Team:MADRID UCM/Hardware

Cloning design - 4C_FUELS

During the early stages of our project we realized that one crucial need for testing our solar production technology was a photobioreactor. Then, following the path of many other iGEM teams we set ourselves out in the challenge of building a DIY system for the automated cultivation of cyanobacteria and data collection.

In this page you will find how we approached the construction of a photobioreactor as a device for the initial prototyping of our technology.

Why to build a photobioreactor?

If you want to study the performance of a microorganism you will require a robust and versatile system capable of controlling a wide range of parameters in order to determine which parameters are optimal for the desired behavior. In our case we are using modified cyanobacteria and biohybrid materials containing them in order to generate an industrially relevant product. Then, a device capable of controlling key parameters such as light irradiation, temperature, aeration rate or culture density as well as registering relevant data is required to study and determine the optimal production conditions. This device is a Photobioreactor.

Furthermore, we have also studied the different approaches for product recovery from the culture media, where the first and common steps to most of the technologies is gas stripping. This operation consists in the removal of our desired product via the injection of a gas which will carry it out of the liquid culture media. This way if we really wanted to fully test our production technology, we are requiring a system capable of performing this gas stripping operation while keeping the culture at their optimal production conditions.

To know more about gas stripping, visit our Implementation page.

The openPBR

Fortunately former iGEM teams have already faced the need of an automated culture system for phototrophic organisms. Among them, the 2019 iGEM Team Humboldt_Berlin worked really hard into the development of a low-cost DIY photobioreactor: The OpenPBR. After carefully reading all the information they provide in their wiki and contacting Paul Herrmann, one of the 2019 team members, we decided that the OpenPBR was a great starting point to cover our needs.

However, as stated by their creators, the OpenPBR is just an initial iteration for the consolidation of cheap, efficient and robust phototrophic cultivation system. Many upgrades can still be done in order to improve its performance or adapt it to specific requirements. This is why we decided to not only replicate the original openPBR design, but to perform some upgrades in their design.

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Improving the openPBR

After carefully reading all the available documentation, we realized which parts of the system could be improved for our needs. We planned the modifications we would like to implement, designed the new blueprints and ordered the required materials to implement them. To simplify the complexity of the building process we decided to build just a single cultivation module.

Temperature control

The main issue we have found in the original design of the openPBR was the absence of an active temperature control system. While some microalgae can be adequately cultivated at room temperature, our chassis grows at an optimal temperature of 38 ┬║C. Then having a temperature control was essential for our purposes.

In order to implement a temperature control system two main elements are required: a measuring device and a heater. For the temperature measuring element, we considered it crucial to control as accurately as possible the temperature in the liquid media, implementing the DS18B20 temperature sensor for liquids. Likewise, to keep the system temperature we decided to use a low impedance carbon fiber resistance coiled around the culture chamber.

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Initially we evaluated the heat loss of our system, filling the chamber with warm water at 42 ┬║C and measuring the temperature drop in time. Also, we tested the ability of different resistance setups to provide heat to the water, identifying the best options.

However, for other openPBR users, the size of the chamber will depend and the thermal transports coefficients will widely vary with the required temperature and construction ending. Then, we decided to install a resistance setup that could offer high heating capacity, providing enough heat in any situation. This resistance setup should be also tightly controlled, in order to release just the necessary amount of heat at any time.

Temperature Sensor

Despite the mention of the DS18B20 temperature sensor within the openPBR documentation, there were no clear guidelines for its implementation within the system. We decided to introduce the temperature measuring probe within the culture chamber in direct contact with the culture media. This can be achieved using a cable gland tightly screwed to the surface of the culture chamber. This way, the temperature sensor could be easily removed, while ensuring the tightness and sterility of the system. However the DS18B20 sensor could be even autoclaved with the growth chamber if required, standing temperatures up to 125 ┬║C, so most of the conventional autoclave programs could be used.

