Hexavalent chromium, or Cr(VI), is a common, toxic heavy metal released by many industrial factories, particularly in electroplating, leather tanning, and other manufacturing industries, in the form of contaminated wastewater. This wastewater may potentially contaminate groundwater sources, affecting the soil quality and drinking water safety for both the ecosystem and the people in nearby areas. However, prolonged proximity or accidental consumption of Cr(VI) may result in cancer, organ failure, and other consequences to one's respiratory and reproductive systems, indicating the dire need for a quick and efficient method of removing Cr(VI) contamination.

In China, specifically, the issue of Cr(VI) emissions and contamination is highly widespread. Studies estimate that China's annual wastewater discharge from electroplating alone is about 4 billion m³ [1], which commonly contains vast quantities of chromium, as well as other heavy metals. When combined with the volumes of wastewater discharged from other industries, the risks of chromium contamination in soils, crops, human drinking water reservoirs, and the ecosystem as a whole escalate drastically. A survey conducted during 2014 concludes that the amount of Cr contaminated soil rose to 15 million tons nationwide [2], threatening not only crop yields but also food safety.

Current methods of ridding wastewater of Cr(VI) tend to be mostly chemical treatments, such as using reducing agents to convert Cr(VI) to Cr(III), and then adding other chemicals to form a precipitation reaction, then filtering out the precipitate to receive Cr-free water. However, these methods are often either overly expensive, especially in the case of reducing agents, or they may cause secondary wastes, such as toxic Chromium sludge, which will require additional costs to treat.

Throughout the developments of synthetic biology, many issues with existing wet lab experiments persist, yet they can be largely resolved with our product. A primary issue in synthetic biology is that a proper mechanism to both detect and degrade contaminants, which, in this case, is hexavalent chromium, does not exist at a cheap and efficient level. Many preexisting products and research projects may contain adequate detection and degradation mechanisms separately, but no proper integration methods of both mechanisms exist in one product. Our product, the Microbial Remediator (MR), answers this problem by serving as a unique microbial hardware device that can both detect the presence of Cr(VI) and degrade said Cr(VI) into Cr(III), using our two strains of E. coli.

Due to the overwhelming presence of toxic Cr(VI) wastewater and the continuous search for a balanced, cheap, and efficient Cr(VI) removal mechanism, iGEM Team Crouton members created the Microbial Remediator (MR), which utilizes two types of distinct genetically-modified E. coli bacteria, containing genes to not only detect, but also degrade Cr(VI) into Cr(III).

Figure 1 Microbial Remediator (MR) Schematic Diagram

Design

The entire structure utilizes beam shaped materials for support, offering high structural flexibility, significant fault tolerance, relatively simple assembly, and keeping costs manageable. The device adopts a top-down layered design, allowing liquids to flow naturally under gravity. This saves energy by reducing the need for water pumps and minimizes space usage, while also limiting structural dead zones and simplifying sterilization procedures.

Additionally, the horizontal layout is divided into functional zones. During the design phase, components of the same type are grouped together as much as possible to minimize interference, for instance, isolating high-noise electrical devices (such as motors and air pumps) from sensitive components (like chips and sensors), containing the environmental impact of heating elements, and improving thermal insulation efficiency.

Figure 2. 1st version of the outer overall structure of our Hardware design

User testing and feedback

We interviewed some experts in the field of wastewater treatment, all of whom offered our team valuable feedback. Organizing the suggestions they gave, we modified our hardware regarding its application, strengths, and areas for improvement.

Our wastewater degradation should take into account concentration, color, sediments, and pollutants, and highlight the importance of visible and easily read results, said Professor Xu, the director of Yangpu District's environmental monitoring station. Furthermore, he stressed that testing the product with real-life wastewater samples to generate precise data will be a necessary step for our product to face real life challenges, recommending using bacteria in industrial wastewater and other human-generated sewage rather than groundwater.

Professor Xu, the director of the environmental monitoring station of the Yangon district of Shanghai, accompanied by Professor Sun, the chief director, also provided some valuable advice. Professor Xu suggested that we refer to relevant standards such as GB/T 5750, GB/T 1576, and GB/T 8538-2008, which set requirements for drinking water safety in China, and recommended pursuing China Metrology Certification (CMA) to build customer trust. In terms of market adoption, she observed that universities and research organizations may not have strict demands due to lower emissions, whereas industrial companies have much higher needs and stricter expectations.

