We thank DGAPA UNAM project number IT100423 for the partial funding. We also thank PROAN and GSI for allowing us to share technical experiences about their photosynthetic biogas upgrading full installations. The technical support of Pedro Pastor Hern&#225;ndez Guerrero, Carlos Martin Sigala, Juan Francisco D&#237;az M&#225;rquez, Margarita Elizabeth Cisneros Ortiz, Roberto Sotero Briones M&#233;ndez and Daniel de los Cobos Vasconcelos is highly appreciated. A part of this research was done at IIUNAM Environmental Engineering Laboratory with an ISO 9001:2015 certificate.

1. System set-up
NOTE: A piping and instrumentation diagram (P&#38;ID) of the system described in this protocol is shown in Figure 2.
<ol>
	<li>Reactor set-up
	<ol>
		<li>Prepare the ground by leveling and compacting it to improve reactor stability.</li>
		<li>On an open field, dig two elongated holes and 3 m from the end, further dig a 3 m2 and 1 m deep hole (known as an aeration well).</li>
		<li>Place two HRAPs (Figure 3) within the space on geomembrane-covered metal supports. Each reactor must have an operating capacity of 22.2 m3.</li>
		<li>Place an air pump per reactor of 1728.42 watts (2.35 hp) close to the point of the HRAPs where the aeration wells were dug.</li>
		<li>Fix a paddle wheel (moved by a 1103.24 watts [1.5 hp] electric motor) across the reactor to promote contact between biomass and media.</li>
	</ol>
	</li>
	<li>Gas treatment set-up (Figure 4)
	<ol>
		<li>Build the desorption column with a 6&#34; polyvinyl chloride (PVC) tube, where the inlet current enters 2 m from the covered top, and the outlet current flows from the bottom (Figure 2).</li>
		<li>Set up the absorption tank (Vt: 2.55 m3), where the gaseous inlet (non-treated biogas) current is bubbled from the bottom through 11 diffuser tubes and comes from the anaerobic digester through a 4&#34; PVC pipeline passing through a biogas blower, a 1&#34; rotameter and a sampling port, while the liquid comes from the media recirculation after the desorption column on the bottom of the tank. The liquid outlet is located on the side of the tank. It transports the CO2-enriched media to the level-control column, and the gas exits from the outlet at the top of the tank, which is connected with a 1&#34; PVC pipeline to conduct obtained biomethane to a burner for its continuous combustion (Figure 2).</li>
		<li>Connect the absorption tank to the desorption column through a 4&#34; PVC tube, passing through a sampling port between both operations (Figure 2).</li>
		<li>Build the level-control column with a 6&#34; PVC tube where the inlet is located at the bottom. It has two outlets (controlled with butterfly valves), depending on the needs of the system; the first one is located at a height of 2.5 m and the second one at 3 m from the ground (Figure 2).</li>
		<li>Connect the HRAP photobioreactors through a 2&#34; PVC pipeline to the 6&#34; desorption column, passing through a recirculation centrifugal pump (1103.24 watts [1.5 hp]) and a 1&#34; rotameter (Figure 2).</li>
		<li>Connect the level-control column through a 4&#34; PVC tube to a schedule 40 PVC tube, passing through a sampling port. Next, connect it to a portion of flexible PVC tubing, followed by another schedule 40 PVC tube, and finally, a 4&#34; PVC tube, which opens to the HRAP photobioreactors (Figure 2).</li>
		<li>Set up the bypass of the desorption column with 2&#34; PVC pipeline and connect it to the main tube before the sampling port (Figure 2).</li>
	</ol>
	</li>
</ol>
2. Functional testing of the system
<ol>
	<li>Recirculation centrifugal pump (1103.24 watts [1.5 hp])
	<ol>
		<li>To determine the maximum flow rate of the pump, prime the interior for at least 10 min to avoid air suction and start it up at 230 V and 1 phase.</li>
		<li>Test the recirculation flow by letting it flow through the 1&#34; rotameter.</li>
	</ol>
	</li>
	<li>Biogas bubbling system
	<ol>
		<li>To determine the force required to bubble at least an air column equivalent to 200 mbar, test at least 3 blowers with different powers (485.52 watts [0.66 hp], 1838.74 watts [2.5 hp], and 3309.74 watts [4.5 hp]) by bubbling air into the absorption tank.</li>
		<li>Visually verify the size and distribution reached by the air bubbles inside the tank. Under the operating conditions described here, the predicted average diameter of the bubbles is 3 mm.</li>
	</ol>
	</li>
</ol>
3. Inoculation and growth under indoor conditions
<ol>
	<li>Transfer a pure strain of Arthrospira maxima from agar plates to 15 mL of aqueous mineral medium17 (NaHCO3 [10 g/L], Na3PO4 &#183;12H2O [0.033 g/L], NaNO3 [0.185 g/L], MgSO4 &#183;7H2O [0.014 g/L], FeSO4 &#183;7H2O [0.0008 g/L], NaCl [0.4 g/L]).</li>
	<li>Scale up the culture to 500 mL flasks with innocuous Jourdan aqueous medium, using 100% of flask volume, and let it grow in 12 h light/ 12 h dark photoperiods using light emitting diode (LED) lamps with surface mount device (SMD) 2835 providing while-cold light at 2000 lm and under continuous mixing by air bubbling (0.3 L/min or 0.6 vvm). (step lasting around 1 month).</li>