Heating element

We used 33 Ohm / m carbon fiber 3 mm diameter flexible resistances. The resistance wire is split into 4 wires 80 cm long in order to circle the whole perimeter of the growth chamber. The resistance contacts were all connected to a common feed wire at each end and adequately connected to the MOSFET control board. The connection in parallel keeps the resistance low enough in each wire, allowing the release of enough heat during its activation.

After that, resistances were coiled around the external surface of the culture chamber, avoiding contact with the tubes and temperature sensor connections. Eventually the resistances were fixed to the chamber using thermal epoxy resin.

The temperature control system was implemented within an arduino code, where a temperature setpoint is established, and a tolerable offset is fixed. Then depending if the temperature is out of the desired range or within the range, the heaters will activate with higher or lower power. In order to perform temperature measurements, OneWire and DallasTemperature libraries were used.

As can be observed in the graphic, the system capacity for heating up the growth media is slow, taking nearly 45 minutes to reach the desired temperature. This is mainly due to the high thermal resistance of the chamber materials and design.

However, once the temperature has been established, the temperature control is nearly perfect, dropping less than ┬▒0.2 ┬║C from the desired setpoint.

In order to study how finely tuned the control could be, during the temperature registration different temperature offset were tested, determining the optimal range between ┬▒0.5 to ┬▒0.2 ┬║C. Lower or higher offset temperatures lead to a more abrupt temperature oscillation.

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Enhanced OD Measurements

In the original design, a single fixed wavelength spectrophotometric unit was implemented in each growth chamber. However, relevant data such as the relative chlorophyll content of the cells can be studied via the ratio between OD680 / OD720. In addition, there is no clear agreement in the OD at which the density of phototrophic microorganism cultures is measured. Because of that, we decided to implement a double OD measuring system, duplicating the LED + Opt101 light measurement system employed by Humboldt.

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Likewise, in the original design, the measurement of OD was performed at the middle point of the chamber, causing the overlap of the light path with the bubbling air column formed by culture aeration. To avoid this issue, we decided to move the Sensors to the sides of the growth chamber.

After comparing the results with the sensors measuring in the middle or in the sides of the chamber, we realized that there were no significant differences when the aeration was off. However, when aeration was turned ON, the measurements performed with the sensor in the middle of the chamber, were only accurate if aeration was turned OFF for a few seconds during the measurement, while the signal of the sensors in the sides was not significantly altered by the aeration.

Eventually, since the Opt101 sensor has maximum sensitivity within the far red to infrared region, we decided to directly implement the potential read to OD conversion within the control program. To do so, we performed a calibration of the sensor, comparing the reads of the DIY spectrophotometric device with the actual reads of a laboratory spectrophotometer. Then the logarithmic correlation coefficients were calculated by non-linear regression and implemented within the control code. The results shown in the Graph 2 demonstrates that the OD measure in the system is reliable even at ODs up to 1.

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Controlling the system

In order to control the openPBR we have adapted the original code developed by 2019 iGEM Team Humboldt_Berlin. We have simplified the code to work as a turbidostat control program. In addition we have expanded its functionality for the integration of the temperature control code and re-structured the structure in order to make it more user-friendly. To know more about the openPBR control, temperature control code or the system calibration procedures, you can visit our GitHub repository.

Construction Issues and recommended modifications

During the building process we found some other aspects of the system that could be problematic to other users, either during the building process or during the utilization of the system. Some of these issues could not be fully solved, however we also implemented some design changes in order to make the system more robust and reliable.

OD Measurements

While assembling the OD measuring devices, we used the same 680 nm LED employed by humboldt (LED680-02AU from Roithner) and wider-angle 720 nm LED (LED720-03AU from Roithner). In both cases, the intensity registered by the Opt101 was too high, even when the growing chamber was filled with cultures with densities up to OD720 > 1.

In order to avoid the saturation of the Opt101 sensor, we had to cover the sensors with a filtering mask made of two layers of transparent tape painted with a black permanent marker. After this modification, the sensor started to offer a sensitive response depending on the OD of the liquid in the culture chamber.