Figure 3. Discussion with Professor Xu, Director of the Yangpu District Environmental Monitoring Station

Understanding Professor Xu's concerns, we implemented new changes to our hardware device, and we tweaked the Color Detection Sensor in our hardware in order to take into account that wastewater possibly being blue in color before the detection of microbes was added, thus greatly disrupting the detection results. Our Color Detection Sensor used to only detect the intensity of the blue light in the sample, but now it's been modified to detect the change in blue light intensity after the detection microbe is added, thus the Cr(VI) concentration detection component of our hardware is still able to work.

Figure 4. Color Detection Sensor

Design

The device is structurally divided into two dedicated zones to sequentially facilitate chromium removal: a biological treatment zone for the microbial reduction of Cr⁶⁺ and subsequent detection, and a chemical treatment zone for the precipitation of Cr³⁺. The entire system is constructed on a 20-mm aluminum profile frame enclosed with acrylic panels, incorporating externally heated plastic reaction vessels. Automated control and real-time environmental monitoring are managed by a central MCU, which supports data transmission via both wired and IoT connectivity.

Within the upper biodegradation chamber, an overhead DC motor-driven impeller ensures homogenization, while the lower detection chamber employs a magnetic stirrer to minimize optical interference. Precise fluid handling is achieved using peristaltic pumps mounted via custom 3D-printed brackets, enabling accurate injection of samples and reagents. In-situ absorbance measurements are performed using a photoelectric sensor to quantify chromium concentration, thereby closing the integrated monitoring and control loop.

Figure 5. 2nd version of the overall outer structure of our Hardware design

User testing and feedback

Professor Shan Huimei is a frontier researcher in the groundwater pollution and environmental remediation field. In our interview with Professor Shan, she underscored that we should include filtering out Cr(III) in our process, noting that sufficient space must be built into the hardware to sustain bacterial activity and allow for their eventual removal.

Figure 6. Discussion with Professor Shan

Professor Shan's concerns inspired our idea of incorporating a filtration chamber into our hardware system. In the chamber, Cr(III) will be precipitated and filtered to remove all chromium content in the water, precluding all possibilities of Cr(III) being converted back into toxic Cr(VI).

Figure 7. Design of the filtration chamber

Principles of Modular Design

Our product adheres to a very systematic and organized flow. We divided the hardware into three main components: degradation, detection, precipitation and filtration. Each component is specialized and is responsible for one specific function, but every component is well interconnected to work as a unified system. We also considered the physical design thoroughly to account for accessibility and ease of use. The first part of the hardware is constructed in a leveled manner; the degradation chamber is located directly on top of the Detection chamber. This allows for samples of the degraded water to flow efficiently to the Detection chamber. We also installed peristaltic pumps to propel the flow of fluids throughout the system. The flexible nature of the tubes and compression from rotors allows for a consistent, pulsating flow of liquid. The Detection chamber acts as an intermediate between Degradation and Precipitation and Filtration; if the water passes the detection, then it flows into the Precipitation and Filtration component.

Figure 8. Final version of the overall structure of our Hardware design

The filtration cup is 3D printed and assembled with 2 parts: the upper cup and lower tubular body, which connects the cup with the container. The two are designed with a spiral screw thread structure for connection, so that it ensures a tight seal between the 2 parts and prevents potential water leakage. The lower tubular body has an extension that connects to a vacuum, designed so that the water will not be directly sucked into the vacuum, but only the air to create a pressure difference that facilitates the downward movement of water. The upper cup also has small extensions around the exterior surface, where rubber bands could be tied from the upper cup down to the container to hold the structure together.

Finally, the waste container and final water container ensure that the water flow has a final destination and provide an overall efficient flow. Each component is also very individual and independent, so the entire model can be rearranged into different structures, such as putting the Degradation chamber down to create a linear structure for more structural stability. The entire structure is well-built and stable, built with aluminum profiles, so that it's easily scalable from the minimized prototype model for industrial use.

