The authors acknowledge the National Science Foundation Emerging Frontiers in Research and Innovation (Award #1332341) for funding this research.&#160;The authors would also like to acknowledge Dr. Andrew Grieshop, as well as the LabJack and DAQFactory online support communities for their assistance and help offered throughout this process.

<ol>
	<li>Liu, X., Clarens, A. F., Colosi, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algae+biodiesel+has+potential+despite+inconclusive+results+to+date.">Algae biodiesel has potential despite inconclusive results to date.</a> Bioresour. Technol. 104, 803-806 (2012).</li><li>Chisti, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiesel+from+microalgae.">Biodiesel from microalgae.</a> Biotechnol. Adv. 25 (3), 294-306 (2007).</li><li>Lardon, L., H&#233;lias, A., Sialve, B., Steyer, J. -. P., Bernard, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life-cycle+assessment+of+biodiesel+production+from+microalgae.">Life-cycle assessment of biodiesel production from microalgae.</a> Environ. Sci. Technol. 43 (17), 6475-6481 (2009).</li><li>Henry, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+Quantity+of+Gases+Absorbed+by+Water,+at+Different+Temperatures,+and+under+Different+Pressures.">Experiments on the Quantity of Gases Absorbed by Water, at Different Temperatures, and under Different Pressures.</a> Philos. Trans. R. Soc. London. 93 (1803), 29 (1803).</li><li>Wang, X., Hao, C., Zhang, F., Feng, C., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+growth+of+two+blue-green+algae+species+(Microsystis+aruginosa+and+Anabaena+spiroides)+by+acidification+treatments+using+carbon+dioxide.">Inhibition of the growth of two blue-green algae species (Microsystis aruginosa and Anabaena spiroides) by acidification treatments using carbon dioxide.</a> Bioresour. Technol. 102 (10), 5742-5748 (2011).</li><li>Juneja, A., Ceballos, R. M., Murthy, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Environmental+Factors+and+Nutrient+Availability+on+the+Biochemical+Composition+of+Algae+for+Biofuels+Production:+A+Review.">Effects of Environmental Factors and Nutrient Availability on the Biochemical Composition of Algae for Biofuels Production: A Review.</a> Energies. 6 (9), 4607-4638 (2013).</li><li>Bernard, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hurdles+and+challenges+for+modelling+and+control+of+microalgae+for+CO+2+mitigation+and+biofuel+production.">Hurdles and challenges for modelling and control of microalgae for CO 2 mitigation and biofuel production.</a> J. Process Control. 21 (10), 1378-1389 (2011).</li><li>Guest, J. S., van Loosdrecht, M. C. M., Skerlos, S. J., Love, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumped+Pathway+Metabolic+Model+of+Organic+Carbon+Accumulation+and+Mobilization+by+the+Alga+Chlamydomonas+reinhardtii.">Lumped Pathway Metabolic Model of Organic Carbon Accumulation and Mobilization by the Alga Chlamydomonas reinhardtii.</a> Environ. Sci. Technol. 47 (7), 3258-3267 (2013).</li><li>Packer, A., Li, Y., Andersen, T., Hu, Q., Kuang, Y., Sommerfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+neutral+lipid+synthesis+in+green+microalgae:+A+mathematical+model.">Growth and neutral lipid synthesis in green microalgae: A mathematical model.</a> Bioresour. Technol. 102 (1), 111-117 (2011).</li><li>Oren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hundred+years+of+Dunaliella+research:+1905-2005.">A hundred years of Dunaliella research: 1905-2005.</a> Saline Systems. 1, 2 (2005).</li></ol>

The authors have nothing to disclose.