Rewiring the MOSFETs control board

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The Arduino AT Mega 2560 board used to control the system is not capable of supplying all the power required by LEDs, Pumps or the Heater, then an auxiliary circuit should be built in order to switch on or off most of the openPBR systems. This power control is achieved using N-channel MOSFETs, which can briefly act as an electronically controlled switch. The MOSFET can be activated by the Arduino output, allowing the current to freely flow from the power source to the required device. In addition, via the arduino PWM output it is possible to regulate the amount of power that would be used by the final device allowing a more precise control of the system.

During the assembly of the MOSFET control board we found many issues with the adequate control of the LEDs or other devices not being reliable. First we found issues with the STP55NF06 N-Channel MOSFETS, which were not capable of providing enough current to the final device. Since the components we acquired probably were faulty, we needed to replace them with IRFZ44N which also worked well. However the biggest challenge was the continued MOSFET saturation. Despite during the first execution of the code the arduino was able to activate the LEDs and other devices, after the switching off the arduino output, the MOSFET remained in saturation. Even after disconnecting the power supply for some time (30 - 90 seconds), after reconnecting the power the MOSFETS remained saturated, keeping all the activated devices permanently ON.

We researched a bit about this problem and after several attempts we found that this is a common problem when working with MOSFETs as control switches. After activation the MOSFET can feedback itself and remain saturated even after ceasing the activation signal in the GATE terminal. To solve this, a pull resistance must be placed between the gate and ground terminals of the MOSFET, allowing it to un-saturate it after the activation signal disconnection.

We have found this additional resistance indispensable for a reliable control of the system using MOSFETS.

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Aeration control

In the original openPBR design, aeration needs to be switched off during certain events like OD measurements. To do so, a solenoid air valve is used. Despite the system works as expected we have found that the pressure drop across the valve is high, significantly reducing the total available pressure for aeration. Since other pressure drops like a 0.22 ╬╝m filter required for air sterilization, and a rotameter valve for flux control, pressure drop must be avoided as much as possible to guarantee enough pressure in the aeration system. In addition, the energy consumption is increased, since most of the solenoid air valves are designed to be normally closed in the absence of an activation signal. This situation also forces the air pump during the periods where the air valve is closed.

Despite not having enough time to test our design, we have planned a better system to efficiently control aeration. In the original design gas supply is provided by an external 220v air pump, then the gas flow could be controlled by the introduction of a 12 v relay normally closed which could be used for connecting or disconnecting the air pump from the current. In addition a 12v air pump could also be used, and normally controlled by the same MOSFET Scheme used to control the rest of the system devices.

Our Experience with openPBR

After working in the construction and testing of the openPBR we have become enthusiasts of the possibility of DIY photobioreactor systems. We believe that the Humboldt_Berlin design is smart and could become the ideal solution for many future iGEM teams willing to work with phototrophic microorganisms in a cheap and well controlled way.

However, some changes and design futures should be improved in order to develop the openPBR as a solid alternative for iGEM and non-iGEM people who would like to use the system within their projects. We have identified some of these aspects, such as the temperature control implementation or the dual OD measurement without the need of switching off aeration. However, many other small details as mentioned above are still waiting to be improved.

Other pending challenges are the overall system usability, either in the physical and digital handling of the openPBR.

In the physical aspect, the system sterilization is still a delicate and time consuming process. Likewise, the tubing connections to the main chamber could be redesigned in order to allow a more efficient emptying of the system. As an example, fine-tuning the provided flow of each pump could allow the distribution of the discharge tube in the bottom of the system, allowing for an eased system drainage without the manipulation of physical valves.

Furthermore, in the digital aspect, despite being a great hardware design, the openPBR still lacks one crucial aspect of every research photobioreactor system: an user-friendly interface that allows to easily set-up the system and collect the required data. During our work with openPBR we have tried to simplify the code and clearly provide the user with guidelines to adapt it to their needs. We have also worked on the development of an automated data processing system using the PySerial library and a python script capable of communicating with the arduino serial, storing the data reads and even plotting them. However more work is required in order to develop software tools for exploiting the real potential of the openPBR as an open-source DIY phototrophic cultivation system.