How Samples Flow Through our Product:

Figure 9. System Design Flowchart

The specific functions of each module

The MR itself consists of three main chambers: Degradation, Detection, Precipitation and Filtration. Initially, the water samples go through a primary filtration zone, where sands and rocks are filtered out of the sample, and the remaining liquid is stored in the Degradation Chamber. Once the hardware is activated, a small portion of the sample flows down to the Detection Chamber. Here, E. coli containing the pET28a-chr-T7-amilCP gene will be released from the bacterial storage zone beside the Detection Chamber, and mixed into the sample, detecting whether or not Cr(VI) exists within the sample. At the same time, the thermistor is activated, which aims to keep the temperature of the Detection Chamber around 37℃, and the heating pads will activate depending on whether or not the temperature is below 37℃. If it is present, the bacteria and thus the sample will turn into a shade of dark blue, which can be recognized by a color detection sensor that only recognizes the sample’s change in blue light intensity, thus reflecting the concentration of Cr(VI) in the sample. If the intensity passes a certain threshold, indicating that there is an excessive amount of chromium that is above international standards, a signal in the Degradation Chamber will be initiated to begin degradation. If not present, or if present at low concentrations, the remaining sample in the Degradation Chamber will flow to the Precipitation and Filtration Chamber. After detection, the E. coli will be quickly disposed of through the UV light present in the Detection Chamber, and the small portion of water sent to the Detection Chamber that could not be degraded will then be transferred to a waste collection zone.

For a sample that has been confirmed to require degradation, E. coli containing Cr(VI) degradation genes will be released from the bacterial storage zone beside the Degradation Chamber, initiating a thermometer, along with heating pads, and mixed thoroughly in the water sample, which thus degrades the Cr(VI) in the water sample to Cr(III). After the degradation process is complete, bacteria will be killed by UV lighting in the Degradation Chamber, and a resulting small portion of the post-treatment sample will flow back down to the Detection Chamber. Once again, the sample is mixed with bacteria released from the bacterial storage zones, and shortly disposed of after detection is complete. Similarly, if Cr(VI) is detected, the degradation process repeats. If not, the valve to the Precipitation and Filtration Chamber will open, allowing the water sample to flow to that area, whilst the previous valve closes.

It must be noted that additional bacteria must be added to the bacterial storage zone whenever the machine is about to be operated, and the bacteria should be subsequently disposed of after each treatment cycle is completed. Therefore, temperature management systems ensuring the bacteria’s survival will only be present within the Degradation and Detection zones, and not in the bacterial storage zones themselves. The temperature management system will contain a thermistor and heating pads, in which if the temperature falls below 37℃, the heating pads will respectively activate, to ensure that the bacteria do not lose their function from unideal temperatures.

The water sample reaching the Precipitation and Filtration Chamber should fulfill one of the two conditions of water flowing to this area. At the preliminary stage of detection, the water that initially was detected not to contain Cr(VI) will flow to this area and immediately pass through the final filter, where this water will be collected. For samples that passed through the secondary detection and were already degraded, a pH detector in the Precipitation and Filtration chamber inspects the sample’s pH. Acidic samples whose pH is below the usual 7~8 value will have NaOH added to them, neutralizing the sample as the mixer mixes the NaOH thoroughly. The remaining precipitate and the dead E. coli containing the degradation genes will be filtered by the final filter, and the resulting water will be free of both bacteria, precipitate, and excessive Chromium.

1. Connection with bacterial storage zones to fulfill Cr(VI) degradation purposes.
2. Thorough mix of degradation bacteria in the sample fluid.
3. Maintenance of bacterial survival in the Degradation Chamber.
4. UV Disinfection of degradation E. coli bacteria.
5. Transport of sample fluid into the Detection Chamber.
6. Transport of sample fluid into the Precipitation and Filtration Chamber.

Figure 10. Close-Up Diagram of the Degradation Chamber

1. Connection with bacterial storage zones to fulfill Cr(VI) detection purposes.
2. Thorough mix of detection bacteria in the sample fluid.
3. Maintenance of bacterial survival in the Detection Chamber.
4. UV Disinfection of detection E. coli bacteria.
5. Retrieval of fluid from Degradation Chamber.
6. Transport of fluid into the waste collection zone.

Figure 11. Close-Up Diagram of the Detection Chamber

1. Retrieval of fluid from Degradation Chamber.
2. Addition of NaOH (aq) into fluid.
3. Thorough mix of NaOH (aq) in the fluid.
4. pH detection of fluid to ensure no Cr(III) ions remain.
5. Filtration of precipitate and dead E. coli out of the fluid.