	<li>Continue the scaling-up process by adding 20% of the previous volume to the new volume until 50 L are reached.</li>
	<li>Adapt the culture to natural light conditions of operation and Jourdan culture media in a greenhouse in 50 L transparent sacks (step lasting around 2 months).</li>
	<li>Continue scaling in these conditions up to 5 m3 HRAP photobioreactors (step lasting around 2 months).</li>
</ol>
4. Operational start of the system under outdoor conditions
<ol>
	<li>Add the full volume of these 5 m3 HRAP photobioreactors to HRAPs photobioreactors of 13 m3 located outdoors and fill the rest of the volume with Jourdan culture medium. Start mixing through a paddle wheel at a speed of 30 cm/s, cultivating in batch mode for 15 days or until it reaches 0.7 g/L (step lasting around 1 month).</li>
	<li>Once growth reaches 0.7 g/L, transfer the volume to the operating 22.2 m3 HRAP, fill the rest with Jourdan media, and set the paddle wheel at a speed of 30 cm/s. Let the biomass grow until it reaches 0.7 g/L and a pH of 10; once these conditions are met, start sampling and harvesting, if needed.</li>
	<li>Start the liquid recirculation from the HRAP photobioreactor to the absorption tank at variable flow to increase biomass productivity. Begin biogas bubbling at an average flow of 3.5 m3/h after 2 h to provide inorganic carbon to the culture. Pay attention to the pH since it must remain above 9. 
	NOTE: Before recirculating the media through the absorption tank, prime&#160;the centrifugal pump described above.</li>
	<li>Nutrient addition: Monitor nutrient conditions weekly through harvesting and the overall nitrogen balance assuming steady state calculated as shown: 
	MNaNO3 = (MBiomass x 0.10)/0.12 [g] 
	Where: 
	MNaNO3 = Sodium nitrate mass [g] 
	MBiomass = Harvested biomass [g] 
	1.10: Mass yield of nitrogen/biomass16 [g/g] 
	1.12: Mass fraction of nitrogen in sodium nitrate [g/g]</li>
	<li>With the nitrogen balance results, reformulate the Jourdan media to add the proportional amount of Na3PO4&#183;12H2O, MgSO4&#183;7H2O, and FeSO4&#183;7H2O. Do not add more sodium bicarbonate or sodium chloride. 
	NOTE: Dissolve the nutrients in clean water before adding them to the reactors.</li>
	<li>Monitor water evaporation and add weekly if needed.</li>
</ol>
5. Sampling and analysis
<ol>
	<li>Biogas
	<ol>
		<li>Sample the biogas from the sampling outlet before the absorption tank and from the sampling outlet after the tank by connecting a 10 L polyvinyl fluoride bag to the outlet with a flexible tube of appropriate diameter; place each one in separate polyvinyl fluoride bags.</li>
		<li>Calibrate the portable gas analyzer by setting the pressure transductor to zero and waiting for stabilization. Do this by pressing Start, then Next, and connecting a clear tube and a yellow tube as instructed by the analyzer. Press Next and finally, Gas Readings.</li>
		<li>Connect each sample contained within the polyvinyl fluoride bags to the analyzer, press Next and measure the CH4, CO2, O2 and H2S concentrations as %vol from both points of the system.</li>
		<li>Determine the volumetric recirculation liquid/biogas ratio (L/G) by dividing the liquid recirculation flow by the biogas production flow. Compute the corresponding gas flow (m3/h) that presents the highest efficiency of CO2 and H2S removal.</li>
	</ol>
	</li>
	<li>Online measuring of system conditions (pH, dissolved oxygen, temperature)
	<ol>
		<li>Calibrate all sensors according to the specifications of the manufacturer.</li>
		<li>Place a pH sensor, a dissolved oxygen (DO) sensor, and a temperature sensor in the liquid of each HRAP. 
		NOTE: For brand and specifications for each of the sensors, review the Table of Materials file.</li>
		<li>Connect the pH and DO sensors to a data-acquisition device consisting of a 1.4 GHz 64-bit quad-core processor connected to a portable screen that stores a pre-made Python program written in Integrated Development and Learning Environment (IDLE) 2.7.
		<ol>
			<li>Open the program through the screen and indicate the time intervals to store each data point (in this case, every 2 min).</li>
			<li>Create a spreadsheet where the program will automatically store the data it collects.</li>
			<li>Click on the button that reads ON, indicating it is ready to start storing data.</li>
			<li>To stop the data acquisition, click on the button that reads OFF.</li>
			<li>To download the information, insert a universal serial bus (USB) and import the spreadsheet.</li>
		</ol>
		</li>
		<li>Connect the temperature sensor to a thermo-recorder to store the data recorded during the experiments.</li>
	</ol>
	</li>
	<li>Short exploratory tests
	<ol>
		<li>Determine the most efficient L/G
		<ol>
