Team:Hong Kong JSS/Demonstrate
Demonstration
Achievement
- From survey and interview results, we designed to construct a biological filtration device to tackle the metal pollution problem
- A prototype was constructed by our design team. Then the prototype was presented to different stakeholders for feedback.
- From the feedback of stakeholders, we improved the design and created the product Bacterial Copper Adsorption Device (B-CAD)
- The function of B-CAD was tested and we demonstrated that it can remove copper pollutants from water as we expected.
Needs of a Device
The goal of our project is to develop a bacteria that can be used as a heavy metal adsorbent. We aimed to lower the cost and environmental impact for cleaning up heavy metal pollution in aquaponics. Based on feedback from the human practice, we noticed that both the consumer and producer for aquaponic products are very negative to the idea of mixing live bacteria with fish and crops. They expressed worries about the health of fish being affected by the bacteria and food safety. Interview with biotechnology experts also reflected that mixing genetically modified bacteria with food crops and fish can be a threat to bio-safety. As a result, we need to build a device that can separate our bacteria from the aquaponic system, while allowing the copper ions to be absorbed by the bacteria.
Principle of Design
To separate bacteria from the aquaponic system yet allowing copper ions to be absorbed by the bacteria, we need a selectively permeable membrane. The pore size of the membrane is large enough for the copper ions, but too small for the bacteria to pass through. In our experiments, we are using dialysis tubing with a molecular weight cut off (MWCO) of 12,000 Da. The concept is illustrated by the diagram below:
We tried to test this idea by putting dialysis tubing filled with bacteria culture into copper solution. However, we do not observe a significant decrease in copper level. This is probably due to the small volume of bacteria culture that can be fitted into dialysis tubing. Therefore, we come up with an idea that we can make use of a reservoir to hold a larger volume of bacterial culture, then circulate the bacteria through the dialysis tubing with a pump. We name this device the Bacterial Copper Absorption Device (B-CAD)
The blue line represent the dialysis tubing. Bacteria cannot pass throught the tube, while copper ions are small enough to enter the tube.
We tried to test this idea by putting dialysis tubing filled with bacteria culture into copper solution. However, we do not observe a significant decrease in copper level. This is probably due to the small volume of bacteria culture that can be fitted into dialysis tubing. Therefore, we come up with an idea that we can make use of a reservoir to hold a larger volume of bacterial culture, then circulate the bacteria through the dialysis tubing with a pump. We name this device the Bacterial Copper Absorption Device (B-CAD)
The initial draft of our Bacterial Copper Adsorption Device (B-CAD).
Phase 1: Prototype Engineering
Before we build a prototype, we interviewed local aquaponics business owners to ask for their opinion (details of the interview are documented in the Human Practice section). Based on their feedback, we need to take the following points into consideration:
Therefore, we revised our design by adding a cage to the dialysis tubing. The figure below shows the design and 3D-printed product of the cage. We use Solidworks, a 3D sketching software, to design all 3D-printed parts of our device. As shown in the figure, there are hooks for holding the dialysis tubing in place in water, further foldings of the dialysis tubing can increase the surface area in contact with water, to increase the diffusion rate of the copper ion into the bacteria culture.
For the peristaltic pump, it is a type of positive displacement pump which is also commonly known as roller pumps. The fluid is contained within a flexible tube fitted inside a circular pump casing. A rotor with a number of "rollers" compresses the tube and as the rotor turns, the part of the tube under compression is pinched closed thus forcing the fluid to be pumped to move through the tube. The advantage of using the peristaltic pump is that the fluids will not be exposed to contamination from exposed pump components.
And for the cost, the major problem is the needed of culture medium. Commonly used bacteria culture such as LB and SOB are too costly. After a literature search, we found that the two major factors affecting bacterial growth are Nitrogen and Carbon source. Using Salifert Nitrate testing kit and the colorimeter from our school laboratory, we tested the Nitrate content of water samples from a local aquaponic system, and it turns out to contain more than 25mg/L nitrate.