This PBR system offers the ability to monitor and control bench-scale algal kinetic growth experiments, allowing for more repeatable results from experimental assays used to quantify growth. However, an understanding of the limitations and uncertainties of sensor measurements is critical to ensure that the sensor readings accurately reflect reactor conditions. This understanding includes basic knowledge of the measurement principles involved with sensors, the process and frequency of calibration, the measurement uncertainty, and what the sensor can and cannot measure. For example, the electrical response for the light sensor described here is not equally distributed across the visible spectrum range, and certain correction factors may need to be applied to the sensor output, depending upon how this sensor data will be analyzed.
Temperature levels and variations are also extremely important, as changes in temperature can drastically influence the sensor response. Understanding potential interferences that can impact the sensor readings is also critically important; this interference can be ambient electrical noise from the building or could stem from the measurement environment (e.g., sodium ions can drastically impact pH readings at pH values over 10)12. Moreover, submerging multiple probes into a solution, especially a highly ionic and conductive salt solution, is also a potential source of interference. Electrodes that measure pH (or ionic strength, dissolved oxygen, dissolved CO2, etc.) are especially sensitive to ambient electrical noise and can be easily perturbed. Signal conditioning used for protecting the electrode signal cannot guarantee that other factors will not interfere with the probe readings. As part of quality control, other laboratory equipment, such as a hand-held pH probe, a hand-held spectrometer, and a thermometer, should be used to verify the sensor readings and to ensure that the system is set up and running properly.
Another limitation that must be addressed is the possible impact of the algae and/or culturing environment on the sensors. For example, if algal debris or bubbles cover the photodiode receptor of the light sensor, the readings will be affected. Similarly, pH electrodes are extremely sensitive and require extra care to ensure accurate readings. These electrodes work by measuring a voltage difference across an internal junction due to the buildup of H+ ions; a hydrated buffer layer within the probe is required to maintain accurate measurements12. Depending upon the conditions within the reactor, this layer will wear off, and the response of the sensor may change over the course of the experiment while the probe is submerged. In preliminary tests, the pH voltage output did not drift by more than ~0.2 pH units over the course of a 20-day experiment, but further assessments should be performed to characterize this change in sensor response and to establish maximum experimental run times, especially if fine pH adjustments/quantifications are needed.
Many current bench-scale PBR systems built to analyze algal growth do not monitor and control the internal culture environment as tightly as needed to discern how different factors impact algal growth, since setting up systems in this way can be challenging. This protocol can help facilitate more controlled experiments by giving step-by-step instructions for constructing a PBR with real-time monitoring. Moreover, this live data can be used not only to better control experimental conditions, but it can potentially be utilized to estimate growth kinetics (e.g., optical density readings as reference for general growth rates).
Controlled experimental systems can help to make algal research more reproducible. Bench-scale PBR setups that are monitored and controlled can increase experimental efficiency by minimizing unintended artifacts in experimental design and can help to advance efforts to make algal biofuels a sustainable, alternative fuel source.