			<li>Regulate the incoming biogas flow to select the L/G value to be tested (0.5, 1, 1.5, 1.6, 2, 2.5, 3.3, 3.4).</li>
			<li>Measure the pH and the inlet and outlet concentrations of each gas (CH4, CO2, H2S, O2, N2) at the start and every 15 min for an hour (60 min), using the instruments described previously.</li>
			<li>Determine the most efficient L/G by comparing the outlet values and choose the one most convenient according to the needs of the experiment.</li>
		</ol>
		</li>
		<li>Relationship between L/G, pH and CO2
		<ol>
			<li>Choose at least two L/G&#39;s to compare.</li>
			<li>For each L/G, measure the pH and the inlet and outlet concentrations of CO2, and of H2S, O2, and N2 as a control at the start, every 15 min for 60 min, and then every hour for a total of 5 h, using the instruments described previously.</li>
			<li>Calculate the CO2 removal percentages using the equation: 
			%CO2 removal = ((CO2in - CO2out)/(CO2in)) x 100</li>
			<li>Graph the results and compare the behavior of the pH and CO2 for each of the L/G&#39;s tested.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calibration curve to correlate biomass weight per liter of culture versus absorbance at 750 nm18
	<ol>
		<li>Sample the algae culture to try and get an absorbance of 1.0. If the culture has an absorbance below 1.0, extract water by filtration (0.45 &#181;m filter) from a culture sample. If the absorbance is greater than 1, it can be decreased by adding a fresh culture medium.</li>
		<li>Prepare five algae cell suspensions using the sample and add fresh culture medium, in volume/volume (V/V) percentage: 100%, 80%, 60%, 40%, and 20%.</li>
		<li>Measure and record the absorbance at 750 nm of the five solutions with a spectrophotometer using plastic cuvettes, where the fresh culture medium is the blank.</li>
		<li>Determine the biomass weight per liter of culture of every suspension by filtering 10 mL through a previously weighed 0.45 &#181;m filter and drying the sample in a silica desiccator for 24 h and later 48 h to ensure a constant weight. Repeat this step for each of the five solutions. 
		NOTE: A higher temperature (above 60 &#176;C) is not recommended for drying due to the loss of certain key compounds that could volatilize and change the sample&#39;s weight.</li>
		<li>Once confirming the weight, calculate the biomass concentration within the reactor with the equation: 
		&#8203;Biomass concentration = (Biomass weight - filter weight) x 1000/Filtered volume [g/L]</li>
		<li>Make a linear regression of the biomass weight data in grams per liter of culture as a function of the absorbance measured at 750 nm using a spreadsheet or any other software. The linear regression coefficient should be greater than 0.95; otherwise, the curve is not useful, and the protocol should be repeated. 
		NOTE: It is described as biomass weight and not as dry weight as most methods because the drying method used does not allow for full removal of water in the sample, leaving a water content of less than 5%19.</li>
	</ol>
	</li>
	<li>Biomass growth
	<ol>
		<li>Monitor the reactors every day. Take a 1 L sample from the halfway point between the paddlewheel and its return from each culture and bring it to the laboratory.</li>
		<li>Check colony growth and purity of the culture under the microscope.</li>
		<li>Measure and record the absorbance at 750 nm of the samples with a spectrophotometer, where the fresh culture medium is the blank.</li>
		<li>Compare with the calibration curve to obtain the estimated biomass weight in grams per liter.</li>
		<li>Record the growth of each raceway reactor.</li>
	</ol>
	</li>
	<li>Biomass production - harvesting
	<ol>
		<li>Monitor the reactors every day. If biomass growth rises above 0.7 g/L during sampling, harvesting is needed.</li>
		<li>Alternating between both HRAPs, place a polyester mesh on top of a section at one end of the reactor and place an end of a flexible PVC tube within the flow of the liquid so that the other end drains the liquid on top of the mesh.</li>
		<li>Drain between 4500 L to 7500 L (depending on the biomass saturation of the reactor) onto the mesh, maintaining a continuous flow back to the corresponding HRAP. The biomass will be retained on the mesh.</li>
		<li>To harvest, remove the mesh from the top of the reactor and place it on a different surface to scrape the biomass off and place it into a funnel.</li>
		<li>Push the biomass through the funnel to create elongated shapes on top of a clean and dry mesh; set the mesh in a warm, covered room (34-36 &#176;C) for 48-72 h.</li>
		<li>Once dry, remove the biomass from the mesh and weigh it. Calculate the biomass harvested concentration in g/L with these equations: 
		Volume of drained liquid = Pump flow rate x Drain time [L] 
		Biomass harvested concentration = Biomass weight of harvested biomass/Volume of drained liquid [g/L]</li>
	</ol>
	</li>
</ol>