Figure 12. Close-Up Diagram of the Precipitation and Filtration Chamber

Before the water sample enters the system, it is first filtrated using filtration layers to remove impurities.

Peristaltic pump: Pumps bacteria from the storage tank into the chambers. The pumps used in our hardware device were peristaltic pumps, which move fluids through a flexible tubing that is continuously compressed by rotating rollers. In order to test the functionality of all of our pumps, we tested whether water was able to flow through each pump.

For each of the pumps, one side of the tubes was elongated by extra tubing and would extend towards one of the water chambers. For instance, a pump would have one of its tubes connecting with the liquid in the Degradation Chamber and another in the Detection Chamber. The testing mechanism was as follows:

1. The pump, once in contact with liquid, is activated.
2. Whirring sounds could be heard; we would observe the pump as rollers began to propel the fluid to another tube.
3. Observe as all the liquid from one Chamber is pumped to another Chamber.

In the process, many of the tubes experienced leakages, but all in all, the problems were simple to fix as the tubes could be replaced easily with better-fitting ones.

Figure 13. Test of pumps

Stirrer: The stirrer is used to mix the bacteria, ensuring even distribution of E. coli and proper bacterial function. The stirrers used in our hardware device are used to mix the microbes and NaOH solution more thoroughly in the liquid. We had two different types of stirrers: a metal one for mixing the NaOH thoroughly, as precipitates will be forming in this mixture, and a plastic one for mixing the microbes more thoroughly in the sample liquid, which also saves costs.

Figure 14. Test of Stirrer

Automatic Temperature Control System: The automatic control system has 4 main components: a thermistor, a variable resistor, a voltage comparator, and a heating pad. The thermistor is a type of temperature-sensitive resistor with a negative temperature coefficient (NTC), meaning its resistance decreases as temperature increases and increases as temperature decreases. When the temperature of the system rises above or falls below the set temperature, the thermistor’s resistance changes, causing a corresponding increase or decrease in the voltage detected by the voltage comparator. The comparator outputs 0 (no signal) or 1 to control the heating pad: the pad remains on when no signal is output, and is turned off when a signal of 1 is sent. The system’s ideal temperature is 37°C, at which the thermistor has a resistance of 10 kΩ. The resistance of the variable resistor is adjusted to match the same resistance of 10 kΩ. With a supply voltage of 3.3V, the total resistance of the system is 20 kΩ. Thus, at the set temperature of 37°C, the voltage between the two resistances is 1.65V. If the temperature drops below 37°C, the resistance increases above 10 kΩ, causing the voltage to drop below 1.65V. The voltage comparator detects the decrease and does not output a signal, keeping the heat pad on. Conversely, if the temperature of the system rises above 37°C, the thermistor’s resistance decreases, causing the voltage to exceed 1.65V. The voltage comparator detects this increase in voltage and outputs 1 to turn off the heating pad. This feedback cycle continuously adjusts the heating pad to maintain the system temperature stably around 37 °C.

The heating of our Detection and Degradation Chambers is necessary to keep the bacteria alive while they perform their functions. Attached to the walls of each Chamber is a heating pad that heats up the liquid collected inside the chamber, if the thermistor in each Chamber shows that the temperature is below 37 °C. Once the temperature is above this threshold, the heating stops. This maintains the temperature of the bacteria's environment constantly at the ideal temperature of 37 °C to bring out the bacteria's optimal functions.

Our procedures for testing are as follows:

1. As shown in Figure 15A, we first initiate our heating pad to heat up the liquid inside.
2. Then, bubbles begin to form on the surface of the thermistor, as shown in Figure 15B, which indicates the increasing temperature. When the temperature is above 37 °C, the heating pad stops heating.
3. We inserted a thermometer to detect whether the thermistor has stopped the heating pad's heating at the right moment.
4. As shown on the thermometer, the results indicate roughly 37.2 °C, which is highly similar to our ideal temperature of 37 °C, indicating the thermometer and heating pad's successful functions.

Figure 15. Test of the Automatic Temperature Control System

UV light: After degradation procedures are completed, the UV light will be turned on to kill all E. coli in the water, preventing bacterial contamination. We added UV lights to dispose of the bacteria after they have completed their functions. We respectively tested whether or not the UV lights in the Detection and Degradation Chamber were able to function and produce UV light. This proves that our wiring is correct and that the UV light can be successfully activated and deactivated.