Due to growing concerns about global climate change and finite fossil fuel resources, governments have been developing policies to reduce fossil fuel consumption and to encourage the development of new, sustainable transportation fuels. The United States Environmental Protection Agency has developed the Renewable Fuel Standard (RFS), which requires that 36 of the annual 140 billion gallons of U.S. transportation fuel mix come from renewable fuel sources by 2022. Innovative and transformational technologies will be necessary to meet these and future renewable energy standards1.
The use of microalgae-based biofuels has the potential to help meet the national RFS while reducing greenhouse gas emissions2. Microalgae-based biofuels have several advantages compared to first-generation biofuels based on terrestrial food crops, such as corn and soybeans. Unlike first-generation biofuels, algae-based biofuels consume fewer land, water, and food-related resources, since algae can be cultivated year-round and on barren land using saltwater or wastewater. Microalgae have high growth rates compared terrestrial crops and can accumulate high levels of lipids, which can be readily converted to biodiesel3. Currently, no industrial-scale algae-to-biofuel plants exist due to the high costs of the energy-intensive production processes, which consist of algal cultivation, lipid separation, and lipid refining into biodiesel. More research is needed to make these processes more efficient and sustainable.
PBRs, which are optically clear, enclosed installations for the production of phototrophic microorganisms in an artificial environment, are considered one of the most promising cultivation methods3. However, current designs still lack the volumetric productivity necessary to make the algae-to-biofuel production process more efficient and economically attractive4. Powerful mathematical models that consider light irradiance and attenuation, the transport of nutrients and CO2, and the growth of the microalgae can greatly facilitate the optimization of PBR design and operation. Bench-scale growth experiments are required to determine species-specific growth parameters for these optimization models.
Kinetic tests require the careful monitoring and control of experimental setups to prevent unintended inhibitors of growth. Given the photosynthetic nature of algae (i.e., their consumption of CO2 and absorption of light), maintaining controlled conditions is especially difficult in bench-scale PBRs. As depicted in Equation 1, the amount of dissolved CO2 in the growth medium, commonly denoted as <img alt="Equation" src="/files/ftp_upload/55545/55545eq1.jpg" /> (Equation 2), will be, at minimum, a function of: 1) the CO2 partial pressure and Henry&#39;s equilibrium constant, which dictates the amount of gas that will dissolve in solution (Equation 3); 2) the initial chemical composition of the growth medium, which impacts the speciation and activity of the carbonate ions and pH (Equations 4 and 5); and 3) the temperature, which impacts Equations 3-55.
<img alt="Equation" src="/files/ftp_upload/55545/55545eq2.jpg" /> 
<img alt="Equation" src="/files/ftp_upload/55545/55545eq3.jpg" /> 
<img alt="Equation" src="/files/ftp_upload/55545/55545eq4.jpg" /> 
<img alt="Equation" src="/files/ftp_upload/55545/55545eq5.jpg" /> 
<img alt="Equation" src="/files/ftp_upload/55545/55545eq6.jpg" />
The various phases and the chemical speciation of carbon create a challenge for measuring and maintaining a consistent dissolved carbon concentration within a PBR while holding other conditions constant (e.g., the pH increases as the algae consume CO2, and increasing the dissolved CO2 substrate can possibly lead to an acidic environment that inhibits growth)6.
An additional layer of complexity for controlling conditions during algal kinetic tests involves the light intensity within the PBR. The average light intensity inside a PBR is a function of not only the incident light intensity, but also the design (e.g., material, shape, depth, and mixing), the absorbance of algal biomass components (particularly chlorophyll), and the light-scattering properties of the algal cells. As the algae grow, the average light intensity will decrease. This change in light intensity, whether caused by an increase in total cells and biomass, an increase in chlorophyll content per cell, or both, can eventually induce a metabolic response, such as an increase in chlorophyll production per cell or the use of carbohydrate and lipid storage products for energy7. Continuous monitoring of the light intensity from within the reactor provides invaluable information. This data can help to ensure that conditions stay within a specified range and can be used to help estimate algal growth and absorbance parameters if combined with other measurements (i.e., biomass, chlorophyll concentration, reactor depth, incident light, etc.).
Understanding how algae grow under a specified set of conditions requires that the pH, dissolved CO2, light intensity, and temperature be monitored in bench-scale kinetic experiments. Many algal growth setups are not equipped to monitor conditions to the extent required for calibrating kinetic models, making the modeling process extremely challenging8. Although many companies offer bench-scale PBRs with automation and control, these bench-scale setups can be extremely expensive (~$20,000) and might not accommodate all experimental considerations of a given research question.
The first step in setting up a control-feedback system for a batch experiment is live data acquisition. This paper aims to demonstrate how to construct and set up a bench-scale PBR equipped with continuous light, pH, and temperature monitoring. This real-time monitoring setup can help to ensure that the experimental conditions stay within desired ranges, at the researcher&#39;s discretion. While this protocol does not detail specific control mechanisms, these step-by-step instructions provide a basic foundation for the data acquisition framework required before more sophisticated control feedbacks can be implemented.