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

<ol><li>Muñoz, R., Meier, L., Diaz, I., Jeison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+on+the+state-of-the-art+of+physical%2Fchemical+and+biological+technologies+for+biogas+upgrading%5BTitle%5D)+AND+%22Rev+Environ+Sci+Biotechnol%22%5BJournal%5D)">A review on the state-of-the-art of physical/chemical and biological technologies for biogas upgrading.</a> Rev Environ Sci Biotechnol. 14, 727-759 (2015).</li>
<li>Karimi, B., Shokrinezhad, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Air+pollution+and+mortality+among+infant+and+children+under+five+years%3A+A+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Atmospheric+Pollut+Res%22%5BJournal%5D)">Air pollution and mortality among infant and children under five years: A systematic review and meta-analysis.</a> Atmospheric Pollut Res. 11 (6), 61-70 (2020).</li>
<li>Koengkan, M., Fuinhas, J. A., Silva, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploring+the+capacity+of+renewable+energy+consumption+to+reduce+outdoor+air+pollution+death+rate+in+Latin+America+and+the+Caribbean+region%5BTitle%5D)+AND+%22Environ+Sci+Pollut+Res%22%5BJournal%5D)">Exploring the capacity of renewable energy consumption to reduce outdoor air pollution death rate in Latin America and the Caribbean region.</a> Environ Sci Pollut Res. 28, 1656-1674 (2021).</li>
<li>Alvarez-Herranz, A., Balsalobre-Lorente, D., Shahbaz, M., Cantos, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+innovation+and+renewable+energy+consumption+in+the+correction+of+air+pollution+levels%5BTitle%5D)+AND+%22Energy+Policy%22%5BJournal%5D)">Energy innovation and renewable energy consumption in the correction of air pollution levels.</a> Energy Policy. 105, 386-397 (2017).</li>
<li>Razmjoo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+technical+analysis+investigating+energy+sustainability+utilizing+reliable+renewable+energy+sources+to+reduce+CO2+emissions+in+a+high+potential+area%5BTitle%5D)+AND+%22Renew+Energy%22%5BJournal%5D)">A technical analysis investigating energy sustainability utilizing reliable renewable energy sources to reduce CO2 emissions in a high potential area.</a> Renew Energy. 164, 46-57 (2021).</li>
<li>Franco-Morgado, M., Tabaco-Angoa, T., Ramírez-García, M. A., González-Sánchez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+for+decreasing+the+O2+content+in+the+upgraded+biogas+purified+via+microalgae-based+technology%5BTitle%5D)+AND+%22J+Environ+Manage%22%5BJournal%5D)">Strategies for decreasing the O2 content in the upgraded biogas purified via microalgae-based technology.</a> J Environ Manage. 279, 111813(2021).</li>
<li>Bailón, L., Hinge, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBail%C3%B3n%2C+L+%22Report%3A+Biogas+and+Bio-Syngas+Upgrading%22">Report: Biogas and Bio-Syngas Upgrading</a>. , Danish Technological Institute, Aarhus. (2012).</li>
<li>Persson, M., Jonsson, O., Wellinger, A. Biogas Upgrading to Vehicle Fuel Standards and Grid Injection. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPersson%2C+M+%22Brochure+of+IEA+Task+37.+Energy+from+Biogas+and+Landfill+Gas%22">Brochure of IEA Task 37. Energy from Biogas and Landfill Gas</a>. , (2006).</li>
<li>Soreanu, G., Béland, M., Falletta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Approaches+concerning+siloxane+removal+from+biogas+--+a+review%5BTitle%5D)+AND+%22Canadian+Biosystems+Engineering%22%5BJournal%5D)">Approaches concerning siloxane removal from biogas -- a review.</a> Canadian Biosystems Engineering. 53, 8.1-8.18 (2011).</li>
<li>Toro-Huertas, E. I., Franco-Morgado, M., de los Cobos Vasconcelos, D., González-Sánchez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photorespiration+in+an+outdoor+alkaline+open-photobioreactor+used+for+biogas+upgrading%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">Photorespiration in an outdoor alkaline open-photobioreactor used for biogas upgrading.</a> Sci Total Environ. 667, 613-621 (2019).</li>
<li>Cozma, P., Wukovits, W., Mămăligă, I., Friedl, A., Gavrilescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+and+simulation+of+high+pressure+water+scrubbing+technology+applied+for+biogas+upgrading%5BTitle%5D)+AND+%22Clean+Technol+Environ+Policy%22%5BJournal%5D)">Modeling and simulation of high pressure water scrubbing technology applied for biogas upgrading.</a> Clean Technol Environ Policy. 17, 373-391 (2015).</li>
