A LOW DILUTION RATE MICROCHEMOSTAT ARRAY WITH
PROGRAMMABLE CELL POPULATION CONTROL
1
Jianzhang Wu1, Michael Polymenis2 and Arum Han1, 3*
Department of Electrical and Computer Engineering, 2Department of Biochemistry and Biophysics,
3
Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA
ABSTRACT
Chemostat is a bioreactor in which microorganisms can be cultured at steady state by controlling the rate of medium inflow and waste outflow. Microfluidic chemostat arrays can provide higher throughput with significantly smaller reagent consumption while still providing accurate chemostatic condition control, all at significantly lower costs compared to conventional chemostats. Here we present a microchemostat array that can provide wide ranges of dilution rates from as low as
0.01/h to 1/h in a 250 nl volume chemostat. The compact size of the device housing 8 arrays on a 4 by 6 cm footprint allows
high-throughput microbial studies. Each chemostat unit also has integrated electrodes for on-line pH sensing and programmable population control mechanism.
KEYWORDS: Microchemostat array, Low dilution rate, Yeast cell culture, pH electrode
INTRODUCTION
Chemostat is a bioreactor in which microorganisms are cultured at steady state by controlling the concentration of available nutrients through culture medium refreshing rate control, and is widely used for quantitative research in microbiology.
Several microfluidic chemostats in array format have been developed to increase the throughput with accurate control over all
units in the array [1-3]. Dilution rate, defined as the volume of replaced medium over the whole volume in a unit time, is a
key controlling factor that determines the concentration of nutrients in the culture medium, which controls the specific
growth rate of microorganisms. Lower dilution rate means less replacement of culture medium in a unit time, causing smaller
fluctuation in nutrient concentration over time. This results in more stable microbial growth state. However, in microchemostats, achieving low dilution rates is difficult due to the already small bioreactor volume. Also, integrating sensors to monitor
the bioreactor parameters such as pH and oxygen become challenging with small microchemostat arrays.
The microchemostat array presented here uses a novel two-depth reactor design to achieve dilution rates as low as 0.01/h
in a 250 nl volume microchemostat unit, one order of magnitude lower than previously achieved by others with the same total
bioreactor volume [1-3]. The array houses 8 microchemostats on a 4 x 6 cm footprint, allowing microbial studies to be conducted at a higher throughput with less reagent consumption and cost. Microfabricated iridium oxide (IrOx) electrodes [4]
were integrated into each of the microchemostat units as pH sensors. LabViewTM programmed syringe pumps and solenoid
valves for pneumatic actuation of microvalve array allow fully automatic operation of the device. Saccharomyces cerevisiae
(baker’s yeast) was cultured as a model microorganism to demonstrate the functionality of this microchemostat array. Cell
growth was analyzed by cell counting in the microchemostat over time.
EXPERIMENTAL
Microchemostat array design and fabrication
Figure 1A illustrates the microchemostat array device having 8 microchemostat units. Each microchemostat unit has a
ring-shaped culture channel, a pneumatically actuated microperistaltic pump to continuously mix culture medium, and two
pneumatic valves for microchannel blockage during culture medium replacement (Figure 2B). The culture channel has two
regions with different depths. The shallow channel between the two microvalves is the culture medium replacement region,
which defines the minimum replacement volume. During a dilution cycle, valve #1 is closed to separate the shallow channel
region (20 µm depth) from the ring-shaped microbe culture channel region (100 µm depth), followed by flushing this isolated
region with fresh medium, and then merging it back to the main channel by opening valve#1 and closing valve#2. Using this
two-depth design, the replacement volume can be significantly reduced with the same footprint to achieve one order of magnitude lower dilution rate, which can be further lowered if needed.
The microchemostat devices were fabricated in PDMS (Sylgard 184, Dow Corning, Inc., Midland, MI) by softlithography technology. The master mold for the two depth culture channel was made by two layers of SU-8 photoresist
(SU-8 2015, 2075, Microchem, Inc., Newton, MA). After two separate exposure with two photomasks, the two layers of SU8 were developed at the same time to form the microstructures with two different heights. After PDMS layer replication, the
culture layer was bonded with the pneumatic actuation PDMS layer with a PDMS membrane (15 µm thick) sandwiched in
between.