Figure 16. Test of UV light

Tubes: The degradation chamber is connected by three tubes to the detection chamber, the precipitation and filtration chamber, and the degradation-bacteria storage tank.

Reaction Container: This is the chamber where all degradation reactions will take place.

Peristaltic pump: same as degradation

Stirrer: same as degradation

UV light: After detection procedures are completed, the UV light will be turned on to kill all E. coli in the water.

Temp system: same as degradation

Tubes: Three tubes connect the detection chamber to the degradation chamber, the waste disposal zone, and the detection-bacteria storage tank.

Color detection sensor: This device can detect and measure changes in the water sample’s color by analyzing the light it reflects or transmits. In our hardware system, this sensor monitors changes in water color to estimate the concentration of Cr(VI) present in the water source. Our color detection system detects the change in blue light intensity, which will be vital in the Detection Chamber, as it displays the concentration of Cr(VI) in our water sample.

In order to verify the precision and accuracy of our color detection system, we tested and calibrated it as follows:

1. We prepared three different solutions of different amilCP protein concentrations: respectively, 0mg/L as displayed in Figure 17A, 10mg/L as displayed in Figure 17C, and 50mg/L as displayed in Figure 17E.
2. We placed our color detection sensor under these three different solutions of different amilCP concentrations, with their results shown aside in the red rectangle

The values fluctuated quite substantially when placed under the color detection sensor due to human error, as rapid movement in the placement or the angle of the tube could disrupt the values shown in the graph. Generally, the value shown for the 0mg/L amilCP is around -100~400; for 10mg/L amilCP, it's around 500~700; for 50mg/L, it's around 500~900.

Figure 17. Test of the Color detection sensor

Waste disposal zone: After detection, the water samples and the dead bacteria will flow into this waste collection area through a tube.

Reaction Container: This is the chamber where the detection reactions will take place. The bacteria will be pumped here for detection.

Water flow is the core of the Microbial Remediator (MR) operation, as it ensures that wastewater can sequentially pass through primary filtration, degradation, detection, precipitation & filtration, and final collection. This test aims to verify whether fluid flows smoothly along the preset path, confirm no leakage at pipe joints, valves, or chamber connections, and ensure that peristaltic pumps and vacuum pumps effectively drive fluid movement. This lays a foundation for subsequent tests involving dyed simulated microbes and Cr(VI) solutions.

Figure 18. Test of Water Flow

pH detector: The pH detector measures the pH value of the liquid. The pH monitor is used in the Precipitation and Filtration Chamber of the hardware device, in order to ensure that the NaOH and the Cr(III) ions sufficiently react and form a precipitate. Both need to react sufficiently in order for the pH detector's value to remain around neutral, or else either NaOH or Cr(III) may have an overly large concentration and result in an overly basic or overly acidic solution.

In order to verify the precision and accuracy of our pH monitor testing system, our test is as follows:

1. Our pH detector has been coded to consider the value of 2471616 as equivalent to the neutral pH. As shown in Figure 19C, when the pH detector was placed in PBS (Phosphate-Buffered Saline)
2. The pH detector is then added to our basic solution of NaOH and acetic acid, respectively, and we observed the changes in the values displayed by the pH detector

The more basic NaOH solution should show a decrease in value due to the decrease in electrode potential brought by the lower H+ concentration in the solution. Thus, when the pH detector is placed in the NaOH solution, the value drops. In acidic acetic acid, the electron potential Sis is more positive due to the higher H+ concentration, thus resulting in an increase in value, as displayed in Figure 19B. However, the value in acetic acid overshoots significantly when compared to the value dropped in NaOH. This is largely because acetic acid is a weak acid that has a much lower ionic strength; thus, the electrode signal is unstable. Yet, this will not significantly affect the purpose of our pH detector, which was to determine if the solution's value is roughly equal to an electrical potential of 2471616.

Figure 19. Test of pH detector

Tubes: Tubes connect the precipitation and filtration chamber with the degradation chamber to allow degraded water to enter the chamber, the NaOH tank, and the storage tank.

Stirrer: The stirrer is used to mix the bacteria with NaOH to accelerate the chemical precipitation reaction.