<li>Sheets, J. P., Shah, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Techno-economic+comparison+of+biogas+cleaning+for+grid+injection%2C+compressed+natural+gas%2C+and+biogas-to-methanol+conversion+technologies%3A+Techno-economic+analysis+of+existing+and+emerging+biogas+upgrading+technologies%5BTitle%5D)+AND+%22Biofuels+Bioprod+Biorefining%22%5BJournal%5D)">Techno-economic comparison of biogas cleaning for grid injection, compressed natural gas, and biogas-to-methanol conversion technologies: Techno-economic analysis of existing and emerging biogas upgrading technologies.</a> Biofuels Bioprod Biorefining. 12, 412-425 (2018).</li>
<li>Toledo-Cervantes, A., Estrada, J. M., Lebrero, R., Muñoz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparative+analysis+of+biogas+upgrading+technologies%3A+Photosynthetic+vs+physical%2Fchemical+processes%5BTitle%5D)+AND+%22Algal+Res%22%5BJournal%5D)">A comparative analysis of biogas upgrading technologies: Photosynthetic vs physical/chemical processes.</a> Algal Res. 25, 237-243 (2017).</li>
<li>Marín, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anaerobic+digestion+of+food+waste+coupled+with+biogas+upgrading+in+an+outdoors+algal-bacterial+photobioreactor+at+pilot+scale%5BTitle%5D)+AND+%22Fuel%22%5BJournal%5D)">Anaerobic digestion of food waste coupled with biogas upgrading in an outdoors algal-bacterial photobioreactor at pilot scale.</a> Fuel. 324, 124554(2022).</li>
<li>Bahr, M., Díaz, I., Dominguez, A., González Sánchez, A., Muñoz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microalgal-biotechnology+as+a+platform+for+an+integral+biogas+upgrading+and+nutrient+removal+from+anaerobic+effluents%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">Microalgal-biotechnology as a platform for an integral biogas upgrading and nutrient removal from anaerobic effluents.</a> Environ Sci Technol. 48 (1), 573-581 (2014).</li>
<li>Franco-Morgado, M., Alcántara, C., Noyola, A., Muñoz, R., González-Sánchez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+study+of+photosynthetic+biogas+upgrading+based+on+a+high+rate+algal+pond+under+alkaline+conditions%3A+Influence+of+the+illumination+regime%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">A study of photosynthetic biogas upgrading based on a high rate algal pond under alkaline conditions: Influence of the illumination regime.</a> Sci Total Environ. 592, 419-425 (2017).</li>
<li>Jourdan, J. P. Manuel de culture artisanale de spiruline. , https://www.scribd.com/document/513003475/Manuel-de-Culture-Artisanale-de-Spiruline (2006).</li>
<li>Lu, L., Yang, G., Zhu, B., Pan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparative+study+on+three+quantitating+methods+of+microalgal+biomass%5BTitle%5D)+AND+%22Indian+J+Geo-Mar+Sci%22%5BJournal%5D)">A comparative study on three quantitating methods of microalgal biomass.</a> Indian J Geo-Mar Sci. 46, 2265-2272 (2017).</li>
<li>Sukarni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermogravimetric+analysis+of+the+combustion+of+marine+microalgae+Spirulina+platensis+and+its+blend+with+synthetic+waste%5BTitle%5D)+AND+%22Heliyon%22%5BJournal%5D)">Thermogravimetric analysis of the combustion of marine microalgae Spirulina platensis and its blend with synthetic waste.</a> Heliyon. 6 (9), e04902(2020).</li>
<li>Kundu, S., Zanganeh, J., Moghtaderi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+on+understanding+explosions+from+methane-air+mixture%5BTitle%5D)+AND+%22J+Loss+Prev+Process+Ind%22%5BJournal%5D)">A review on understanding explosions from methane-air mixture.</a> J Loss Prev Process Ind. 40, 507-523 (2016).</li>
<li>Serejo, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+biogas+flow+rate+on+biomass+composition+during+the+optimization+of+biogas+upgrading+in+microalgal-bacterial+processes%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">Influence of biogas flow rate on biomass composition during the optimization of biogas upgrading in microalgal-bacterial processes.</a> Environ Sci Technol. 49 (5), 3228-3236 (2015).</li>
<li>Toledo-Cervantes, A., Madrid-Chirinos, C., Cantera, S., Lebrero, R., Muñoz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+the+gas-liquid+flow+configuration+in+the+absorption+column+on+photosynthetic+biogas+upgrading+in+algal-bacterial+photobioreactors%5BTitle%5D)+AND+%22Bioresour+Technol%22%5BJournal%5D)">Influence of the gas-liquid flow configuration in the absorption column on photosynthetic biogas upgrading in algal-bacterial photobioreactors.</a> Bioresour Technol. 225, 336-342 (2017).</li>
<li>Posadas, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimization+of+biomethane+oxygen+concentration+during+biogas+upgrading+in+algal-bacterial+photobioreactors%5BTitle%5D)+AND+%22Algal+Res%22%5BJournal%5D)">Minimization of biomethane oxygen concentration during biogas upgrading in algal-bacterial photobioreactors.</a> Algal Res. 12, 221-229 (2015).</li>