We also tried to evaluate the need of additional carbon source by adding glucose to aquaponic water and measure the growth of bacteria. Surprisingly, bacteria can grow well without adding any glucose and the grow is even inhibited in higher glucose concentration. It indicated that there are organic substance in aquaponic water and it is enough for growing bacteria. As a result, we concluded that aquaponic water contain adequate Nitrogen and Carbon source so that E. coli can grow in it without the addition of any growth medium, the cost is therefore very low.
Reference:
- If the dialysis tubing is free lying in the aquaponic system, the fish could come into contact with the tubing and break it.
- It is better to separate the pump from the bacteria culture. We can use the peristaltic pump instead of the conventional one.
- The cost of maintaining the system need to be lower or at least equal to current methods.
Therefore, we revised our design by adding a cage to the dialysis tubing. The figure below shows the design and 3D-printed product of the cage. We use Solidworks, a 3D sketching software, to design all 3D-printed parts of our device. As shown in the figure, there are hooks for holding the dialysis tubing in place in water, further foldings of the dialysis tubing can increase the surface area in contact with water, to increase the diffusion rate of the copper ion into the bacteria culture.
The design of the cage. It contains hooks to fix the position of the dialysis tubing.
The diagrams on the right show the 3D design and printed object.
The diagrams on the right show the 3D design and printed object.
For the peristaltic pump, it is a type of positive displacement pump which is also commonly known as roller pumps. The fluid is contained within a flexible tube fitted inside a circular pump casing. A rotor with a number of "rollers" compresses the tube and as the rotor turns, the part of the tube under compression is pinched closed thus forcing the fluid to be pumped to move through the tube. The advantage of using the peristaltic pump is that the fluids will not be exposed to contamination from exposed pump components.
The peristaltic pump we used in the B-CAD device.
And for the cost, the major problem is the needed of culture medium. Commonly used bacteria culture such as LB and SOB are too costly. After a literature search, we found that the two major factors affecting bacterial growth are Nitrogen and Carbon source. Using Salifert Nitrate testing kit and the colorimeter from our school laboratory, we tested the Nitrate content of water samples from a local aquaponic system, and it turns out to contain more than 25mg/L nitrate.
We use Salifert Nitrate testing kit and colorimeter
to quantify Nitrate concentration in aquaponic water samples.
to quantify Nitrate concentration in aquaponic water samples.
We also tried to evaluate the need of additional carbon source by adding glucose to aquaponic water and measure the growth of bacteria. Surprisingly, bacteria can grow well without adding any glucose and the grow is even inhibited in higher glucose concentration. It indicated that there are organic substance in aquaponic water and it is enough for growing bacteria. As a result, we concluded that aquaponic water contain adequate Nitrogen and Carbon source so that E. coli can grow in it without the addition of any growth medium, the cost is therefore very low.
E. coli can grow in aquaponic water without adding any glucose.
Reference:
1. Role of Carbon and Nitrogen Sources in Bacterial Growth and Sporulation
2. Nutrition and Growth of Bacteria
Phase 2: Evolution and Fixes
After the test run, we evaluate and modified the design.
In our test run, we discovered that bacteria can easily accumulate to the bottom of the reservoir. As a result, the efficiency of our device will be decreased. Therefore, we added a magnetic stirrer to prevent bacteria from settling to the bottom.
Based on feedback from aquaponic business owners, they want to have more control over the speed of the pump and magnetic stirrer. In human practice, environmental science expert Prof. Wong Woon Chung also suggested that we may need to switch between low and high flow rate to facilitate copper adsorption by the bacteria as well as to prevent the accumulation of biofilm on the dialysis membrane. (For details, see Integrated Human Practice) As a result, we designed a simple circuit using switches, rheostats to control the current, and DC circuit board to draw electricity from USB plug socket and increase the voltage to 12V for the peristaltic pump and magnetic stirrer. The circuit diagram of our device is shown in the diagram below.