• Filtration layer: This layer filters out the impurities, including dead E. coli tissues and Cr(III) sediments. Filtration plays an important role in ensuring the biosafety of our hardware product, and that the wastes and contaminants are not released into the Final Collection Zone. Our process for assembling and testing the filtration mechanisms is as follows:

1. As per Figure 20A, we added one microbiological membrane filter into our 3D-printed filter component, which is later added to the wider component used to carry the water, as shown in Figure 20B.
2. Water was added to the wider component and confined to the metal railing, which holds the filter.
3. After waiting for filtration to occur, we see the steady, albeit small, flow of filtered water through the filtration component in Figure A and end up in a tube, where the filtered water can be collected. This successful flow of water signifies successful filtration.

Figure 20. Test of the Filtration Layer

Vacuum pump: This pump removes air under the filtration layer to accelerate the speed of filtration into the storage tank.

Storage tank: After the full cycle finishes, the treated water will be stored in the storage tank.

We selected the gene chr-T7-amilCP as our indicator for Cr(VI) detection. p_Chr is the operator of the sequence that produces RNA polymerase that binds with the T7 promoter. The ChrB gene continuously creates ChrB repressors that bind to the p_Chr operator. The T7 promoter is the TATA box sequence that the RNA polymerase, recruited by the p_Chr operator, binds to and amplifies the signal of protein production. The RBS is the ribosome binding site, which summons nearby ribosomes to gather and initiate translation, and amilCP CDS is the gene that codes for the blue pigment protein. The RNA polymerase leaves the strand once it reaches the terminator sequence. In the absence of Cr(VI), the ChrB gene produces ChrB repressor, which is able to bind onto p_Chr operator, inhibiting the production of RNA polymerase. The inability to produce RNA polymerase fails to activate amilCP and will not produce blue proteins.

Figure 30. chrB Gene Without Cr(VI) Present, Focusing On: p_chr Operator, chrB Gene, and T7 Promoter in the Regulatory Module chr

However, in the presence of Cr(VI), Cr(VI) binds with the ChrB repressor, altering the shape of the repressor. As a result, the repressor is not able to bind with the p_Chr operator, leaving the p_Chr operator vacant. The higher the concentration of Cr(VI) is, the higher the possibility of activating the p_Chr operator (since the repressor can be seen as a competitive inhibitor). With the majority of produced ChrB repressors occupied by Cr(VI), the p_Chr operator initiates production of RNA polymerase. The RNA polymerase then recognizes the TATA sequence located on the T7 promoter, starting to bind to the T7 promoter sequence. RNA polymerase then creates a strand of mRNA, which combines with ribosomes at the RBS. T7 promoter thus amplifies the protein-producing signal, creating more amilCP proteins. Under higher concentrations of Cr(VI), the more blue amilCP proteins are made, and thus the higher blue intensity as shown in the hardware.

Figure 31. chrB Gene with Cr(VI) Present, Focusing On: p_chr Operator, chrB Gene, and T7 Promoter in the Regulatory Module chr

For the Cr(VI) reduction system, ChrR is a dimeric flavoprotein that catalyzes both one- and two-electron transfer to Cr(VI) with the momentary formation of Cr(III). Starting from Cr(VI), Chromate reductase can catalyze the reaction of transferring two electrons: one electron to Cr(VI) to form Cr(V), while the other electron generates ROS. The second electron transfer fully reduces Cr(V) to Cr(III). This process can generate reactive oxygen species, or ROS, unstable molecules that are oxidative.

Figure 32. Diagram Display of ChrR Degrading Cr(VI) to Cr(III)

YieF catalyses direct Cr(VI) reduction to Cr(III) via four electron transfer, in which three electrons are utilised in the reduction of Cr(VI) and the rest are transferred to oxygen. This also generates ROSs, but comparatively less than those generated by ChrR.

Figure 33. Diagram Display of YieF Degrading Cr(VI) to Cr(III)

For the Cr(VI) detection system, we test the effectiveness of pET28a-chr-T7-amilCP in detecting Chromium VI at different concentrations ( 0, 10, 50, 80, 100 mg/L ). The results show that the blue color indicates that amilCP protein is present, with the higher intensity of blue(amilCP) prevalent, which indicates the higher chromium concentration in the solution. However, if the levels of Cr(VI) concentration in the solution are too high, the bacteria may die and cannot express the amilCP gene, leading to the absence of amilCP protein, which leads to the absence of blue color. 30mg/L is discarded due to some practical errors, which lead to some unreliable results.