<li>González Sánchez, A., FloresMárquez, T. E., Revah, S., Morgan Sagastume, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enrichment+and+cultivation+of+a+sulfide-oxidizing+bacteria+consortium+for+its+deploying+in+full-scale+biogas+desulfurization%5BTitle%5D)+AND+%22Biomass+Bioenergy%22%5BJournal%5D)">Enrichment and cultivation of a sulfide-oxidizing bacteria consortium for its deploying in full-scale biogas desulfurization.</a> Biomass Bioenergy. 66, 460-464 (2014).</li>
<li>González-Sánchez, A., Posten, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fate+of+H2S+during+the+cultivation+of+Chlorella+sp.+deployed+for+biogas+upgrading%5BTitle%5D)+AND+%22J+Environ+Manage%22%5BJournal%5D)">Fate of H2S during the cultivation of Chlorella sp. deployed for biogas upgrading.</a> J Environ Manage. 191, 252-257 (2017).</li>
<li>Hussain, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microalgae+an+ecofriendly+and+sustainable+wastewater+treatment+option%3A+Biomass+application+in+biofuel+and+bio-fertilizer+production.+A+review%5BTitle%5D)+AND+%22Renew+Sustain+Energy+Rev%22%5BJournal%5D)">Microalgae an ecofriendly and sustainable wastewater treatment option: Biomass application in biofuel and bio-fertilizer production. A review.</a> Renew Sustain Energy Rev. 137, 137(2021).</li>
<li>lvarez-González, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Can+microalgae+grown+in+wastewater+reduce+the+use+of+inorganic+fertilizers%5BTitle%5D)+AND+%22J+Environ+Manage%22%5BJournal%5D)">Can microalgae grown in wastewater reduce the use of inorganic fertilizers.</a> J Environ Manage. 323, 116224(2022).</li>
<li>Deepika, P., MubarakAli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+and+assessment+of+microalgal+liquid+fertilizer+for+the+enhanced+growth+of+four+crop+plants%5BTitle%5D)+AND+%22Biocatal+Agric+Biotechnol%22%5BJournal%5D)">Production and assessment of microalgal liquid fertilizer for the enhanced growth of four crop plants.</a> Biocatal Agric Biotechnol. 28, 101701(2020).</li>
<li>Huguen, P., Le Saux, G. Perspectives for a european standard on biomethane: a Biogasmax proposal. , https://trimis.ec.europa.eu/sites/default/files/project/documents/20120601_135059_69928_d3_8_new_lmcu_bgx_eu_standard_14dec10_vf__077238500_0948_26012011.pdf (2010).</li>
<li>Gas Networks, Ireland. Biomethane - Oxygen Content Assessment. , https://www.gasnetworks.ie/docs/corporate/gas-regulation/Oxygen-concentration-report-17985-AI-RPT-001-Rev-5-Biomethane-review-Penspen.pdf (2018).</li>
<li>Wellinger, A. European biomethane standards for grid injection and vehicle fuel use. , European Biogas Association. https://www.biosurf.eu/wordpress/wp-content/uploads/2015/06/9.-Arthur_Wellinger.pdf (2017).</li>
<li>Diario Oficial de la Federación. NORMA Oficial Mexicana NOM-001-SECRE-2010, Especificaciones del gas natural (cancela y sustituye a la NOM-001-SECRE-2003, Calidad del gas natural y la NOM-EM-002-SECRE-2009, Calidad del gas natural durante el periodo de emergencia severa). , https://www.dof.gob.mx/normasOficiales/3997/sener/sener.html (2010).</li>
<li>Sharifian, R., Wagterveld, R. M., Digdaya, I. A., Xiang, C., Vermaas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrochemical+carbon+dioxide+capture+to+close+the+carbon+cycle%5BTitle%5D)+AND+%22Energy+Environ+Sci%22%5BJournal%5D)">Electrochemical carbon dioxide capture to close the carbon cycle.</a> Energy Environ Sci. 14, 781-814 (2021).</li>
<li>Masojídek, J., Torzillo, G., Koblížek, M. Photosynthesis in Microalgae. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMasoj%C3%ADdek%2C+J+%22Handbook+of+Microalgal+Culture%22">Handbook of Microalgal Culture</a>. , John Wiley & Sons. (2013).</li>
<li>Rendal, C., Witt, J., Preuss, T. G., Ashauer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+framework+for+algae+modeling+in+regulatory+risk+assessment%5BTitle%5D)+AND+%22Environ+Toxicol+Chem%22%5BJournal%5D)">A framework for algae modeling in regulatory risk assessment.</a> Environ Toxicol Chem. 42 (8), 1823-1838 (2023).</li>
<li>Alami, A. H., Alasad, S., Ali, M., Alshamsi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigating+algae+for+CO2+capture+and+accumulation+and+simultaneous+production+of+biomass+for+biodiesel+production%5BTitle%5D)+AND+%22Sci+Total+Environ%22%5BJournal%5D)">Investigating algae for CO2 capture and accumulation and simultaneous production of biomass for biodiesel production.</a> Sci Total Environ. 759, 143529(2021).</li>
</ol>

