Hardware
In this page you will find the main parts of our bioreactor system such as it's function and future implementations.
![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector.png)
![](https://static.igem.wiki/teams/4460/wiki/img/illustrations/rum-igem/component-vector-2.png)
Overview
A bioreactor is a vessel where a biochemical reaction occurs involving living systems. Specifically, a stirred-batch bioreactor functions as a container with rotational movement to maintain a homogenized mixture. In our case, RDX will be biodegraded into formaldehyde and nitrite within this type of bioreactor. The shaft and impellers will maintain a continuous flow of the mixture to achieve the same concentration of reactants and products throughout the entirety of the vessel.
Afterwards, the water with the reaction byproducts is discharged through the effluent tube and flows toward the second phase of the system. It consists of an ozone bubble diffuser for denitrification and an activated carbon filter for capturing the formaldehyde. Then, the water passes on to a third and final phase where aquatic plants will denitrify and remove formaldehyde as a second step to make sure none is left in the water.
![Bioreactor System Overview](https://static.igem.wiki/teams/4460/wiki/our-bioreactor-system.png)
Function
The bioreactor begins to perform its function once a certain amount of contaminated water enters through the entrance of the medium. It then encounters our bacterial culture, Escherichia coli, contained in the container. The bioreactor vessel allows the biological reaction to occur effectively, since it has linked different sensors that ensure optimal conditions to decompose the RDX into formaldehyde and nitrite.
Among these sensors and controls are those of the temperature, pH, and pressure. The bioreactor will use its sensors to corroborate a specific value of the measurements and send this information to their respective control units. The temperature control unit ensures that the temperature is maintained between the temperature set for the bioreactor. When a drastic change in temperature is measured, the control unit sends signals to heat or cool water in the thermal jacket of the bioreactor and, consequently, regulate the temperature. The pressure control unit ensures that the pressure inside the vessel is near 101.3 kPa. If the pressure exceeds the established parameter, an escape will be opened for the air to equalize the pressure. On the other hand, if pressure decreases, oxygen will automatically be added to the container through the sprayer to provide them with the necessary oxygenation, and simultaneously regulate the pressure. For the pH measurement, the control unit is programmed to corroborate that this value is 7; if not, there are two ways to achieve it. If the value of the pH measurement is greater than desired, an acidic solution shall be added; if less than desired, air shall be added to the crop through the sprayer to remove the added CO2. To ensure that these functions can be carried out, the bioreactor also has an input for the acid/base supplies for the pH control unit. If the bioreactor has its controls calibrated and its parts in optimal conditions, the decomposed RDX into nitrite and formaldehyde is released through the effluent outlet.
First Phase: RDX Biodegradation
![First Bioreactor](https://static.igem.wiki/teams/4460/wiki/vessel-phase-1.png)
Parts
The first bioreactor is divided into three main parts: vessel and thermal jacket, shaft, and lid:
- Vessel and Thermal Jacket:
- Shaft, Impellers, and Foam Blades:
- Lid:
The vessel is the container where the biodegradation will occur. To optimize the volume of the vessel with the least amount of manufacturing material, it was determined that the height and radius are of equal length. Using a volume of 1.5 L, the height and radius measure 80 mm. The vessel is surrounded by a thermal jacket that is 5 mm from the vessel all around it. The thermal jacket functions as a temperature control to maintain the optimum temperature at 37°C. The jacket has two lateral holes, an inlet for cooling water and the outlet for the same water.
The primary function of the shaft is to transmit rotation from the stirrer motor to the impeller; it also resists bending forces and supports the impeller’s weight (Doran, 2013). The motor-driven central shaft, which supports one or more agitators, receives power from the stirrer motor and transmits torque to the blades, ensuring an even mixture of the medium.