<table border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Cast acrylic sheets</td>
			<td style="width:133px;">McMaster Carr</td>
			<td style="width:135px;">8560K244</td>
			<td style="width:345px;">7/8&#39;&#39; thick, 12x36&#39;&#39;, optically-clear, the size of sheets purchased will depend on reactor dimensions.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:169px;">Acrylic cement</td>
			<td style="width:133px;">McMaster Carr</td>
			<td style="width:135px;">7517A4</td>
			<td style="width:345px;">Scigrip plastic pipe cement, #4SC nonwhitening for acrylic. Not needed if gaskets and screws are used for PBR assembly.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Acrylic cement applicator needle</td>
			<td style="width:133px;">McMaster Carr</td>
			<td style="width:135px;">75165A136</td>
			<td style="width:345px;">Acrylic cement applicator needle, 25 Gauge, 1&#34;, Stainless steel, PTFE lined.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Plastic dispensing bottle for acrylic cement</td>
			<td style="width:133px;">McMaster Carr</td>
			<td style="width:135px;">7544A67</td>
			<td style="width:345px;">Plastic dispensing bottle, 2-oz size, packs of 5.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Viscous acrylic cement</td>
			<td style="width:133px;">McMaster Carr</td>
			<td style="width:135px;">7515A11</td>
			<td style="width:345px;">Scigrip plastic pipe cement. Medium-bodied acrylic cement to seal in any gaps within PBR body.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">PG-13.5 thread tap</td>
			<td style="width:133px;">McMaster Carr</td>
			<td style="width:135px;">2485A14</td>
			<td style="width:345px;">Can be used to help secure pH electrode to lid (if applicable).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">PBR and lid</td>
			<td style="width:133px;">NCSU Precision Machine Shop</td>
			<td style="width:135px;">Karam Algae 3.2L Reactor Revision E</td>
			<td style="width:345px;">This machine shop is open to public for business. Contact shop manager.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">pH sensor</td>
			<td style="width:133px;">Hamilton</td>
			<td style="width:135px;">238643</td>
			<td style="width:345px;">EasyFerm Plus 120, autoclavable, millivolt output.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Light sensor</td>
			<td style="width:133px;">Apogee Instruments</td>
			<td style="width:135px;">SQ-225</td>
			<td style="width:345px;">Amplified 0-5 volt electric calibration quantum sensor, water-proof.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Temperature sensor</td>
			<td style="width:133px;">LabJack</td>
			<td style="width:135px;">EI1034</td>
			<td style="width:345px;">Stainless steel, water-proof temperature sensor.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:169px;">pH transmitter wire with BNC end</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:135px;">HAM355173-1EA</td>
			<td style="width:345px;">This wire will vary with type of pH probe. Make sure wire is compatible with pH probe and has BNC connector end.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Unity gain pre-amplifier</td>
			<td style="width:133px;">Omega Engineering</td>
			<td style="width:135px;">PHTX-21</td>
			<td style="width:345px;">Signal processing amplifier for pH electrode needed for high-impedance pH readings.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Coaxial adapter, BNC female-to-binding post</td>
			<td style="width:133px;">Amazon</td>
			<td style="width:135px;">SMAKN B00NGD5K80</td>
			<td style="width:345px;">For connecting pH signal from pre-amplifier to microcontroller.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Capacitor (1000 uF)</td>
			<td style="width:133px;">Amazon</td>
			<td style="width:135px;">Nichicon BCBI4950</td>
			<td style="width:345px;">For low-pass filter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Resistor (1000 ohm)</td>
			<td style="width:133px;">Radio Shack</td>
			<td style="width:135px;">2711321</td>
			<td style="width:345px;">For low-pass filter.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Hookup wire</td>
			<td style="width:133px;">RadioShack</td>
			<td style="width:135px;">2781222</td>
			<td style="width:345px;">For making low-pass filters, connecting sensors to microcontroller, and wiring motor.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Heat shrink tubing</td>
			<td style="width:133px;">RadioShack</td>
			<td style="width:135px;">2781611</td>
			<td style="width:345px;">For low-pass filter assembly.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:169px;">Data acquisition and control unit</td>
			<td style="width:133px;">LabJack</td>
			<td style="width:135px;">LabJack U6</td>
			<td style="width:345px;">To process electrical signal from sensors and communicate with data acquisition and control software.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:169px;">DAQFactory data acquisition software</td>
			<td style="width:133px;">DAQFactory</td>
			<td style="width:135px;">DAQFactory Express Release 5.87c Build: 2050</td>
			<td style="width:345px;">Free to download, for up to 10 channels.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Mini DC-gearmotor</td>
			<td style="width:133px;">McMaster Carr</td>
			<td style="width:135px;">6331K31</td>
			<td style="width:345px;">Motor for mixer impeller.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Impeller and shaft</td>
			<td style="width:133px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:345px;">Email authors for 3D files.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Variable DC power supply</td>
			<td style="width:133px;">Amazon</td>
			<td style="width:135px;">Tekpower HY1803D</td>
			<td style="width:345px;">Variable DC power supply, 0-18V @ 0-3A.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Grow Lamp</td>
			<td style="width:133px;">HydroGrow</td>
			<td style="width:135px;">SOL-1</td>
			<td style="width:345px;">This exact model is no longer available.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:169px;">Incubator</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:135px;">Precision Model 818</td>
			<td style="width:345px;">This particular incubator can withstand an internal heat source since this unit&#39;s cooling compressors run non-stop regardless of temperature setting.</td>
		</tr>
	</tbody>
</table>