Since the connections between the tubing and different parts of the device tend to be leaky. We designed some 3D printed adaptors with a convex pattern to tighten the connections between the rubber tube and the adaptors. The pattern designed on the adaptors is used to tighten the connections of the tubings.
After all the modifications, the diagram below shows a 3D model of our final design. Since we do not have enough time and resources to build a large scale one, the device we built and tested are miniatured version. We made several prototypes and tested them in aquaponic water dosed with copper ions to simulate real-life copper polluted aquaponic systems. We understand this is still far from reality, but we hope our B-CAD design and testing can provide preliminary data and shed light on the idea of using bacteria as a biological adsorbent for pollutants.
The copper removal effects of the B-CAD were studied with different variables. In the experiment, 50 mL of CgMT-expressing E. coli together with 950ml aquaponic solution were loaded into the B-CAD and used to filter 5L of aquaponic water with 10 mg/L copper ion. Based on the result, after IPTG induction, our B-CAD system could remove ~25% and ~38% of the copper inside the water after 24 hours and 48 hours respectively.
We also investigate how our B-CAD performs under different conditions. First, we checked the efficiency of copper removal by CgMT-expressing E. coli when the pump was on with maximum flow rate (~400 ml/min) compared with the pump was off. The result shows that the speed of the flow is an important factor determining the copper removal efficiency. This is probably due to the fact that when the flow is turned off, bacteria in the reservoir cannot interfuse with the copper ion diffusing into the dialysis tubing, thus reducing the efficiency of B-CAD.
On the other hand, we also tested the efficiency of B-CAD under different copper pollution level. In our experiments, we tested the device under 5mg/L, 10mg/L and 15mg/L initial copper concentration. Our results showed that B-CAD can work well in these concentrations as it can remove 25% - 55% of the copper from the system in 48 hours. The data collected from these experiments also helps to structure our model.
In our experiments, due to limited resources, we are only able to build a miniature version of our B-CAD for testing. In addition, due to biosafety and animal care, we cannot test dose a real aquaponic system with copper and test our device in it. We tried our best by using water from an aquaponic system to perform our experiments in a close system. Yet, we understand that there could still be many differences between our test and a real aquaponic system.
In our human practice, although we had successfully convinced some potential users (aquaponics / aquaculture business owner) to try our product. Yet, there are still more than half of them being skeptical about the efficiency of our device. Although we believed that our method is cheaper and more environmental friendly, we agree that our device is not as effective as other currently using methods for removing heavy metals. Therefore in the future, we had to further improve the copper adsorption ability of the bacteria.
In addition, we also need to scale up our B-CAD in further testing so that it could be more comparable to real-life situations.
- The bacteria in the device precipitate to the bottom of the reservoir instead of suspending in the culture. A magnetic stirrer is needed.
- It would be a good idea to allow more control over the device, for example the speed of the pump and magnetic stirrer.
- The connection between tubings and the tank tends to be leaky. We need to design some connectors to prevent leakage.
In our test run, we discovered that bacteria can easily accumulate to the bottom of the reservoir. As a result, the efficiency of our device will be decreased. Therefore, we added a magnetic stirrer to prevent bacteria from settling to the bottom.
The magnetic stirrer we added to the B-CAD.
Based on feedback from aquaponic business owners, they want to have more control over the speed of the pump and magnetic stirrer. In human practice, environmental science expert Prof. Wong Woon Chung also suggested that we may need to switch between low and high flow rate to facilitate copper adsorption by the bacteria as well as to prevent the accumulation of biofilm on the dialysis membrane. (For details, see Integrated Human Practice) As a result, we designed a simple circuit using switches, rheostats to control the current, and DC circuit board to draw electricity from USB plug socket and increase the voltage to 12V for the peristaltic pump and magnetic stirrer. The circuit diagram of our device is shown in the diagram below.
The circuit diagram of the B-CAD and the electronic components used.
