobalt (II) nitrate hexahydrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Compressor</td>
			<td style="width:204px;">Makita</td>
			<td>MAC700</td>
			<td>This equipment is used for the injection CO2 system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Control Valve</td>
			<td style="width:204px;">Sierra Instruments</td>
			<td style="width:161px;">SmartTrak 100</td>
			<td>This item needs to be customized for your application. In our case, it was used a 5% CO2 and 95% air mixture.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Copper (II) Sulfate Pentahydrate (CuSO4*5H2O)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:161px;">7758-99-8</td>
			<td>Trace Elements: Copper (II) Sulfate Pentahydrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Data Logger: Campbell unit CR3000</td>
			<td style="width:204px;">Scientific Campbell</td>
			<td style="width:161px;">CR3000</td>
			<td>This equipment is used for controlling all the system, motoring and recording data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Dissolvde Oxygen Solution</td>
			<td style="width:204px;">Campbell Scientific</td>
			<td style="width:161px;">14055</td>
			<td>Dissolved oxygen electrolyte solution DO6002 - Lot No. 211085</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:285px;">Dissolved Oxygen probe</td>
			<td style="width:204px;">Sensorex</td>
			<td>&#65279;</td>
			<td>DO6400/T Dissolved Oxygen Sensor with Digital Communication</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Electroconductivity calibration solution</td>
			<td style="width:204px;">Ricca Chemical Company</td>
			<td style="width:161px;">2245 - 32 ( R2245000-1A )</td>
			<td>Conductivity Standard, 5000 uS/cm at 25C (2620 ppm TDS as NaCl)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Electroconductivity probe sensor</td>
			<td style="width:204px;">Hanna Instruments</td>
			<td>HI3003/D</td>
			<td>Flow-thru Conductivity Probe - NTC Sensor, DIN Connector, 3m Cable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:285px;">Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA*2H2O)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>6381-92-6</td>
			<td>Medium Preparation: Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Filters</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td>09-874-48</td>
			<td>Whatman Binder-Free Glass Microfiber Filters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Flasks</td>
			<td style="width:204px;">Fisher scientific</td>
			<td>09-552-40</td>
			<td>Pyrex Fernbach Flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Furnace</td>
			<td style="width:204px;">Hogentogler</td>
			<td style="width:161px;">Model: F6020C-80</td>
			<td>Thermo Sicentific Thermolyne F6020C - 80 Muffle Furnace</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Glass dessicator</td>
			<td style="width:204px;">VWR International LLC</td>
			<td style="width:161px;">75871-430</td>
			<td>Type 150, 140 mm of diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Glass funnel</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td style="width:161px;">FB6005865</td>
			<td>Fisherbrand Reusable Glass Long-Stem Funnels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow hood</td>
			<td>Fisher Hamilton Safeair</td>
			<td></td>
			<td>Fisher Hamilton Stainless Safeair hume hood</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Magnesium sulfate heptahydrate (MgSO4*7H2O)</td>
			<td>Fisher Scientific</td>
			<td>10034 - 99 - 8</td>
			<td>Medium Preparation: Magnesium sulfate heptahydrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Methanol</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>67-56-1</td>
			<td>Lipid extraction solvent</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:285px;">Micro bubble Diffuser</td>
			<td style="width:204px;">Pentair Aquatic Eco-Systems</td>
			<td>1PMBD075</td>
			<td>This equipment is used for the injection CO2 system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Microalgae: Chlorella Sorokiniana</td>
			<td style="width:204px;">NAABB</td>
			<td style="width:161px;">DOE 1412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microoscope</td>
			<td></td>
			<td></td>
			<td>Carl Zeiss 4291097</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Microwave assistant extraction</td>
			<td style="width:204px;">MARS, CEM Corportation</td>
			<td style="width:161px;"></td>
			<td>CEM Mars 5 Xtraction 230/60 Microwave Accelerated Reaction System. Model: 907601</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">MnCl2*4H2O</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>13446-34-9</td>
			<td>Manganese(II) chloride tetrahydrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Mortars</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td>FB961B</td>
			<td>Fisherbrand porcelein mortars</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Nitrogen evaporator</td>
			<td style="width:204px;">Organomation</td>
			<td style="width:161px;"></td>
			<td>N-EVAP 112 Nitrogen Evaporatpr (OA-SYS Heating System)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Oven</td>
			<td style="width:204px;">VWR International LLC</td>
			<td style="width:161px;">89511-410</td>
			<td>Forced Air Oven</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Paddle Wheel</td>
			<td style="width:204px;"></td>
			<td style="width:161px;"></td>