Figure 34. pET28a-chr-T7-amilCP Turning Blue with Cr(VI) Presence

The petri dish results match our results of the test tubes after the centrifuge. 0mg/L Cr(VI) is the condition in which the petri dish was spread with pET28a-chr-T7-amilCP only, acting as a control group for the petri dish examination. Starting from 10mg/L, there are already signs of bacteria possessing blue color, which signifies that the protein amilCP has been expressed and produced. Bacteria exposed to 50mg/L of Cr(VI) displayed the darkest blue color among all of the plates, indicating that pET28a-chr-T7-amilCP can be activated to the greatest extent among our selected concentrations. Moreover, concentrations above 50mg/L failed to incubate pET28a-chr-T7-amilCP.

Figure 35. Cell Culture Display of Bacterial Strains that Expressed amilCP Protein

For the Cr(VI) reduction system, results demonstrate that there is a decreasing trend for Cr(VI) with the increase in time. Moreover, it can be observed that the optimum time for degrading Cr(VI) is the 5th hour, as the slope of the line showing the change in Cr(VI) concentration over time plummeted to the greatest extent. The graphs below indicate our results.

Figure 36. Cr(VI) concentrations in ChrR, YieF, and ChrR+YieF protein solutions after degradation

We then calculated the rate of reduction (degradation rate) for every target protein (ChrR, YieF, and ChrR-YieF) for every concentration. Based on our previous result of 5 hours being the optimum time, we measured degradation at the 5-hour mark. The graph below shows the reduction (degradation) rates for ChrR, YieF, and ChrR+YieF.

After 5 hours of reaction, YieF protein demonstrated significantly superior degradation efficacy compared to ChrR and ChrR+YieF, achieving a higher degradation rate. This indicates that YieF is the most potent chromium-degrading protein with substantial application value.

Figure 37. Graph for the degradation of Cr(VI) at different concentrations after 5 hours

Subsequently, we further calculated the actual efficiency of the three proteins in reducing chromium. YieF’s reduction rate reached 78.6%, which was higher than ChrR, which was higher than ChrR+YieF. Paired with our previous conclusion, we came to our third conclusion: YieF exhibits the highest rate of Cr(VI) degradation at 12 hours, so it is the most optimal protein for hexavalent chromium degradation.

Table 1: Calculation of Reduction Rates for Three Types of Protein Chromium

Building upon our current integrated system, future iterations will enhance intelligence and adaptability through advanced IoT sensors for monitoring additional contaminants like cadmium and lead, enabling multifunctional remediation within a single platform. Our development roadmap includes implementing machine learning algorithms for predictive maintenance and autonomous adjustments, complemented by upgraded wireless communication modules supporting 5G and satellite connectivity to ensure reliable operation in remote areas. These advancements will transform the system into an adaptive water treatment solution capable of self-optimization based on real-time water quality data, while providing a practical platform for implementing engineered biological systems in environmental remediation.

The project demonstrates effective translation of synthetic biology into functional hardware through a modular architecture that separates degradation, detection, precipitation, and filtration into independent units, enabling other teams to adapt components based on their specific needs. By open-sourcing hardware designs, control software, and biological parts, we provide a replicable framework for researchers working on bioremediation and biosensing applications, with our safety protocols setting a precedent for responsible innovation in field applications of synthetic biology. The developed genetic constructs for heavy metal reduction, particularly the synergistic use of multiple reductase enzymes, offer new design paradigms for metabolic engineering in environmental biotechnology, helping bridge academic research and industrial implementation.

This integrated system shows significant promise across commercial and educational domains, offering industries such as electroplating, textiles, and mining a cost-effective wastewater treatment solution, especially as environmental regulations increase demand for reliable remediation technologies. Its modular design supports both emergency deployment in spill scenarios and continuous use in remote locations. In educational settings, the system serves as an immersive demonstration tool for teaching synthetic biology, environmental engineering, and automation principles, enabling hands-on learning about wastewater treatment processes and inspiring future innovators through practical experience with biological systems and real-time monitoring. The collected operational data will contribute to understanding the stability and efficiency of engineered organisms in controlled environments, potentially accelerating innovation in bioremediation and fostering collaboration between synthetic biologists and environmental engineers.