Name: microalgae cultivation and biomass quantification in a bench-scale photobioreactor with corrosive flue gases
Uploaded: 2019-12-19T15:00:00
Description: Watch this Scientific Journal Video about Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases at JoVE.com

This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1546595. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The work was also supported by a University of Iowa Graduate and Professional Student Government research grant, and the University of Iowa Foundation, Allen S. Henry endowment. Research was conducted in the W. M. Keck Phytotechnologies Laboratory. The authors would like to thank the University of Iowa power plant staff, especially Mark Maxwell, for expertise and financial support for the simulated flue gases. The authors would also like to acknowledge Emily Moore for her assistance with sampling and analysis and Emily Greene for her assistance and participation in the protocol video.

1. Safe use and sampling of a photobioreactor sparged with corrosive gases
NOTE: This method does not describe appropriate procedures for safe sampling of microalgal cultures that produce or consume highly flammable gases.
<ol>
	<li>Manage toxic gas as a risk to human health. 
	NOTE: Per the University of Iowa's Chemical Hygiene Plan, the authors worked with the University Fire Safety Coordinator and University Environmental Health &#38; Safety Industrial Hygiene Officer to develop a sa...

<ol>
	<li>Obom, K. M., Magno, A., Cummings, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operation+of+a+Benchtop+Bioreactor.">Operation of a Benchtop Bioreactor.</a> Journal of Visualized Experiments. (79), e50582 (2013).</li><li>Cheah, W. Y., Pau Loke, S., Chang, J. -. S., Ling, T., Juan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosequestration+of+atmospheric+CO2+and+flue+gas-containing+CO2+by+microalgae.">Biosequestration of atmospheric CO2 and flue gas-containing CO2 by microalgae.</a> Bioresource Technology. 184, 190-201 (2014).</li><li>Xu, L., Weathers, P. J., Xiong, X. -. R., Liu, C. -. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microalgal+bioreactors:+Challenges+and+opportunities.">Microalgal bioreactors: Challenges and opportunities.</a> Engineering in Life Sciences. 9 (3), 178-189 (2009).</li><li>Tsang, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Photobioreactors: Advancements, Applications and Research. , (2017).</li><li>Molitor, H. R., Moore, E. J., Schnoor, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+CO2+Utilization+by+Nutritious+Microalgae.">Maximum CO2 Utilization by Nutritious Microalgae.</a> ACS Sustainable Chemistry &amp; Engineering. 7 (10), 9474-9479 (2019).</li><li>Khan, M. I., Shin, J. H., Kim, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promising+future+of+microalgae:+current+status,+challenges,+and+optimization+of+a+sustainable+and+renewable+industry+for+biofuels,+feed,+and+other+products.">The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products.</a> Microbial Cell Factories. 17 (1), 36 (2018).</li><li>Benemann, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+production+by+microalgae.">Hydrogen production by microalgae.</a> Journal of Applied Phycology. 12 (3), 291-300 (2000).</li><li>. IH MOD Available from: <a target="_blank" href="https://aiha.org/public-resources/consumer-resources/topics-of-interest/ih-apps-tools">https://aiha.org/public-resources/consumer-resources/topics-of-interest/ih-apps-tools</a> (2019)</li><li>Centers for Disease Control and Prevention, Immediately Dangerous To Life or Health (IDLH) Values. The National Institute for Occupational Safety and Health Available from: <a target="_blank" href="https://www.cdc.gov/niosh/idlh/intridl4.html">https://www.cdc.gov/niosh/idlh/intridl4.html</a> (2019)</li><li>Nakanishi, K., Deuchi, K., Kuwano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+four+valuable+strains+of+microalgae,+including+viability+and+characteristics+during+15+of+cryostorage.">Cryopreservation of four valuable strains of microalgae, including viability and characteristics during 15 of cryostorage.</a> Journal of Applied Phycology. 24 (6), 1381-1385 (2012).</li><li>Bischoff, H. W., Bold, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Some soil algae from Enchanted Rock and related algal species. , (1963).</li><li>Mata, T. M., Martins, A. A., Caetano, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microalgae+for+biodiesel+production+and+other+applications:+A+review.">Microalgae for biodiesel production and other applications: A review.</a> Renewable and Sustainable Energy Reviews. 14 (1), 217-232 (2010).</li><li>Wood, A. M., Everroad, R. C., Wingard, L. M., Andersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+growth+rates+in+microalgal+cultures.">Measuring growth rates in microalgal cultures.</a> Algal Culturing Techniques. , 270-272 (2005).</li></ol>

Batch, axenic photobioreactor experiments with regulated pH, temperature, gas flow rate, and gas concentration promote meaningful results by eliminating contamination by non-target algal strains and variability in culture conditions. Accurate pure culture growth kinetics can be obtained even in the presence of corrosive gases (CO2, SO2, NO2), which serve as nutrients, turning waste gases into a valuable product such as animal feed.
Prior to beginning any microa...