Conflict of interest. The authors declare that they have no conflict of interest.

Throughout the years, this algal technology has been tested and used as an alternative to the harsh and expensive physicochemical techniques to purify biogas. Particularly, the Arthrospira genus is widely used for this specific purpose, along with Chlorella. There are few methodologies, however, that are made on a semi-industrial scale, which adds value to this procedure.
It is critical to maintain lower O2 concentrations by using the proper L/G ratio; however, thi...

In recent years, several technologies have emerged to purify biogas to biomethane, promoting its use as non-fossil fuel, therefore mitigating undesairable methane emissions1. Air pollution is a problem that affects most of the world&#39;s population, particularly in urbanized areas; ultimately, around 92% of the world&#39;s population breathe polluted air2. In Latin America, the air pollution rates are mostly created by the use of fuels, whereby in 2014, 48% of the air pollution was brought on by the electricity and heat production sector3.
In the last decade, more and more studies on the relationship between pollutants in the air and the increase in mortality rates have been proposed, arguing that there is a strong correlation between both datasets, particularly in children populations.
As a way to avoid the continuation of air pollution, several strategies have been proposed; one of these is the use of renewable energy sources, including wind turbines and photovoltaic cells, which diminish the CO2 release into the atmosphere4,5. Another renewable energy source comes from biogas, a byproduct of the anaerobic digestion of organic matter, produced along with a liquid organic digestate6. This gas is composed of a mixture of gasses, and their proportions depend on the source of organic matter used for anaerobic digestion (sewage sludge, cattle manure, or agro-industrial biowaste). Generally, these proportions are CH4 (53%-70%vol), CO2 (30%-47%vol), N2 (0%-3%vol), H2O (5%-10%vol), O2 (0%-1%vol), H2S (0-10,000 ppmv), NH3 (0-100 ppmv), hydrocarbons (0-200 mg/m3) and siloxanes (0-41 mg/m3)7,8,9, where the scientific community is interested in the methane gas since this is the renewable energetic component of the mixture.
However, biogas cannot be simply burned as obtained because the byproducts of the reaction can be harmful and contaminant; this raises the need to treat and purify the mixture to increase the percentage of methane and decrease the rest, essentially converting it into biomethane10. This process is also known as upgrading. Even though, currently, there are commercial technologies for this treatment, these technologies have several economic and environmental drawbacks11,12,13. For example, systems with activated carbon and water washing (ACF-WS), pressure water washing (PWS), gas permeation (GPHR), and pressure swing adsorption (PSA) present some economic or other drawbacks of environmental impact. A viable alternative (Figure 1) is the use of biological systems such as those that combine microalgae and bacteria grown in photobioreactors; some advantages include the simplicity of design and operation, the low operating costs, and its environmentally friendly operations and byproducts10,13,14. When biogas is purified to biomethane, the latter can be used as a substitute for natural gas, and the digestate can be implemented as a source of nutrients to support microalgae growth in the system10.
A method widely used in this upgrading procedure is the growth of microalgae in open raceway photoreactors coupled with an absorption column due to the lower operation costs and the minimal investment capital needed6. The most used type of raceway reactor for this application is the high-rate algal pond (HRAP), which is a shallow raceway pond where the circulation of the algal broth occurs via a low-power paddle wheel14. These reactors need large areas for their installation and are very susceptible to contamination if used in outdoor conditions; in biogas purification processes, it is advised to use alkaline conditions (pH &#62; 9.5) and the use of algal species that thrive in higher pH levels to enhance the removal of CO2 and H2S while avoiding contamination15,16.
This research aimed to determine the biogas treatment efficiencies and final production of biomethane using HRAP photobioreactors coupled with an absorption-desorption column system and a microalgae consortium.