The foam blades are axial flow, pitched blade turbine impellers. These mechanical foam breakers split up agglomeration and impart solids effectively while converting motor power into shear energy (Mixer Direct, n.d.). These blades have an excellent mixing ability provided by their radial and axial flow (Pitch Blade Turbine mixing impeller, n.d.). The foam blades serve as gas-dispersing tools that maintain aerobic conditions for the cells to decompose and divide efficiently; they ensure efficient gas transfer to growing cells and good mixing of the contents. The foam blades significantly reduce the formation of excess foam, decreasing the probability of handling and pumping difficulties. These pitched blade turbine impellers provide circulation of air in the headspace area of the bioreactor, preventing the creation of residual organic matter.
The bioreactor contains double-suction airfoil impellers as the source of homogeneous mixing inside the vessel. These blades have an airfoil design because our mixture has a low viscosity; this design produces the maximum pumping with the lowest shear (James, 2011). Our double-suction design reduces the force exerted on the shaft since the medium enters the center of the impeller blades from both sides simultaneously (Impeller - types of impellers, 2016). The pumping of the airfoil impellers promotes aeration and necessary heat transfer; thus, we achieve efficient cell proliferation and denitrification of RDX inside the vessel.
![Bottom Lid](https://static.igem.wiki/teams/4460/wiki/bottom-lid.png)
Nevertheless, in the research process, examples of each one of the sensors were found. The guidelines established for finding the parts were simply to be cost-effective, accessible and with similar parameters to our set standard size. The pressure sensor is lollipop-shaped, where the diameters of the bottom part are thirty millimeters horizontally and twenty five millimeters vertically. In continuation, there are three other holes with a diameter of eighteen millimeters, these ones are destined for the air supply, acid supply, and base supply. As for the remaining two, their diameter is twenty-four millimeters; one is destined for the medium and the other is in charge of the input of water from the lagoon into the device.
It must be noted that the diameter of these three will change as there is not a specific sensor that is going to be used in the bioreactor, the diameter of twelve millimeters is solely a standard size. Nevertheless, in the research process, examples of each one of the sensors were found. The guidelines established for finding the parts were simply to be cost-effective, accessible and with similar parameters to our set standard size.
Sensors
Table 1. Possible Sensors to be implemented in the first bioreactor
Sensor | Company Manufacturer | Dimensions | Measurement Range | Price |
---|---|---|---|---|
Pressure | Walfront | 30.00 x 25.00 mm | 0-30 kg | $11.19 |
pH | GAOHOU | 42.01 x 32.00 x 19.99 mm | 0-14 | $34.99 |
Temperature | HiLetgo | Probe Diameter: 5 mm; Cable Length: 1 m | -55-125°C | $10.99 |
The idea of the lid is to prevent anything not desired to come in or out of the bioreactor. For this part, in addition and in unison to the two halves of the lid, the top half has two circle-like gaps underneath. These two rings are six millimeters deep into the lid and five millimeters thick; the first one has an inner diameter of one hundred and sixty millimeters and the second one of one hundred and ninety millimeters. Primarily, the idea is for the rings to have a thin layer of rubber, silicone, or something similar to make the lead more leakproof and prevent undesired motion. The top half will fit into the vessel (inner ring) and the thermal jacket (outer ring), then the bottom half and the top half will be joined together through the thermal jacket with screws and bolts, sealing everything tightly.
Second Phase: Denitrification and Formaldehyde Removal
Second Bioreactor
The second bioreactor is a copy of the first bioreactor’s vessel. It consists of a granular activated carbon filter (GAC) at the inlet for formaldehyde removal and an ozone bubble diffuser to convert the nitrite into nitrate.
Figure 3. Second-Phase Bioreactor
Third Bioreactor
The third bioreactor is identical to the previous one. However, this one consists of denitrifying aquatic plants. Different methods will be used to reduce the concentration of nitrite and formaldehyde, the by-products of our genetic circuit. One innovative approach is phytoremediation, a plant-based method, which involves the use of plants to extract and remove elemental pollutants (Yan et al., 2020). A greenhouse that provides ideal conditions for Pistia stratiotes’ growth will be implemented since Pistia stratiotes can effectively remove nitrite from the contaminated water, achieving a removal of 72.28% (Rawat et al., 2012). This plant is native to the State Forest of Guánica,
Figure 4. Third-Phase Bioreactor
Puerto Rico, making its possession legal and accessible while guaranteeing Puerto Rico’s environmental conditions are convenient for the plant. After growing these plants in the greenhouse, they will be transferred to the nitrite and formaldehyde degradation vessel.