Photobioreactors are useful for controlled experiments and cultivation of purer microalgal products than can be achieved by open ponds. Microalgal cultivation in bench-scale photobioreactors supports the development of fundamental knowledge that may be used for process scale-up. Slight changes to environmental conditions can significantly alter microbiological experiments and confound the results1. A sterile process with temperature, pH, and gas sparging control is advantageous for studying microalgal properties and performance under varied conditions. Additionally, the control over input gas concentrations, temperature, shear force from mixing, and medium pH can support diverse species that are otherwise challenging to cultivate. Photobioreactors can be run as a batch process with continuous gas feed and sparging, or as a chemostat flow-through system with continuous gas feed and sparging plus influent and effluent wastewater nutrient inputs. Here, we demonstrate the batch process with continuous gas feed and sparging.
The use of photobioreactors addresses several microalgal cultivation and production challenges. The field generally struggles with concerns of contamination by other microorganisms, efficient substrate utilization (which is especially important in the case of CO2 mitigation or wastewater treatment)2, pH control, illumination variability, and biomass productivity3. Photobioreactors enable researchers to study a wide range of phototrophs in closely-controlled batch systems, where even slow growing species are protected from predators or competing microorganisms4. These batch systems are also better at facilitating greater CO2 utilization rates and biomass productivity because they are closed systems that are more likely to be in equilibrium with supplied gases. Photobioreactor technology also offers pH control, the lack of which has hindered high biomass productivity in past studies5. At bench scale, the level of control offered by photobioreactors is advantageous to researchers. At larger industrial scales, photobioreactors can be used to maintain commercial bioproduct purity and improve production efficiency for nutraceutical, cosmetic, food, or feed applications6.
Microalgae are of great interest for biosequestration of CO2 because they can rapidly fix CO2 as biomass carbon. However, most anthropogenic sources of CO2 are contaminated with other corrosive and toxic gases or contaminants (NOx, SOx, CO, Hg), depending on combustion process fuel source. Growing interest in sustainable CO2 sequestration has prompted development of photobioreactor technologies to treat CO2-rich emissions, such as those from coal-fired power plants (Table 1). Unfortunately, there is inherent risk of human and environmental exposure to the corrosive and toxic contaminants during research and scale-up processes. As such, describing the safe assembly and operation of bioreactors using corrosive gases is necessary and instructive.
This method is for the use of a 2 L bench-scale photobioreactor for the growth of microalgae under carefully controlled experimental conditions. The protocol describes microalgal storage, inoculum preparation, and photobioreactor setup and sterilization. Beyond basic operation, this work describes microalgal biomass measurements and biomass productivity calculations, and adaptation of the equipment for microalgal cultivation with corrosive gases. The protocol described below is appropriate for researchers seeking to exert greater experimental control, optimize microalgal growth conditions, or axenically culture a range of phototrophic microbes. This method does not describe appropriate materials for cultivation of microbes that produce or consume flammable gases (e.g. CH4, H2, etc.)7.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biostat A bioreactor</td>
			<td>Sartorius Stedim</td>
			<td></td>
			<td>2-liter bioreactor for microbial fermentation; designed to be autoclaved; pH, temperature, gas flow rate control</td>
		</tr>
		<tr>
			<td>Bump test NO2 gas</td>
			<td>Grainger</td>
			<td>GAS34L-112-5</td>
			<td>Calibration gas for MultiRAE gas detector</td>
		</tr>
		<tr>
			<td>Bump test O2, CO, LEL gas</td>
			<td>Grainger</td>
			<td>GAS44ES-301A</td>
			<td>Calibration gas for MultiRAE gas detector</td>
		</tr>
		<tr>
			<td>Bump test SO2 gas</td>
			<td>Grainger</td>
