Name: power input measurements in stirred bioreactors at laboratory scale
Uploaded: 2018-05-16T12:30:00.000+00:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about Power Input Measurements in Stirred Bioreactors at Laboratory Scale at JoVE.com

The authors would like to thank Dieter H&#228;ussler and Beat Gautschi for their assistance during the experimental set up. We are also grateful to Caroline Hyde for English proof reading.

1. Preparation of sucrose solutions
NOTE: The sucrose solutions are used as cheap, Newtonian model media with elevated viscosity and density for reduced turbulence conditions (see Table 1).
<ol>
	<li>Fill a Duran glass bottle with water and sucrose of different concentrations (20 - 60 %w/w).</li>
	<li>Mix the content with a magnetic stirrer until the sucrose has completely dissolved.
	<ol>
		<li>For sucrose concentrations in excess of 40 %w/w, add the sucrose intermittently and heat the glass bottle slightly (~ 50 &#176;C). Let the sucrose solution cool down to room temperature before use.</li>
	</ol>
	</li>
</ol>
2. Preparation of a measurement recipe and the data logging

<ol>
	<li>After starting the software, initiate the communication with the control unit by selecting the correct serial COM port from the dropdown menu and clicking the Connect button. 
	Note: The Connect button will change the colour to green and the LED below the dropdown menu will switch on, once the communication with the control unit is initiated.</li>
	<li>Set up the data file path inside the bioreactor control unit software in order to store the data on the operator PC.
	<ol>
		<li>Open the Settings tab page and hit the folder symbol next to the Data file location text field.</li>
		<li>In the file dialog window, browse to the desire folder, type a file name into the File name text field and click the OK button. 
		Note: The data log file path and name are displayed in the text box and the DAQ start button is enabled, once a valid file path is defined.</li>
	</ol>
	</li>
	<li>Set up a routine inside the recipe manager of the bioreactor control unit software in order to automate the measurement procedure.
	<ol>
		<li>Open the Recipe tab page and type the desired input values for the recipe phase elapsed time (min) and the corresponding impeller speed (rpm) into the text field boxes. The profile is automatically displayed in the chart. 
		Note: For example, the agitator speed is increased stepwise by 20&#160;rpm from 100&#160;rpm to 300&#160;rpm, and each value is maintained for 4&#160;minutes in order to guarantee a stable torque signal (see discussion below). The minimum and maximum speeds as well as the amount of the increase can be adjusted for different agitators and vessels. 
		Note: Select the speed range carefully with respect to the torque sensor resolution, the nominal torque and vortex formation. The latter often occurs in unbaffled bioreactors agitated at higher speeds and can cause damage to the torque meter.</li>
		<li>Click the Save button, browse to the desired file path and type a file name in the text field. Hit the OK button to save the file.</li>
	</ol>
	</li>
</ol>
3. Installation of the torque sensor
NOTE: The experimental setup is shown schematically in Figure 1.
<ol>
	<li>Install the torque transducer in a specially designed holder that incorporates the air bearing (see Figure 1) using screws to fix the sensor into place. The air bearing used in this study has a porous carbon bushing material with an inner diameter of 13 mm.
	<ol>
		<li>Mount the brushless servo agitator motor onto the top of the holder. Fix the torque transducer to the vertical holder mounting using four screws.</li>
		<li>Connect the motor shaft to the drive shaft of the torque transducer using a metal bellow coupling that can compensate small axial misalignments of the shafts and tighten the coupling using screws. Connect the agitator shaft to the measurement shaft of the torque transducer using another metal bellow coupling. 
		NOTE: In this study, specifically designed impeller shafts with a diameter of 13 mm (tolerance: - 0.0076 mm) and with lengths of between 270 mm and 520 mm were used for the different vessels investigated.</li>
	</ol>
	</li>
	<li>Mount the sensor holder onto the bioreactor head plate and install the impellers on the agitator shaft with the desired off-bottom clearance. Mount baffles and additional installations (e.g. sampling and harvest tubes, electrochemical sensors, etc.) inside the bioreactor if required.</li>
	<li>Install the desired bioreactor in the vessel holder if required (bioreactors #1, #3 to #10) or place the head plate onto the bioreactor tank (bioreactor #2) and tighten the head plate with screws.
	<ol>
		<li>For investigations of glass bioreactors, place the bioreactor glass vessel into the holder.</li>
		<li>For investigations of single-use bioreactors, disassemble the top mounted tubing ports and impeller shaft housing from the plastic head plates by using appropriate cutting tools. Place the plastic vessel into the holder.</li>