			<td>8-blade horizontal axis propeller. This usually comes as part of the paddlewheel reactor.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paddle wheel motor</td>
			<td>Leeson</td>
			<td>M1135042.00</td>
			<td>Leeson, Model: CM34025Nz10C; 1/4 HP; Volts 90; FR 34; 62 RPM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Pestles</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td>FB961M</td>
			<td>Fisherbrand porcelein pestles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">pH and EC Transmitter</td>
			<td style="width:204px;">Hanna Instruments</td>
			<td>HI98143</td>
			<td>Hanna Instruments HI98143-04 pH and EC Transmitter with Galvanic isolated 0-4V.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">pH calibration solutions</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td style="width:161px;">13-643-003</td>
			<td>Thermo Scientific Orion pH Buffer Bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">pH probe sensor</td>
			<td style="width:204px;">Hanna Instruments</td>
			<td>HI1006-2005</td>
			<td>Hanna Instruments HI1006-2005 Teflon pH Electrode with matching pin 5m.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pippete tips</td>
			<td>Fisher Scientific</td>
			<td>1111-2821</td>
			<td>1000 ul TipOne graduated blue tip in racks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pippetter</td>
			<td>Fisher Scientific</td>
			<td>13-690-032</td>
			<td>Eppendorf Reserch plus Variable Adjustable Volume Pipettes: Single-channel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Plastic cuvettes</td>
			<td style="width:204px;">Fisher scientific</td>
			<td>14377017</td>
			<td>BrandTech BRAND Plastic Cuvettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Plates</td>
			<td style="width:204px;">Fisher scientific</td>
			<td style="width:161px;">08-757-100D</td>
			<td>Corning Falcon Bacteriological Petri Dishes with Lid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potash</td>
			<td></td>
			<td></td>
			<td>This chemical is used for the optimazed medium preparation. It was bought in a fertilizer local company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Potassium phosphate dibasic (K2HPO4)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>7758 -11 - 4</td>
			<td>Medium Preparation: Potassium phosphate dibasic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Pyrex reusable Media Storage Bottles</td>
			<td style="width:204px;">Fisher scientific</td>
			<td>06-414-2A</td>
			<td>1 L and 2 L bottels - PYREX GL45 Screw Caps with Plug Seals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Raceway Pond</td>
			<td></td>
			<td></td>
			<td>Similar equipment can be bought at https://microbioengineering.com/products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Real Time Optical Density Sensor</td>
			<td>University of Arizona</td>
			<td></td>
			<td>This equipment was design and build by a member of the group</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">RS232 Cable</td>
			<td style="width:204px;">Sabrent</td>
			<td style="width:161px;"></td>
			<td>Sabrent 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)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker Table</td>
			<td></td>
			<td></td>
			<td>Algae agitation 150 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Sodium Carbonate (Na2CO3)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:161px;">497-19-8</td>
			<td>Sodium carbonate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Sodium molybdate dihydrate (Na2MoO4*2H2O)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:161px;">10102-40-6</td>
			<td>Medium Preparation: Sodium molybdate dihydrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Sodium nitrate (NaNO3)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>7631-99-4</td>
			<td>Medium Preparation: Sodium nitrate</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:285px;">Spectophotometer</td>
			<td style="width:204px;">Fisher Scientific Company</td>
			<td style="width:161px;">14-385-400</td>
			<td>Thermo 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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Test tubes</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td style="width:161px;">14-961-27</td>
			<td>Fisherbrand Disposable Borosilicate Glass Tubes with Plain End (10 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Thermocouples type K</td>
			<td style="width:204px;">Omega</td>
			<td>KMQXL-125G-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Urea</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:161px;">2067-80-3</td>
			<td>Urea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Vacuum filtration system</td>
			<td style="width:204px;">Fisher Scientific</td>
			<td style="width:161px;">XX1514700</td>
			<td>MilliporeSigma Glass Vacuum Filter Holder, 47 mm. The system includes: Ground glass flask attachment, coarse-frit glass filter support, and flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Vacuum pump</td>
			<td style="width:204px;">Grainger</td>
			<td style="width:161px;"></td>
			<td>Marathon Electric AC Motor Thermally protected G588DX - MOD 5KH36KNA510X. HP 1/4. RPM 1725/1425</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Zinc sulfate heptahydrate (ZnSO4*7H2O)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:161px;">7446-20-0</td>
			<td>Zinc sulfate heptahydrate</td>
		</tr>
	</tbody>
</table>