			<td>GAS34L-175-5</td>
			<td>Calibration gas for MultiRAE gas detector</td>
		</tr>
		<tr>
			<td>Corrosion resistant tubing for NO2 gas</td>
			<td>Swagelok</td>
			<td>SS-XT4TA4TA4-6</td>
			<td>PTFE Core Hose Smooth Bore X Series&#8212;Fiber Braid and 304 SS Braid Reinforcement</td>
		</tr>
		<tr>
			<td>Corrosion resistant tubing for SO2 gas</td>
			<td>QC Supply</td>
			<td>120325</td>
			<td>Reinforced Braided Natural EVA Tubing - 1/4&#34; ID</td>
		</tr>
		<tr>
			<td>cozIR 100% CO2 meter</td>
			<td>Gas Sensing Solutions Ltd.</td>
			<td>CM-0121 at CO2meter.com</td>
			<td>CO2 meter for concentrations up to 100%</td>
		</tr>
		<tr>
			<td>cozIR 20% CO2 meter</td>
			<td>Gas Sensing Solutions Ltd.</td>
			<td>CM-0123 at CO2meter.com</td>
			<td>CO2 meter for concentrations up to 20%</td>
		</tr>
		<tr>
			<td>Durapore Membrane Filter, 0.45 &#956;m</td>
			<td>Millipore Sigma</td>
			<td>HVLP04700</td>
			<td>Hydrophilic, plain white, 47 mm diameter, 0.45 &#956;m pore size, PVFD membrane filters</td>
		</tr>
		<tr>
			<td>Gas cylinder regulators</td>
			<td>Praxair</td>
			<td>PRS 40221331-660</td>
			<td>Single-stage stainless steel regulator configured for 0-15 psi outlet assembly diaphragm valve with 1/4&#34; MNPT threads, Stainless steel to resist corrosion from NOx and SOx</td>
		</tr>
		<tr>
			<td>Gas cylinders</td>
			<td>Praxair</td>
			<td>Ulta-zero air, high purity CO2, or custom gas composition</td>
			<td>Dependent on study objectives</td>
		</tr>
		<tr>
			<td>Gas monitoring and leak detection system</td>
			<td>RAE Systems by Honeywell</td>
			<td>MAB3000235E020</td>
			<td>Pumped model that detects O2, SO2, NO2, CO, and LEL</td>
		</tr>
		<tr>
			<td>GasLab software</td>
			<td>GasLab</td>
			<td>v2.0.8.14</td>
			<td>Software for CO2 meter measurements and data logging</td>
		</tr>
		<tr>
			<td>Hose barb</td>
			<td>Grainger</td>
			<td>Item # 3DTN3</td>
			<td>Used to adapt regulators to tubing, Stainless steel to resist corrosion from NOx and SOx</td>
		</tr>
		<tr>
			<td>K30 1% CO2 meter</td>
			<td>Senseair</td>
			<td>CM-0024 at CO2meter.com</td>
			<td>CO2 meter for concentrations less than 1%</td>
		</tr>
		<tr>
			<td>LED grow panels</td>
			<td>Roleadro</td>
			<td>HY-MD-D169-S</td>
			<td>Red &#38; blue LED light panels</td>
		</tr>
		<tr>
			<td>Memosens dissolved oxygen probe</td>
			<td>Endress+ Hauser</td>
			<td>COS22D-19M6/0</td>
			<td>Autoclavable (with precautions) dissolved oxygen probe for bioreactor</td>
		</tr>
		<tr>
			<td>Memosens pH probe</td>
			<td>Endress+ Hauser</td>
			<td>CPS71D-7TB41</td>
			<td>Autoclavable (with precautions) pH probe for bioreactor</td>
		</tr>
		<tr>
			<td>Oven, Isotemp 500 Series</td>
			<td>Fisher Scientific</td>
			<td>13246516GAQ</td>
			<td>Small oven for drying</td>
		</tr>
		<tr>
			<td>Prism GraphPad software</td>
			<td>GraphPad Software</td>
			<td>Version 7.03 or 8.0.1</td>
			<td>Graphing software for data organization, data analysis, and publication-quality graphs</td>
		</tr>
		<tr>
			<td>Stem to hose barb fitting</td>
			<td>Swagelok</td>
			<td>SS-4-HC-A-6MTA</td>
			<td>Stainless Steel Hose Connector, 6 mm Tube Adapter, 1/4 in. Hose ID</td>
		</tr>
		<tr>
			<td>Tubing, dilute acid/base transfer</td>
			<td>Allied Electronics and Automation</td>
			<td>6678441</td>
			<td>Silicone TP Process Tubing; 1.6mm Bore Size; 3000mm Long; Food Grade</td>
		</tr>
		<tr>
			<td>Tubing, gas transfer</td>
			<td>Allied Electronics and Automation</td>
			<td>6678444</td>
			<td>Silicone TP Process Tubing; 3.2mm Bore Size; 3000mm Long; Food Grade</td>
		</tr>
	</tbody>
</table>

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.

A calibration curve for the green microalgae, S. obliquus, harvested in the exponential phase, was established with OD750 and dried biomass concentrations (Figure 2). The linear regression had an R2 value of 0.9996.
An S. obliquus culture was started in a 250 mL Erlenmeyer flask from a culture stored on a refrigerated agar plate. The microalga was inoculated in 3N-BBM with 10 mM HEPES buffer and sparged with 2.2% CO2</s...

Watch this Scientific Journal Video about Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases at JoVE.com

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

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 ...

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