construction and setup of a bench-scale algal photosynthetic bioreactor with temperature, light, and ph monitoring for kinetic growth tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

Data from this real-time monitoring system show the dynamic culturing environment for algae within a bench-scale PBR and highlight the need for monitoring and controlling the system. The logged temperature data (Figure 6) demonstrates how light illumination, incubator air temperature, and energy dissipation associated with algal growth can change the temperature within the PBR and how the real-time data can be used to adjust incubator temperature controls, as needed.
The measured light over the course of the experiment further emphasizes the dynamic nature of this growing environment. As observed in Figure 7, the light sensor reading, measured as photosynthetic photon flux density (PPFD; &#181;E-m&#8722;2s&#8722;1), was ~100 PPFD before algae was added and dropped immediately to 85 PPFD after inoculating the reactor with the algal culture. The light continued to drop to less than 5 PPFD on day 7. This decrease in light intensity is due to increasing biomass and cell counts, and/or to increasing absorption by increased chlorophyll content, showing that algae are active through day 7, despite low light levels. Additional biological measurements are required to make further inferences.
The continuously logged pH data show that, overall, the pH was adequately controlled during this experiment with the implemented pH control algorithm (Figure 8). This data, showing both minute-by-minute readings and hour-long averages, demonstrate a few key points about culturing algae and monitoring pH in real time. First, the pH increased above the desired set point of 7.6 immediately after inoculating the PBR with algae. This change was expected, as the culture seed that was added to the PBR had a pH value higher than the set point, since the flask used to grow the inoculum was not pH-controlled. Secondly, this live data highlights how sensitive pH electrodes are to external electrical noise. This sensitivity is noted by a drastic jump in the electrode values between day 1 and day 2. These sudden changes in pH values were likely created by electrical noise from a solenoid valve from an adjacent experimental setup. This electrical disturbance prematurely triggered the pH control algorithm to inject CO2 into the PBR. Consequently, the pH dropped below the desired set point. The sensitivity of the pH electrodes can lead to extreme outliers and can potentially disrupt control systems.