coupling carbon capture from a power plant with semi-automated open raceway ponds for microalgae cultivation

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.

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...

Watch this Scientific Journal Video about Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation at JoVE.com

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

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.

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

coupling-carbon-capture-from-power-plant-with-semi-automated-open

Research

JoVE Journal

Environment

4.9K Views.  University of Arizona. 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.

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

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

Rhizobial  bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host  legume plants.  One  of the most well-developed model systems ...

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The system comprises a high-pressure rheometer which is connected to a circulation loop. The rheometer has a rotational flow-through measurement cell with two alternative geometries: coaxial cylinder and double gap. The circulation loop contains a mixer, to bring the crude oil sample into equilibrium with CO2, and a gear pump that transports the mixture from the mixer to the rheometer and recycles it back to the mixer. The CO2 and crude oil are brought to equilibrium by stirring and circulation and the rheology of the saturated mixture is measured by the rheometer. The system is used to measure the rheological properties of Zuata crude oil (and its toluene dilution) in equilibrium with CO2 at elevated pressures up to 220 bar and a temperature of 50 &#176;C. The results show that CO2 addition changes the oil rheology significantly, initially reducing the viscosity as the CO2 pressure is increased and then increasing the viscosity above a threshold pressure. The non-Newtonian response of the crude is also seen to change with the addition of CO2.

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented.

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The ...

Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. However, they are vulnerable parts of the landscapes that are often poorly understood and understudied. Land use and management practices, as well as climate change are thought to be a contribution to the global amphibian decline. However, more research is needed to understand the extent of these impacts. Here, we present methodology for characterizing a vernal pond's morphology and detail a monitoring station that can be used to collect water quantity and quality data over the duration of a vernal pond's hydroperiod. We provide methodology for how to conduct field surveys to characterize the morphology and develop stage-storage curves for a vernal pond. Additionally, we provide methodology for monitoring the water level, temperature, pH, oxidation-reduction potential, dissolved oxygen, and electrical conductivity of water in a vernal pond, as well as monitoring rainfall data. This information can be used to better quantify the ecosystem services that vernal ponds provide and the impacts of anthropogenic activities on their ability to provide these services.

Understanding the ecosystem services and processes provided by vernal ponds and the impacts of anthropogenic activities on their ability to provide these services requires intensive hydrologic monitoring. This sampling protocol using in-situ monitoring equipment was developed to understand the impact of anthropogenic activities on water levels and quality.

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and increasing the recovery of coalbed methane. A visualized and constant-volume gas-solid coupling system is introduced here to investigate the influence of CO2 sorption on the physical and mechanical properties of coal. Being able to keep a constant volume and monitor the sample using a camera, this system offers the potential to improve instrument accuracy and analyze fracture evolution with a fractal geometry method. This paper provides all steps to perform a uniaxial compression experiment with a briquette sample in different CO2 pressures with the gas-solid coupling test system. A briquette, cold-pressed by raw coal and sodium humate cement, is loaded in high-pressure CO2, and its surface is monitored in real-time using a camera. However, the similarity between the briquette and the raw coal still needs improvement, and a flammable gas such as methane (CH4) cannot be injected for the test. The results show that CO2 sorption leads to peak strength and elastic modulus reduction of the briquette, and the fracture evolution of the briquette in a failure state indicates fractal characteristics. The strength, elastic modulus, and fractal dimension are all correlated with CO2 pressure but not with a linear correlation. The visualized and constant-volume gas-solid coupling test system can serve as a platform for experimental research about rock mechanics considering the multifield coupling effect.

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal&#8217;s physical and mechanical properties induced by CO2 adsorption.

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and ...