	</ol>
	</li>
	<li>Place a temperature sensor inside the bioreactor and connect it to the control unit. Connect the tubing for the pressurized air to the gas inlet port of the air bearing and apply a pressure of around 5.5 bar provided by a compressor. Connect the torque transducer to the A/D converter and power on the transmitter.</li>
</ol>
4. Configurations in the data acquisition software
<ol>
	<li>Open the software for the data acquisition of torque sensor signal and configure the measurement preferences.
	<ol>
		<li>Make sure that the first two channels in the DAQ channels window are initialized and active. In this study, the torque signal was set on channel 0 and the rotational speed signal was set on channel 1.
		<ol>
			<li>Click the Live update button to display the current measurement values.</li>
		</ol>
		</li>
		<li>Set the torque channel signal to zero if the absolute torque signal without rotation is larger than 0.1 mN&#183;m by using the right-mouse click on the channel item in the channel list and selecting the Zero balance option.</li>
		<li>Navigate to the DAQ job tab page and define a data acquisition rate of 2 Hz from the dropdown menu list. Use the options Immediately at job start and Duration from the dropdown lists to set the Start and Stop of the data acquisition, respectively.</li>
		<li>Define a time span for the Sample duration that is longer than the time required to finish the measurement (for example, use 1 h 0 m 30 s for a one hour recipe defined in the second step).</li>
		<li>Navigate to the Data storage settings page and select the option ASCII + channel info from the dropdown list to set the File format for the data save file. Set a file path on the PC hard drive for the measurement Output file.</li>
	</ol>
	</li>
</ol>
5. Perform the torque measurement
<ol>
	<li>Start the data acquisition for the torque signal in the control and data acquisition software for the torque meter by clicking the Start button on the DAQ job menu page.</li>
	<li>Start the data acquisition for the agitator speed and the temperature in the bioreactor control unit software by clicking the DAQ start button on the Settings tab page.</li>
	<li>Start the agitator control in the control unit software with a manual set-point or the pre-defined recipe scheme.
	<ol>
		<li>If a single measurement is conducted, use the control box entry on the Main tab page of the bioreactor control software. Type the desired set-point into the text box and click on the &#8216;Agitator control on&#8217; item.</li>
		<li>If multiple measurements with a recipe are conducted, navigate to the Phases tab page and click the Start button. 
		Note: The software will automatically disable all manual entry boxes for the duration of the recipe and a window opens automatically to confirm the end of the process.</li>
	</ol>
	</li>
	<li>In the data acquisition software, a window opens automatically after the pre-defined measurement duration. Save the data for each measurement on the operator PC, preferably on the hard drive, by clicking the Save data now button.</li>
	<li>Repeat the measurement for each desired agitator speed without and with liquid inside the bioreactor vessel.
	<ol>
		<li>Pour water (or the sucrose solution) through a funnel into the bioreactor. 
		Note: Make sure that the liquid completely covers the impellers since (partially) exposed impellers can result in undesired axial forces that could damage the torque sensor.</li>
	</ol>
	</li>
</ol>
6. Data evaluation
NOTE: The obtained torque values in the empty vessel (dead torque) correspond to the residual friction losses of the bearing and must be subtracted from the values determined in the liquid in order to obtain the effective torque values (see Eq. 1).
<ol>
	<li>Average the torque values for each agitator speed measured after a quasi-stable signal has been achieved (see discussion below). Ideally, calculate the mean value over a period of at least 2 min for each condition, corresponding to 240 data points at a measurement rate of 2 Hz.</li>
	<li>Use a Matlab code for the data processing by running the code from the software command line. 
	NOTE: The code is provided for download in the supplement section of this manuscript. This script imports the raw data file from the data recording, calculates the phase average rotational speed, Reynolds number (from Eq. 3 based on user inputs) and torque values for each of the phases, visualizes the outcomes, and stores the results in a second text file, which then can be used to further process the data.</li>
	<li>Subtract the torque values obtained in the empty vessel from those measured in the liquid to obtain the effective torque values.</li>
	<li>Calculate the power input and dimensionless power number from the time-averaged torque values according to Eq. 1 and Eq. 2.</li>
</ol>