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55545/55545fig3.jpg" /> 
Figure 3: pH Response and Calibration Example Graphs. (a) Example response graph of the pH sensor (b) Example calibration graph of the pH sensor, with an equation to use for the conversion. Regression analysis shows a 95% confidence interval. Error bars are not visible (standard error less than 0.03%). These graphs show that the pH sensors was connected properly and that its signal was very steady. <a href="http://cloudflare.jove.com/files/ftp_upload/55545/55545fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55545/55545fig6.jpg" /> 
Figure 6: Temperature Measurements Within the PBR During a 7 day Experiment. Dark blue points represent 1-h averages of sensor data, and light blue points represent sensor readings acquired over 1 min (acquisition timing of 1 s, average length of 60) and converted to temperature using manufacturer-supplied conversion factors. Black arrows show when the incubator temperature setting was adjusted to maintain the culture temperature around 25 &#176;C (this desired set point is designated with a red, dotted line). Fluctuations in temperature are due to algal growth and changes in incubator temperature. <a href="http://cloudflare.jove.com/files/ftp_upload/55545/55545fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55545/55545fig7.jpg" /> 
Figure 7: Light Measurements Within the PBR During a 7 day Experiment. Dark blue points represent 1 h averages of sensor data, and light blue points represent sensor readings acquired over 1 min (acquisition timing of 1 s, average length of 60) and converted into PPFD using default light-sensor factory calibration values. <a href="http://cloudflare.jove.com/files/ftp_upload/55545/55545fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55545/55545fig8.jpg" /> 
Figure 8: pH Measurements Within the PBR During a 7 day Experiment. Dark blue points represent 1-h averages of sensor data, and light blue points represent sensor readings logged every 1 min (acquisition timing of 0.1 s, average length of 600) and converted into pH using conversion equation established via calibration. The pH was maintained between 7.6 and 7.5 using a 99% CO2 gas injection. The red, dotted lines indicate the desired pH range. <a href="http://cloudflare.jove.com/files/ftp_upload/55545/55545fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests at JoVE.com

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

construction-setup-bench-scale-algal-photosynthetic-bioreactor-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

16.1K Views.  North Carolina State University. This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

Video: Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or non-acidic refluxate. This paper describes a method for the development, manufacturing, and implantation of a miniature wireless-enabled pH sensor. The sensor is designed to be implanted endoscopically with a single hemostatic clip. A fully passive rectenna-based receiver based on a zero-bias Schottky diode is also constructed and tested. To construct the device, a two-layer printed circuit board and off-the-shelf components were used. A miniature microcontroller with integrated analog peripherals is used as an analog front end for the ion-sensitive field-effect transistor (ISFET) sensor and to generate a digital signal which is transmitted with an amplitude shift keying transmitter chip. The device is powered by two primary alkaline cells. The implantable device has a total volume of 0.6 cm3 and a weight of 1.2 grams, and its performance was verified in an ex vivo model (porcine esophagus and stomach). Next, a small footprint passive rectenna-based receiver which can be easily integrated either into an external receiver or the implantable neurostimulator, was constructed and proven to receive the RF signal from the implant when in proximity (20 cm) to it. The small size of the sensor provides continuous pH monitoring with minimal obstruction of the esophagus. The sensor could be used in routine clinical practice for 24/96 h esophageal pH monitoring without the need to insert a nasal catheter. The &#34;zero-power&#34; nature of the receiver also enables the use of the sensor for automatic in-vivo calibration of miniature lower esophageal sphincter neurostimulation devices. An active sensor-based control enables the development of advanced algorithms to minimize the used energy to achieve a desirable clinical outcome. One of the examples of such an algorithm would be a closed-loop system for on-demand neurostimulation therapy of gastroesophageal reflux disease (GERD).

The manuscript presents a miniature implantable pH sensor with ASK modulated wireless output together with a fully passive receiver circuit based on zero-bias Schottky diodes. This solution can be used as a basis in the development of in vivo calibrated electrostimulation therapy devices and for ambulatory pH monitoring.

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

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Chapters in this video

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

Operation of a Benchtop Bioreactor

Construction and Characterization of a Novel Vocal Fold Bioreactor

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver