Hardware

Bioproduction is expensive and inaccessible.

In a world where resources are limited and the global population is growing rapidly, there is an increasing demand for many substances we use every day, from food flavourings to pharmaceuticals, fuels, and even the dyes in our clothing. Unfortunately, these are still often produced through unsustainable industrial processes, leading to chemical pollution, high carbon emissions, and harmful farming practices.

As demand continues to rise, bioproduction offers a promising alternative. In particular, continuous bioproduction can ensure a more sustainable future by allowing long-term cultivation in stable conditions, which results in higher product quality, process reproducibility, and resource efficiency compared to traditional batch or fed-batch methods (see below for more details).

As our project aims to enhance biomanufacturing of complex pathways, our team was soon facing a problem. The core vessel of continuous bioproduction - the chemostat - is very expensive. Commercial systems often cost tens thousands of euros and require complex infrastructure and specialized expertise. This makes continuous bioproduction inaccessible to many researchers and, notably, to numerous iGEM teams and small labs worldwide. This financial and technical barrier limits the exploration of sustainable and innovative approaches in biotechnology. While many teams can design and model biological systems for steady-state operation, many lack the tools to experimentally test them. We were convinced that these circumstances need to change to make research more accessible and facilitate impactful insights on a sustainable future, so we set out on a mission to develop our own open-source, low-cost, and modular chemostat. Our design drastically reduces costs while maintaining functionality and flexibility. It can be built using readily available components and our detailed assembly guide, allowing anyone, anywhere, to experiment with continuous bioproduction. By lowering the barrier to entry, we aim to democratize science and make sustainable biotechnological research more accessible to all. After all, isn’t that what iGEM is all about?

How does a chemostat work?

Continuous bioreactors, like chemostats, are powerful tools for research and application in many different fields such as biotechnology, evolutionary studies, metabolic engineering and of course synthetic biology. We find another example in microbiology, where scientists use chemostats to simulate realistic environmental conditions for microbes. Typical cultivation systems are based on a one-time only substrate addition (batch), which is not the reality outside of a lab. Unlike fed-batch systems, which continuously supply nutrients, but still eventually face culture decline, chemostats maintain a steady state by imposing controlled substrate limitation and continuously removing part of the culture. This minimizes metabolic burden and other stress factors, enables stable growth as we keep the cells at a constant growth rate and work towards reproducible experimental outcomes (Bioprocess Operation Modes: Batch, Fed-Batch, and Continuous Culture - Eppendorf Deutschland, n.d.).

Our chemostat

Figure 1: Setup of the chemostat.

Our chemostat is made up of various components that all contribute to the perfect setup! The glassware consists of two parts (vessel and lid), which are held together by a clamp mechanism. The clamp is made of autoclavable plastic material. The glassware is supplemented with various sensors. Additionally, the system contains four peristaltic pumps with tubing attached, and a metal stirrer moved by a motor. The sensors are read out and controlled via a circuit composed of an ESP8266 module, an Arduino microcontroller, and other small electronic components.

Take a look at all components and get inspired!

Glassware

We designed our glassware with consideration to both technical limitations and adaptability. By using standardized sizes for outlet and inputs, we ensure a broad compatibility across the board. Generally, chemostats are double-walled: the outer cavity is filled with heated water to maintain optimal temperatures, while the inner cavity contains the culture liquid, probes, and other experimental components. Refer to Figure 2 for technical details. Not shown on the scheme is the condensator (spiral glass body on top of the lid), this prevents excessive evaporation, condenses vapor back into the culture, and maintains culture volume and sterility. The lid is equipped with four valves, each sealed with a thin silicone plate and screw cap. This design allows for easy customization - whether you want to add extra analytical tools, additional inlets, or outlets, the setup is flexible enough to accommodate your specific requirements. The opening in the middle of the lid is used for the stirrer, see more info below.

The final fabrication was done by Tanja Noch, a glassblower at our university.

Figure 2: Technical drawing of the chemostat glassware.

Sensors and Probes

To ensure we cover and record all relevant parameters, we equipped our glassware with various sensors. This included sensors for:

• Temperature: Maintaining and monitoring temperature ensures optimal microbial growth and metabolic activity, as deviations can stress cells and affect experimental outcomes.
• Dissolved Oxygen (DO): Measuring DO allows control over aerobic conditions, which directly affects respiration, metabolism, and product formation in the culture.
• pH: Monitoring pH is critical for microbial health, as many microbes have narrow pH ranges for optimal growth and metabolic stability.
• Gas flow: Controlling gas flow ensures proper aeration and mixing, prevents oxygen limitation, and maintains a stable environment for microbial growth.
• Liquid flow: Tracking and controlling liquid flow maintains the steady state of the chemostat, regulates nutrient supply, and ensures waste removal.
• CO₂ concentration: Measuring CO₂ provides insights into metabolic activity and respiration rates, helping assess microbial growth and overall culture health. To connect the tubing to the sensor, we 3D printed a case (see Figure 3).
• Fluorescence. This is a key step, and the connection between our engineered cells and the hardware. It allows us to detect and quantify fluorescence, and therefore the evaluation of the biosensor.

Figures 3: CAD files of the 3D CO₂ sensor housing (top and base).

Pumps

We included 4 peristaltic pumps to spike in substrate, base and feed and remove gas flow out. These pumps allow precise, real-time control over culture conditions, enabling customized experimental protocols and improving reproducibility.

Stirrer and Motor

The stirrer is held in a teflon case to not damage the glass. It is made of stainless steel and moved by a motor. The motor shaft is connected to the stirrer by a 3D printed adapter that is secured by 4 screws. The motor itself is hung on a stand, to achieve vertical stirring. While initially we worked with a bigger propeller, after consultation with Prof. Dr. Nick Wierckx, we changed our design to a smaller, pitch-bladed one (see Figure 4). This allows better mixing and prevents the formation of dead zones.

Figure 4: Different stirring propellers. Top: pitch-bladed stirrer used in the final chemostat design. Bottom: alternative larger designs.

Arduino and ESP

To communicate with the sensors, we set up an electric circuit using Arduino Mega 2560 and an ESP8266 as microcontrollers. The ESP8266 serves primarily as a Wi-Fi access point, enabling remote interaction with the system, while the Arduino Mega 2560 manages the sensor connections and input/output control. The circuit was further equipped with the necessary level shifters, capacitors, and other supporting components, ensuring stable communication and reliable data transmission between the sensors and the software interface.

Curious, fascinated, and ready to build your own? If you’re as excited about exploring continuous cultures as we are, check out our Chemostat Assembly Guide below. It has everything you need to get started on your own project!

Assembly Guide

Chemostat Parts List and Cost Analysis

Part	Quantity	Function	In which step used?	Link	Price per unit (EUR)
Atlas Scientific Dissolved Oxygen Kit	1	Measuring dissolved oxygen	Taking measurements in chemostat	https://atlas-scientific.com/kits/surveyor-analog-do-kit/	183.37
Atlas Scientific SMA PT-1000 Temperature Kit	1	Measuring temperature	Taking measurements in chemostat	https://atlas-scientific.com/kits/sma-temperature-kit/	56.28
Atlas Scientific pH Kit	1	Measuring pH	Taking measurements in chemostat	https://atlas-scientific.com/kits/ph-kit/	136.46
Sensair K30 CO2 Sensor	1	Measuring CO2	Taking measurements in chemostat	https://senseair.com/product/k30/	57.58
Sensirion SFM3200 Flow Sensor	1	Measuring gas flow	Taking measurements in chemostat	https://sensirion.com/products/catalog/SFM3200-AW	169.06
Digikey 18080 4 Position Cable Assembly	1	Wiring with adapter	Wiring with adapter (JST style) the gas flow sensor	https://www.digikey.be/en/products/detail/sparkfun-electronics/CAB-18080/14322719	1.51
AZ ESP8266MOD 12-F	1	Wifi communication	Communicating with chemostat sensors wirelessly via I2C	exact model not sold anymore; can be substituted with any esp8266	0.00
Adafruit Assembled Feather HUZZAH w/ESP8266	1	Wifi communication	Communicating with (INSERT)	https://eu.mouser.com/ProductDetail/Adafruit/3213	17.16
SainSmart Arduino MEGA 2560 R3	1	Connecting, communicating, powering	Connecting sensors and pumps	https://de.sainsmart.com/products/mega-2560-r3-atmega2560-16au-arduino-kompatibel	18.99
T100-SE01/WX10 OEM Peristatic Pump	4	Dosing	Pumping in chemostat	https://de.drifton.eu/shop/98-oem-schlauchpumpen/2633-t100-se01wx10-oem-peristaltikpumpe-mit-integriertem-treiber/	177.30 per pump
LONGER Platinum-cured silicone extension tubing, WT 1.00 mm, ID 1.00 mm	1	Tubing	Tubing for pumps in chemostat	https://www.drifton.eu/shop/107-extension-tubing/2489-platinum-cured-silicone-extension-tubing-15-m/	39.90
LONGER LongerPump(r) silicone tubing #24 5m	1	Tubing	Tubing for pumps in chemostat	https://www.drifton.eu/shop/107-extension-tubing/2489-platinum-cured-silicone-extension-tubing-15-m/	39.90
SparkFun Triad Spectroscopy Sensor - AS7265x	1	Spectrophotometer	Measuring fluoresence	https://www.sparkfun.com/sparkfun-triad-spectroscopy-sensor-as7265x-qwiic.html	69.95
Adafruit Qwiic JST SH 4-pin to Premium Male Headers Cable	1	Qwiic adapter	Adapter for Sparkfun spectrophotometer	https://eu.mouser.com/ProductDetail/Adafruit/4209	0.82
LED yellow	1	LED	take blue one	https://www.amazon.de/-/en/dp/B0CZ3YSHZB	7.99
Dispensing tubes	3	Tubing	10G	https://www.amazon.de/-/en/Cannulas-Stainless-Syringes-Refilling-Adhesives/dp/B0D661MTV6	10.79
Luer Lock Connectors	3	Tubing addon	Adapter for dispensing tubes	https://www.amazon.de/-/en/Connectors-Transparent-Connector-Syringe-Coupling/dp/B0BMDVDT1T	7.19
SparkfunROB-13302 servo motor	1	Stirring	Motor for stirrer	https://www.mouser.de/ProductDetail/SparkFun/ROB-13302	6.76
EP1WSSS47RJ	1	Resistor	Resistor	https://www.mouser.de/ProductDetail/TE-Connectivity-Neohm/EP1WSSS47RJ	0.41
TXS0108E 8 Channel Bi-Directional Logic Level Converter	1	Level conversion	Level conversion for sensors	https://components101.com/modules/txs0108e-bi-directional-logic-level-converter-module	1.14
Sunny SYS1638-0605-W2E Switching Adapter 5V	1	Power adapter	Providing power to breadboard	https://www.soselectronic.com/de-de/products/sunny/sys1638-0605-w2e-341498	6.29
Adafruit Qwiic JST SH 4-pin to Premium Male Headers Cable	1	Qwiic adapter	Adapter for Sparkfun spectrophotometer	https://eu.mouser.com/ProductDetail/Adafruit/4209	0.82
BPS Jumper Wires ZIPWIRE 10cm MALE TO FEMALE	120	Wiring	Connecting parts	https://eu.mouser.com/ProductDetail/BusBoard-Prototype-Systems/ZW-MF-10	4.43
LDO Voltage Regulator 3.3V 1.0A Positive	1	Voltage regulation	Protoboard	https://eu.mouser.com/ProductDetail/STMicroelectronics/LD1117AV33	0.78
Renkforce JKMF403 Jumper-Kabel	1	Jumper cables	Connecting parts	https://www.conrad.com/en/p/renkforce-jkmf403-jumper-cable-arduino-banana-pi-raspberry-pi-40x-wire-jumper-40x-wire-jumper-socket-30-00-cm-mul-2299844.html	4.29
Renkforce JKMM403 Jumper-Kabel	1	Jumper cables	Connecting parts	https://www.conrad.com/en/p/renkforce-jkmm403-jumper-cable-arduino-banana-pi-raspberry-pi-40x-wire-jumper-40x-wire-jumper-30-00-cm-multi-colo-2299846.html	4.29
200 V resistor	1	-	-	-	0.00
KETOTEK Digital Temperature Controller Thermostat 230V	1	Temperature control	Temperature control in chemostar	https://www.amazon.de/KETOTEK-Digitaler-Thermostat-Temperaturregler-F%C3%BChler/dp/B08H4RK4D5	29.74
AQUA MEDIC Titanium Heater 100W	1	Temperature control	Heating the chemostat	https://teichbedarf-discount.de/AQUA-MEDIC-Titanium-Heater-Titan-Aquarium-Heizer-Alternative-zu-Glasheizstaeben-von-100-250-Liter-Laenge-ca-27-cm-Leistung-100-Watt-110010	34.90
AQQA Aquarium Water Pump, 1000 L/H	1	Water pumping	Pumping water into chemostat	https://www.amazon.de/-/en/Aquarium-Diving-Adjustable-Nozzles-Hydroponics/dp/B08SHK8ZR3	25.99
MEAN WELL DRP-240-24 24V Power supply	1	Power	Supplying 24V power to pumps	https://eu.mouser.com/ProductDetail/MEAN-WELL/DRP-240-24	70.23
Flow-Through Cell, Type 131-QS	1	Cuvette	Fully automatic fluorescence detection (optional)	https://www.analytics-shop.com/de/hl131-10-40	439.00
Glassware	1	Bioreactor glassware	Glass components of chemostat	Tanja Noch, glassblowing workshop at HHU	300.00
Stirrer	1	Stirring	Stirring cultures in chemostat	Waldemar Seidel, workshop at HHU	100.00
Filters for air	-	-	-	-	0.00

Cost summary:

Total cost: 2556,34€

Notes:

• The cost for "AZ ESP8266MOD 12-F", "200 V resistor", and "Filters for air" were not included in the total as no prices were provided in the original document.
• Prices are listed in EUR unless otherwise stated.
• Some items are listed as packs (e.g., dispensing tubes come in pack of 10 for 10.79 EUR, Luer Lock Connectors come in 30 pcs for 7.19 EUR).

Base assembly

1. Insert sensors into the base.
2. Place silicone ring and secure with clamp
3. Insert Teflon insert + stirring rod.
4. Attach the stirrer
5. Assemble top and bottom parts of glassware
6. Tighten screws

Motor assembly (for Sparkfun TB6612FNG Motor Driver)

1. Remove white plastic cover from motor - Always unscrew caps before inserting tubes
2. Screw the cap onto the glass vessel.
3. Ensure the correct silicone sealing tube sizes match the respective sensors - Always fill with electrolyte where required for the sensors
4. Attach the screw cap with the harder side facing the bottom

Sensor installation

Make sure sensors do not touch the stirrer directly. If they do, pull the sensor outward slightly!

Example: Dissolved oxygen sensor

1. Insert the dissolved oxygen sensor through the funnel port
2. Assemble the sensor mount
3. Ensure the dissolved oxygen sensor cap is completely submerged
4. The more the black ring is submerged, the better the reading
5. If needed, remove rubber ring → adjust → reattach before reconnecting the cap
6. Make sure you have selected the correct port name

Figure 5: Sensor wiring schematic

Spectrophotometer Assembly

1. Wire as in Figure 6
2. Place LED and sensor into 3D printed housing (3D housing taken from Laganovska et al., 2020)
3. Add either cuvette or cuvette with in and out flow to chamber
4. Make sure to change the provided python code to match the port of your device

Figure 6: Spectrophotometer wiring schematic

Figure 7: Spectrophotometer 3D housing

Condenser Assembly

1. Apply a thin layer of grease to the condenser (should always be a bit greasy)
2. Attach condenser as shown in schematic
3. Lower port = outflow; upper port = inflow
4. Position tubing so the end of the outflow tube is at water level

Pump Assembly (for LONGER T100-SE01 peristaltic pumps)

1. Cut and clamp the power and communication interface wires such that they are compatible with Arduino and your power supply
2. Use a 24V DC power supply:
   • DC24V+ → Connect to +24V from your power supply
   • DC24V- → Connect to GND (negative terminal of power supply)
3. Connect communication wires to Arduino as follows:

Table 1: Arduino wiring scheme for one pump

Pump Pin	Function	Arduino Connection	Notes
START (Pin 6)	Start/Stop control	Digital pin (22)	HIGH = Run, LOW = Stop (default logic)
DIR (Pin 4)	Direction control	Digital pin (23)	HIGH = CW, LOW = CCW
V/F (Pin 3)	Speed control (0–5V)	PWM pin (5)	Use analogWrite() for speed control
GND	Signal ground	GND on Arduino	Common ground essential

Figure 8: Arduino wiring scheme for 4 pumps

4. Set the DIP switches on the pump as follows:
   • SW2: ON-OFF-OFF
   • SW1: OFF-OFF-OFF
5. Upload code to Arduino
6. Place on a large supporting box (for stability)
7. If using multiple pumps, ground them via a breadboard
8. Connect Arduino, and then pumps to power supply
9. Attach pump tubes to designated in/outflow lines
10. Confirm tubing direction: From chemostat → pump → waste container

Video 1: Running chemostat

Sterilization Protocol

To ensure full lab compatibility, when choosing out materials and components, we made sure that they are all either autoclavable or easily sterilizable.

Components that are autoclavable:

• Glassware
• Clamp
• Pump tubing
• Stirrer

Components that are sterilized in bleach for 10min:

• pH, temperature and dissolved oxygen sensor

Other components:

• Pumps, Gas flow and CO₂ sensor
• Tubing
• Power supply 5V and 24V
• Microcontrollers and corresponding cables

Procedure:

1. Swabs taken from inside of the chemostat, pH sensor, temperature sensor, stirrer before sterilization are plated, as well as a control plating. Incubation for 24 hours at 37°C.
2. The temperature sensor and pH probe will be sterilized in bleach for 10 minutes.
3. Glassware, plastic and metals will be autoclaved.
4. Swabs from the same locations are taken and plated after sterilization and incubated for 24h at 37°C.

Further Perspectives

While our current setup already allows stable continuous cultures and real-time monitoring, there are several exciting directions for future development:

• Automation and feedback control: Integrating automated control systems such as PID loops or adaptive algorithms would allow the chemostat to autonomously maintain target parameters like dissolved oxygen, or optical density. This would minimize manual intervention and further improve process stability and reproducibility.

• Scalability: Expanding the system to include multiple parallel vessels could enable multiplexed experiments, allowing researchers to test different environmental conditions, substrates, or genetic constructs simultaneously. Such a modular, scalable setup would be ideal for optimization studies or evolutionary experiments.

• Advanced sensing and data integration: Incorporating additional analytical capabilities, such as optical density sensors, biosensing modules, or online metabolite detection, could significantly expand the system’s functionality. Linking these data streams to a cloud-based dashboard would allow real-time visualization, remote monitoring, and easy data sharing between collaborators.

• Community-driven development: Since all our CAD files (you can find them in our gitlab here), circuit designs, and Arduino code are open-source, we invite others to build upon our work. Through collaboration and shared improvement, we hope to foster an open hardware ecosystem that supports innovation and accessibility in synthetic biology.

Conclusion

With our open-source chemostat, we took a step toward making continuous bioproduction more accessible, affordable, and adaptable. By combining standardized glassware, 3D-printed components, and low-cost electronics, we created a system that can be assembled and operated by virtually anyone: from academic labs to fellow iGEM teams. Our design demonstrates that quality continuous cultivation does not have to rely on expensive commercial systems. Instead, it can be achieved through creativity, collaboration, and a commitment to open science. Beyond the hardware itself, we hope our project inspires others to question existing barriers and reimagine what biotechnology tools can look like when designed for everyone, everywhere.

After all, democratizing access to scientific tools doesn’t just make research more inclusive - it makes innovation more sustainable.

References

Bioprocess Operation Modes: Batch, Fed-Batch, and Continuous Culture - Eppendorf Deutschland. (n.d.). https://www.eppendorf.com/de-de/lab-academy/applied-industries/bioprocessing/introduction-to-bioprocessing/batch-fed-batch-and-continuous-culture/

Laganovska, K., Zolotarjovs, A., Vázquez, M., Mc Donnell, K., Liepins, J., Ben-Yoav, H., Karitans, V., & Smits, K. (2020). Portable low-cost open-source wireless spectrophotometer for fast and reliable measurements. HardwareX, 7, e00108. https://doi.org/10.1016/j.ohx.2020.e00108