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Eng. Res. Des. 79, 811-818 (2001).</li><li>Werner, S., Greulich, J., Geipel, K., Steingroewer, J., Bley, T., Eibl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+propagation+of+Helianthus+annuus+suspension+cells+in+orbitally+shaken+bioreactors:+Improved+growth+rate+in+single-use+bag+bioreactors.">Mass propagation of Helianthus annuus suspension cells in orbitally shaken bioreactors: Improved growth rate in single-use bag bioreactors.</a> Eng. Life Sci. 14, 676-684 (2014).</li></ol>

The authors have declared no conflicts of interest.

Despite the importance of the (specific) power input for the engineering characterization and scaling-up/down of bioreactors, only a few publications on experimental investigations in benchtop scale bioreactors, particularly single-use systems in the one-digit liter volume range, can be found in the literature. One reason for this lack of data can be seen in the difficulties of accurate power input measurements in such small scales. In order to overcome some of these difficulties, the present study provides a detailed protocol for torque based power input measurements that are supported by an air bearing to minimize the friction losses in the bearing. The applicability of the method was demonstrated using three commercially available single-use bioreactors as well as multi-use bioreactors in scales between 1 L and 10 L working volume.
Based on our experience with the torque based measurements, the most critical factors to address are: 1) reducing the dead torque by minimizing the friction losses inside the bearings and seals, in particular in laboratory scale bioreactors, and 2) the selection of a suitable torque meter for the desired bioreactor size and agitation conditions. As has been shown earlier35, the dead torque can be dramatically reduced by the use of an air bearing. In the present study, a low cost air bushing made of porous carbon material was used. The residual torque in the empty vessels tested were typically below 0.5 mN&#183;m with agitation rates of up to 900 rpm, corresponding to impeller tip speeds of up to 3 m&#183;s-1. In contrast, the dead torque of the bioreactor #6 with the built-in mechanical shaft bearing was, for example, between 9.4 mN&#183;m and 20 mN&#183;m, and comparable values of around 3 mN&#183;m have been also reported for the bioreactor #732. This is about one order of magnitude higher than the values obtained in the proposed experimental setup.
Besides the air bearing, the torque meter used is the most critical component. A commercially available torque meter that is designed for measuring static and dynamic torque, rotation speed and angle of rotation was selected for this study. Considering the bioreactors of interest with maximum working volumes of 10 L and the corresponding agitators, a nominal torque of 0.2 N&#183;m was chosen. It was found that high reproducibility with relative standard deviation of replicates &#60; 5% and reliable measurements can be obtained for effective torques as low as 2 mN&#183;m, corresponding to only 1 % of the nominal torque. Hence, the measurement range of the sensor applied in the present study was significantly wider than results which have been published based on an inter-laboratory study of members of the German GVC-VDI working group on mixing41.
Nevertheless, the range of the agitator speed should be carefully selected with respect to the torque sensor resolution, the nominal torque and vortex formation. The latter often occurs in unbaffled bioreactors agitated at higher speeds and can cause damage to the torque meter. Both the minimum and maximum feasible agitator speeds can be limiting factors of the method described in this study. In addition to our previous work35, this study involved also the bioreactor #3, the smallest member in glass bioreactor family provided by the manufacturer, which is agitated by two-stage impellers with diameters of 42 mm. A comparable power characteristic to that in the geometrically similar bioreactor #4 was obtained with the presented experimental setup. This is notable since the torque scales with M <img alt="Proportional to" src="/files/ftp_upload/56078/56078pro.jpg" /> d5 for a given liquid density, impeller geometry (i.e. power number) and rotational speed (see Eq. 1 and Eq. 2). Consequently, an approximately 40% lower impeller torque results from a 10% smaller impeller diameter, for example. Nevertheless, higher rotational speeds in the 1 L scale than in the 2 L scale were required during operation to resolve the produced torque with the available torque meter. Due to the built-in baffles of the bioreactor #3, no vortex formation was observed, but this can become an issue with unbaffled vessels. It should be emphasized that the constant offset in the power numbers that was found between the two scales could result from measurement inaccuracies caused by the limited sensor resolution (in addition to geometrical differences). Further investigations are required to draw final conclusions on the minimum scale at which the proposed setup is still feasible.
Nevertheless, the same protocol was used for power input measurements in various glass vessels from different manufacturers with working volumes of between 1 L and 10 L in our laboratory. This highlights the transferability of the used method for the characterization of different bioreactor systems. The experimental effort could be reduced by automated measurements using the recipe management within the automation system provided by the control unit software and the automated data processing based on the universal Matlab language.
Further, it should be noted that, by using the sucrose containing, cheap Newtonian model media, a wide range of Reynolds numbers (100 &#60; Re &#60; 6&#183;104), depending on the agitator and scale, was covered. It should be also emphasized that the lower limit of the turbulence range is usually irrelevant for animal cell cultures with water-like media, even if very low impeller speeds are used. However, significant increases in the broth viscosity, which results in turbulence damping, and even non-Newtonian behavior have been described for fungi- and plant cell-based cultures. For example, apparent viscosities in plant cultures of up to 400-fold compared to water have been reported42, which leads to much lower Reynolds numbers.
Finally, using the bioreactor #7 as a first case study, it has been demonstrated that the proposed experimental setup can be used to study the effect of design modifications on the power input at laboratory scale. In combination with rapid prototyping techniques, this can be a powerful tool for impeller design studies, which will form parts of future work.