Formaldehyde is slightly persistent in water, with a half-life of 2-20 days; about 99% of emitted formaldehyde will eventually end up in the air, and the rest will end up in the water (Formaldehyde (methyl aldehyde), 2022). Therefore, the main focus for formaldehyde reduction will be on the air. Plantago asiatica has a strong tolerance to formaldehyde in the air and good formaldehyde removal ability; its high oxidative potential and lower defensive enzyme activity lead to a high formaldehyde removal rate (Zhao et al., 2019). Currently, Plantago asiatica is not found in Puerto Rico, but, with the implementation of this project, the corresponding permits will be requested for this plant to grow throughout the Anones Lagoon.
Future Implementation
Manufacturing
During our cycle in IGEM we managed to digitally model and 3D-print our proposed stirred-batch bioreactor. For our future projections we would like to be able to buy some components, 3D-print others, manufacture it a larger scale, and assemble it. This way we would be able to do our proof of concept with our bioreactor and be a step closer to implementing our project in order to diminish the RDX contamination on the island.
We would not only impact our municipal island Vieques, but other areas that have been contaminated by RDX as well or that could potentially be contaminated in a similar way in order to lessen and clean up the contaminants that affect food production, human health and environmental health. To this day, there are still many sites contaminated by RDX because of the explosions from World War ll in which our system could be implemented. That’s why our goal as a team is not only to help out through our project but to also educate the people being affected by it, so that the people outside the scientific community can also understand what’s happening around them and the consequences of these harmful practices.
Greenhouse
As the project aims for an attainable solution, it is important to consider how sustainable it will be. Seen as the project depends on scheduling and costs, system sustainability must be a crucial factor to account for when developing a solution for a particular problem. Importing the plants mentioned above will entail legal procurement, monetary actions and a considerable amount of time. Therefore, some questions have to be raised and addressed: How will plants be tested? How can we ensure they will be prepared under the optimal environment conditions? How often will these be imported? By taking into account the previously mentioned problems, and evaluating the benefits and risks of different solutions, the implementation of a greenhouse best suits the needs of the project. By general definition, a greenhouse is a glass building used to grow plants in adequate conditions while preserving adjacent ecosystems. This particular element of the system will be placed in the surroundings of the Anones Lagoon. Once plants are imported to Vieques, Puerto Rico, it is imperative to temper and adjust the habitat to the desired conditions for process execution while simultaneously reducing the probability of potential damages caused by local wildlife. Furthermore, legal processes and importation costs will be greatly reduced by being able to grow and test these plants in the local area.
Engineering goes beyond design and innovation as it involves analysis of human and social practices. As a fundamental aspect of human society, it is a driving force on how groups of people interact with each other on political, economical and material-cultural levels. The greenhouse implementation can be a great opportunity to defray the social-economic deficit caused by RDX contamination. An integral part of having such a beneficial and successful project for the community is for Vieques people to feel part of it. Therefore, the maintenance recurrent work can be achieved by giving community leaders the freedom of managing the greenhouse themselves, delegating responsibilities across the community while promoting workforce and economic growth. Our greenhouse will not only provide an optimal environment for the growth of Pistia stratiotes and Plantago asiatica, but it will also contribute to the renovation and restoration of Vieques’ environment and social development.
References
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Australian Government Department of Climate Change, Energy, the Environment and Water. (2022, June 30). Formaldehyde (methyl aldehyde). DCCEEW. Retrieved October 11, 2022, from https://www.dcceew.gov.au/environment/protection/npi/substances/fact-sheets/formaldehyde-methyl-aldehyde
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