Since the connections between the tubing and different parts of the device tend to be leaky. We designed some 3D printed adaptors with a convex pattern to tighten the connections between the rubber tube and the adaptors. The pattern designed on the adaptors is used to tighten the connections of the tubings.
Custom made adaptors used in the B-CAD to prevent leakage.
After all the modifications, the diagram below shows a 3D model of our final design. Since we do not have enough time and resources to build a large scale one, the device we built and tested are miniatured version. We made several prototypes and tested them in aquaponic water dosed with copper ions to simulate real-life copper polluted aquaponic systems. We understand this is still far from reality, but we hope our B-CAD design and testing can provide preliminary data and shed light on the idea of using bacteria as a biological adsorbent for pollutants.
3D model of our B-CAD (Generated by Blender, a free open source 3D rendering software).
The diagram on the right is the actural prototype we built.
The diagram on the right is the actural prototype we built.
Phase 3: Testing of Device
Our team developed a Bacterial Copper Adsorption Device (B-CAD) that can utilize the metal absorption property of the GM E. coli to remove copper in a fish tank or aquaponic tank. We tested the device with actual aquaponic water added with copper (II) sulphate to a final copper ion concentration of 10mg/L. As discussed above, aquaponic water contains the nutrients that E. coli needed and no extra nutrient broth is required for the growth of the bacteria. Therefore, in practical situation, the cost of growing our CgMT expressing E. coli would be minimized.
Testing the B-CAD with real aquaponic water added with copper.
The copper removal effects of the B-CAD were studied with different variables. In the experiment, 50 mL of CgMT-expressing E. coli together with 950ml aquaponic solution were loaded into the B-CAD and used to filter 5L of aquaponic water with 10 mg/L copper ion. Based on the result, after IPTG induction, our B-CAD system could remove ~25% and ~38% of the copper inside the water after 24 hours and 48 hours respectively.
The copper removal effect of B-CAD when hosting different E. coli. The B-CAD system with CgMT-expressing E. coli showed significantly higher efficiency in lowering the copper concentration in the system.
We also investigate how our B-CAD performs under different conditions. First, we checked the efficiency of copper removal by CgMT-expressing E. coli when the pump was on with maximum flow rate (~400 ml/min) compared with the pump was off. The result shows that the speed of the flow is an important factor determining the copper removal efficiency. This is probably due to the fact that when the flow is turned off, bacteria in the reservoir cannot interfuse with the copper ion diffusing into the dialysis tubing, thus reducing the efficiency of B-CAD.
The efficiency of copper removal of CgMT-expressing E. coli when the pump was on with maximum flow rate (~400 ml/min) compared with the pump was off. This shows that the speed of the flow is an important factor determining the copper removal efficiency.
On the other hand, we also tested the efficiency of B-CAD under different copper pollution level. In our experiments, we tested the device under 5mg/L, 10mg/L and 15mg/L initial copper concentration. Our results showed that B-CAD can work well in these concentrations as it can remove 25% - 55% of the copper from the system in 48 hours. The data collected from these experiments also helps to structure our model.
The efficiency of copper removal of CgMT-expressing E. coli with different initial copper concentration.
Limitation and Improvement
In our experiments, due to limited resources, we are only able to build a miniature version of our B-CAD for testing. In addition, due to biosafety and animal care, we cannot test dose a real aquaponic system with copper and test our device in it. We tried our best by using water from an aquaponic system to perform our experiments in a close system. Yet, we understand that there could still be many differences between our test and a real aquaponic system.
Future Direction
In our human practice, although we had successfully convinced some potential users (aquaponics / aquaculture business owner) to try our product. Yet, there are still more than half of them being skeptical about the efficiency of our device. Although we believed that our method is cheaper and more environmental friendly, we agree that our device is not as effective as other currently using methods for removing heavy metals. Therefore in the future, we had to further improve the copper adsorption ability of the bacteria.
In addition, we also need to scale up our B-CAD in further testing so that it could be more comparable to real-life situations.