Power input is a key engineering parameter for the characterization and scaling-up of bioreactors because it relates to many unit operations, such as homogenization1,2,3, gas-liquid dispersion2,4,5, heat transfer6 and solid suspension7. Power input is associated also with shear stress, which can particularly affect growth and product formation in shear sensitive cell cultures8,9,10,11.
The most common techniques for the measurement of the power input in stirred bioreactors are based on electrical power draw12,13,14, calorimetry12,15 (i.e. stationary heat balance or dynamic heating through the agitation) or the torque upon the agitator. The latter can be experimentally determined by dynamometers, torque meters or strain gauges, which have been applied for a variety of agitators, including single or multi-stage Rushton turbines1,16,17,18,19,20,21,22,23,24,25, pitched blade impellers19,20,23,26,27, InterMig19,21 and Scaba impellers28,29. A detailed review is provided by Ascanio et al. (2004)30.
From the torque (T), the power input (P) can be estimated from Eq. 1, where N is the rotational speed of the agitator.
<img alt="Equation 1" src="/files/ftp_upload/56078/56078eq1.jpg" /> (1)
In order to account for losses occurring in the agitation (in bearings, seals and the motor itself), the effective torque (Teff) should be determined as the difference between the value measured in the empty vessel (TD) and in the liquid (TL). Finally, the dimensionless power number (P0, also known as Newton number), which is defined by Eq. 2 where &#961;L denotes the liquid density and d represents the impeller diameter, can be used to compare different agitators.
<img alt="Equation 2" src="/files/ftp_upload/56078/56078eq2.jpg" /> (2)
It is well-known that the power number is a function of the Reynolds number (i.e. the turbulence) and becomes constant under fully turbulent conditions. The impeller Reynolds number is defined by Eq. 3, where &#951;L is the liquid viscosity.
<img alt="Equation 3" src="/files/ftp_upload/56078/56078eq3.jpg" /> (3)
Nevertheless, power input measurements in small scale bioreactors are still challenging due to the relatively high friction losses inside mechanical bearings of the impeller shafts and the limited accuracy of most commercially available torque meters. Consequently, only a few reports about power input measurements in benchtop scale bioreactors have been published17,18,22,24,31,32. There is also a lack of data about the power input in single-use bioreactors, which are delivered by the manufacturers preassembled, sterilized and ready-to-use33,34. In contrast to their reusable counterparts, most single-use bioreactors are agitated by specially designed impellers, making comparisons difficult.
In order to close this gap, a reliable method for power input measurements with special focus on laboratory scale stirrers has been developed recently35. The torque values measured in the empty vessels, which were caused by the friction losses, were effectively reduced by the use of an air bearing. Consequently, a wide range of operational conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104) could be investigated and the power input of several multi-use and single-use bioreactors has been provided.
The present study provides a detailed measurement protocol of the previously developed method and describes how to set up, conduct and evaluate a torque-based power input measurement in laboratory scale bioreactors. Special focus is on commercially available single- and multi-use systems. An automated measurement procedure is used to reduce the experimental effort.