<table><tbody><tr><td>1&quot; rotameter</td><td>CICLOTEC</td><td>N/A</td><td></td></tr><tr><td>1&quot; rotameter</td><td>GPI</td><td>A10-LMA100IA1</td><td></td></tr><tr><td>Absorption tank</td><td>EFISA</td><td>Made under previous design</td><td></td></tr><tr><td>Air blower (2.35 HP)</td><td>Elmo Rietschle</td><td>2BH11007AH01</td><td></td></tr><tr><td>Biogas blower (2 HP)</td><td>Elmo Rietschle</td><td>2BH11007AH01</td><td></td></tr><tr><td>Biogas composition measure</td><td>Geotech</td><td>BIOGAS 5000</td><td></td></tr><tr><td>Data-acquisition device</td><td>LabJack Co.</td><td>U3-LV</td><td></td></tr><tr><td>Diffuser tubes</td><td>Aero-Tube</td><td>C3060AR</td><td></td></tr><tr><td>DO sensor</td><td>Applisens</td><td>Z10023525</td><td></td></tr><tr><td>Dodecahydrated trisodium phosphate&amp;nbsp;</td><td>Quimica PIMA</td><td>N/A</td><td>Fertilizer grade (greenhouse and experior use)</td></tr><tr><td>Dodecahydrated trisodium phosphate&amp;nbsp;</td><td>Fermont</td><td>35963</td><td>Analytical grade (Used in cultures inside the laboratory)</td></tr><tr><td>Durapore membrane (45 &amp;micro;m)</td><td>MerckMillipore</td><td>HVLP04700&amp;nbsp;</td><td></td></tr><tr><td>Electric motor 1.5 HP</td><td>Weg</td><td>00158ET3ERS56C</td><td></td></tr><tr><td>Ferrous sulfate heptahydrate</td><td>Agroquimica Samet</td><td>N/A</td><td>Fertilizer grade (greenhouse and experior use)</td></tr><tr><td>Ferrous sulfate heptahydrate</td><td>Fermont</td><td>63593</td><td>Analytical grade (Used in cultures inside the laboratory)</td></tr><tr><td>Geomembrane</td><td>GEOSINCERE</td><td>N/A</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Tepeyac</td><td>N/A</td><td>Fertilizer grade (greenhouse and experior use)</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Fermont</td><td>63623</td><td>Analytical grade (Used in cultures inside the laboratory)</td></tr><tr><td>Paddle wheel</td><td>GSI</td><td>Made under previous design</td><td></td></tr><tr><td>pH sensor</td><td>Van London pHoenix</td><td>715-772-0041</td><td></td></tr><tr><td>Portable screen</td><td>Rasspberry</td><td>Pi 3 B+</td><td></td></tr><tr><td>Recirculation centrifugal pump (1.5 HP)</td><td>Aquapak&amp;nbsp;</td><td>ALY 15</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Industria del alcali</td><td>N/A</td><td>Fertilizer grade (greenhouse and experior use)</td></tr><tr><td>Sodium bicarbonate</td><td>Fermont</td><td>12903</td><td>Analytical grade (Used in cultures inside the laboratory)</td></tr><tr><td>Sodium chloride</td><td>Sal Colima</td><td>N/A</td><td>Fertilizer grade (greenhouse and experior use)</td></tr><tr><td>Sodium chloride</td><td>Fermont</td><td>24912</td><td>Analytical grade (Used in cultures inside the laboratory)</td></tr><tr><td>Sodium nitrate</td><td>Vitraquim</td><td>N/A</td><td>Fertilizer grade (greenhouse and experior use)</td></tr><tr><td>Sodium nitrate</td><td>Fermont</td><td>41903</td><td>Analytical grade (Used in cultures inside the laboratory)</td></tr><tr><td>Storing program (pH, DO)</td><td>&amp;nbsp;Python Software Foundation&amp;nbsp;</td><td>Python IDLE 2.7</td><td></td></tr><tr><td>Tedlar bags</td><td>SKC Inc.</td><td>232-25</td><td></td></tr><tr><td>Temperature recorder</td><td>T&amp;amp;D</td><td>TR-52i</td><td></td></tr><tr><td>UV-Vis Spectrophotometer</td><td>ThermoFisher Scientific instrument</td><td>GENESYS 10S&amp;nbsp;</td><td></td></tr><tr><td>Vacuum pump</td><td>EVAR</td><td>EV-40</td><td></td></tr></tbody></table>

biogas purification through the use of a microalgae-bacterial system in semi-industrial high rate algal ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

Following the protocol, the system was built, tested, and inoculated. The conditions were measured and stored, and the samples were taken and analyzed. The protocol was performed a year, starting in October 2019 and lasting until October 2020. It is important to mention that from here onwards, the HRAPs will be referred to as RT3 and RT4.
Biomethane productivity 
In order to determine the conditions that promote the highest H2S and CO2 removal and, c...

Watch this Scientific Journal Video about Scaling Microalgal Biotechnology for Enhanced Biomethane Production at JoVE.com

Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

biogas-purification-through-use-microalgae-bacterial-system-semi

Research

JoVE Journal

Environment

2.4K Views.  Universidad Nacional Autónoma de México. Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

Video: Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ammonia removal, nitrogen removal and algal growth are compared over an 8-week period in paired sets of small (100 L) and large (1,000 L) reactors designed for algal remediation of landfill wastewater. Contents of the small and large scale reactors were mixed before the beginning of each weekly testing interval to maintain equivalent initial conditions across the two scales. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted to better equalize conditions occurring at both scales. During the short 8-week representative time period, starting ammonia and total nitrogen concentrations ranged from 3.1-14 mg NH3-N/L, and 8.1-20.1 mg N/L, respectively. The performance of the treatment system was evaluated based on its ability to remove ammonia and total nitrogen and to produce algal biomass. Mean &#177; standard deviation of ammonia removal, total nitrogen removal and biomass growth rates were 0.95&#177;0.3 mg NH3-N/L/day, 0.89&#177;0.3 mg N/L/day, and 0.02&#177;0.03 g biomass/L/day, respectively. All vessels showed a positive relationship between the initial ammonia concentration and ammonia removal rate (R2=0.76). Comparison of process efficiencies and production values measured in reactors of different scale may be useful in determining if lab-scale experimental data is appropriate for prediction of commercial-scale production values.

An experimental methodology is presented to compare the performance of small (100 L) and large (1,000 L) scale reactors designed for algae remediation of landfill wastewater. System characteristics, including surface area to volume ratio, retention time, biomass density, and wastewater feed concentrations, can be adjusted based on application.

An experimental methodology is presented to compare the performance of two different sized reactors designed for wastewater treatment. In this study, ...

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

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 ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...

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.

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.