Yeast cell growth analysis in the microchemostat
Saccharomyces cerevisiae (CEN.PK) was cultured in the microchemostat device with synthetic carbon limited medium
(100 ml with Yeast Nitrogen Base 0.17 g, Ammonium Sulfate 0.5 g, Dextrose 0.08% m/v and MES 0.426 g). A programma-
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001
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15th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
October 2-6, 2011, Seattle, Washington, USA
ble syringe pump (HA33, Harvard Apparatus, Holliston, MA) and solenoid valves (SMC, Noblesville, IN) connected to a
digital I/O module (DO-16(USB)GY, Contec Co., Sunnyvale, CA) are automatically controlled by a custom LabViewTM (National Instruments, Austin, TX) program to culture yeast cell in the microchemostat device with adjustable dilution rates. Microscopic pictures of cell growth in the shallow channel were automatically captured by an optical microscope (Eclipse
TS100, Nikon America Inc., Melville, NY) every 10 min and then counted and converted into cell density.
A B Figure 1. (A) Illustration of the 8-unit microchemostat array composed of three PDMS layers. Top layer shows the
pneumatic bridge layer for interconnecting the pneumatic channels, middle layer shows the pneumatic actuation layer for
microperistaltic pump and microvalve control, and bottom layer shows the microbe culture channel layer. (B) A single
chemostat unit composed of a two-depth channel structure and a pneumatically actuated peristaltic pump.
pH electrode fabrication and testing
The pH electrodes were fabricated by an IrOx electrochemical deposition process [4]. First, a Cr/Au (30/300 nm) layer
was deposited on a glass substrate by thermal evaporation and then the electrodes were pattern by lithography. The electrolyte solution was prepared by diluting 85 mg IrCl4, 1 ml 35% H2O2 and 250 mg oxalic acid into 150 ml DI water. Before electroplating, pH of the solution was adjusted to 10.5 by adding K2CO3. A pulse power supply (DuPR10-1-3, Dynatronix, Inc.
Amery, WI) was used in voltage cycling mode to apply +0.7V (250 ms) and -0.5V (250 ms) on the Au seed layer for 10 min.
A platinum electrode (CH Instruments, Inc. Austin, TX) worked as a counter electrode during electroplating. The IrOx pH
electrodes were tested by measuring the potential difference between a Ag/AgCl reference electrode (Orion 900100, Thermo
Scientific Inc. Barrington, IL) in standard pH buffers (4, 7, 10).
RESULTS AND DISCUSSION
The assembled device of the microchemostat array with color dye filling the microchannels for visualization is shown in
Figure 2. Red tubings indicate culture medium refreshing tubings connected to syringe pumps. Blue and green tubings indicate
pneumatic control layers connected to solenoid valves to actuate the microperistaltic pumps and microvalves. With LabViewTM
control, fully automatic operation of the device for over 6 days was successfully conducted.
Yeast cells were culture in the microchemostat device for over 140 hours (6 days). Figure 3 shows that the cell density in
the device responds well to the change in dilution rate from 0.1/h to 0.25/h. Cell density stabilization time varies for different
dilution rates, however was in the range of 12 to 36 hours.
Figure 2. A microchemostat array device filled with color dye for visualization, showing the microbe culture channel
(red), the pneumatic actuated peristaltic pumps (blue), and the pneumatic valves (green).
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A B Figure 3. (A) Yeast cell density inside the microchemostat at four different dilution rates over a 140h run. (B) Example
microscopic images of yeast cell growth inside the microchemostat at different time point.
The pH electrodes were continuously tested for a period of 3 weeks. Extremely good linearity (R2=0.9999) could be
achieved over a broad pH range during the test period (Figure 4 A). Sensitivity (Figure 4B) and baseline (E0, Figure 4C) fluctuated in the first week, however then stabilized after about 10 days. This result demonstrates that IrOx microelectrodes are
suitable for long-term on-line pH sensing.
Figure 4. (A) The voltage response of a microfabricated Iridium Oxide pH electrode (R2=0.9999). (B) Variation of sensitivity and (C) variation of E0 (base line) over a 3-week period.
CONCLUSION
We have developed a low dilution rate microchemostat array device with programmable control system, in which longterm and high throughput yeast cell culture with adjustable dilution rates were successfully demonstrated. Initially testing of
the pH sensing electrodes shows good linearity and stable sensitivity and baseline, and will be integrated into the microchemostat units in the future.
REFERENCES
[1] F. Balagadde, et al., "Long-term monitoring of bacteria undergoing programmed population control in a micro chemostat," Science, 309, 5731 (2005).
[2] Z. Zhang, et al., "Microchemostat-microbial continuous culture in a polymer-based, instrumented microbioreactor," Lab
Chip, 6, 7 (2006).
[3] A. Groisman, et al., "A microfluidic chemostat for experiments with bacterial and yeast cells," Nature methods, 2, 9
(2005).
[4] K. Pásztor, et al., "Iridium oxide-based microelectrochemical transistors for pH sensing," Sensors and Actuators B, 12
225-230 (1993)
CONTACT
*A. Han, tel: +1-979-8459686; arum.han@ece.tamu.edu
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