<table><tbody><tr><td>T20WN torque meter</td><td>HBM Hottinger Baldwin&lt;br/&gt; Messtechnik GmbH</td><td></td><td>Nominal torque 0.2 Nm</td></tr><tr><td>Spider-8</td><td>HBM Hottinger Baldwin&lt;br/&gt; Messtechnik GmbH</td><td></td><td>HBM Spider8 is no longer available for sale. QuantumX&lt;br/&gt; DAQ system (especially the QuantumX modules MX840A and MX440A) are recommended.</td></tr><tr><td>Catman easy software</td><td>HBM Hottinger Baldwin&lt;br/&gt; Messtechnik GmbH</td><td></td><td>Version 4.2.2</td></tr><tr><td>Air bearing</td><td>IBS precision engineering</td><td></td><td>13 mm air bushing</td></tr><tr><td>Stainless steel impeller shaft</td><td>Bioengineering AG</td><td></td><td>Shaft tolerance -0.0076 mm</td></tr><tr><td>Brushless motor AKM2</td><td>Kollmorgen</td><td></td><td></td></tr><tr><td>Metal bellow coupling</td><td>Uiker AG</td><td></td><td></td></tr><tr><td>Finesse RDPDmini control unit</td><td>Finesse, a part of Thermo Fisher Scientific</td><td></td><td>No longer supported (the replacement product G3Lab universal controller can be used)</td></tr><tr><td>Sucrose</td><td>Migros Schweiz AG</td><td></td><td>Food grade</td></tr><tr><td>Matlab software</td><td>Mathworks</td><td></td><td>Version R2017a</td></tr><tr><td>Finesse &amp;mu;TruBio PC software</td><td>Finesse, a part of Thermo Fisher Scientific</td><td></td><td>Version 3.1 (no longer supported)</td></tr><tr><td>SmartGlass 1L</td><td>Finesse, a part of Thermo Fisher Scientific</td><td></td><td>referred to as Bioreactor 1L in Table 2</td></tr><tr><td>SmartGlass 3L</td><td>Finesse, a part of Thermo Fisher Scientific</td><td></td><td>referred to as Bioreactor 3L in Table 2</td></tr><tr><td>SmartVessel 3L</td><td>Finesse, a part of Thermo Fisher Scientific</td><td></td><td>referred to as Single-Use 3L Bioreactor in Table 2</td></tr><tr><td>Mobius CellReady 3L</td><td>Merck Millipore</td><td></td><td>referred to as Cell Ready Single-Use 3L Bioreactor in Table 2</td></tr><tr><td>UniVessel SU 2L</td><td>Sartorius Stedim Biotech</td><td></td><td>referred to as Single-Use 2L Bioreactor in Table 2</td></tr></tbody></table>

power input measurements in stirred bioreactors at laboratory scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

Watch this Scientific Journal Video about Power Input Measurements in Stirred Bioreactors at Laboratory Scale at JoVE.com

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

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17.7K Views.  Thermo Fisher Scientific. The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

Video: Power Input Measurements in Stirred Bioreactors at Laboratory Scale

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, ...

Environment

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 ...

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 ...

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

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.

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.

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

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 ...

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.

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 ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

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Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

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Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

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The Use of Chemostats in Microbial Systems Biology

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Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...