The application development of our project spanned from ideation to finish. Thanks to the many experts and stakeholders we contacted and provided help to us, we were able to develop a refined project pipeline and future development plan in the end. With this we would like to show our process and the human practices involved, pitching the story of this journey.
Initial design
Initial design
The initial design came from one of our teammates during ideation. At that time, our idea was to remove cadmium from sludge ash to reduce pollution and recycling it due to there being a “growing market for cadmium consumption” according to the initial research. The general steps as follows:
- Sludge ash is produced by incineration, which is currently done by the local sludge facility.
- Citric acid is added to the sludge ash to leach solid cadmium compounds out of the solution into ions.
- Our chassis is added to bind to cadmium.
- The binded chassis is filtered to be obtained, separating sludge ash and cadmium.
- Cadmium is separated from our chassis through elution using EDTA/ biological releasing triggers.
- Cadmium is obtained, sludge ash may be disposed into landfills while the chassis and citrate may be reused.
The design focused on cadmium recovery from sludge ash and suggested to have included eco-friendliness through application of biological methods, but was considerarbly challenged by our project instructors with the amount of cadmium that could be recovered and use of EDTA as an elutant.
This design was certainly not the best, but marked the start of development.
Second design
Second design 1
Second design 2
The second design amended the complicated flow of the initial diagram and included more environmental friendly elutants EDDS. The 3 key steps marked in the diagram being:
- Citric acid leaching of heavy metal
- Addition of chassis
- Recovery of cadmium and chassis with elutant
Project focus alteration 1
After presenting our second design, we acknowledged from internal feedback that the actual amount of cadmium we could revolver would be very low in the local context. Therefore, we agreed that it was not ideal to continue to focus on recovery. We switched our project focus from cadmium recovery to sludge remediation. Our rationale for using sludge ash being that it was how the local sewage sludge was in the form of.
However, from our consultation with Prof. Terence Wong, it was brought to our concern that this was not a good rationale if we plan to use our project to address the sewage sludge pollution problem, even more so when the local regulations of this problem are strict and sludge ash does not pose a problem worth implementing our solution to solve. To prevent targeting a problem that does not exist, we amended the focus again from providing a better method for sludge remediation to solving a problem in cadmium pollution, with recovery as an addition to benefit. In addition, we widened the scope to extend beyond the local context, providing us a better rationale for implementing the project.
To gain better insight on the application and industrial processes involved in our project, we contacted the start up BioMetallica, to which we presented our new focus of using our project to target cadmium pollution. We were suggested to include more data on cadmium pollution if we were to target it and a potential recovery method of cadmium - incineration. BioMetallica offered to assist our project development in the long term.
Third design
Third design
- Removed chassis recycling step
- Temporarily changed cadmium recovery method to incineration
After consultation with BioMetallica, we removed the process of recovering the chassis and recycling it with consideration of the cost effectiveness of the process and how surface protein expression would be hindered after the first round of processes. As a result, we could consider incineration as our recovery method and we thought it would be a better method of cadmium recovery which caused less secondary pollution compared with chemical eluting agents. Therefore we included it into our project pipeline.
As suggested by the company, we contacted Prof. Zhiguo Yuan AM in seeking validation on the advantages and feasibility of our project, who’s main criticism towards the process was that sludge ash was not a good remediation target. Sludge ash is already the product of a remediation process, namely incinerating sludge to greatly reduce its volume and removing the water to allow pollutants to maintain in a relatively stable form. In contrast, by targeting sludge, we might be able to add an additional market point to our project by including its potential to become fertilizer.
Project focus alteration 2
After the above alterations, we conducted our second meeting with BioMetallica, presenting our project focus of targeting cadmium pollution, with addition of enhancing sludge potential and heavy metal recycling. However, the main question brought up was “why only cadmium?” If our focus was to target pollution, it would certainly make more sense to have an end goal of targeting heavy metals as a whole rather than specifically cadmium. Moreover, the addition of heavy metal recycling of cadmium would not make sense. The cost effectiveness for remediating cadmium alone would not justify the process of doing so.
In response, we included remediating all heavy metals from sludge in our future cycles as a long term end goal. We decided to retain the metal recycling process because the other heavy metals recovered may have a higher market value than cadmium. This potential revenue improves the project's cost-effectiveness while furthering our primary goal of pollution removal. We also started searching for alternative methods to replace incineration as the recovery method.
In an attempt to connect with an end user facility, we were fortunately able to contact Shenzhen Shen Shui Ecological Environmental Technology CO.,LTD to gain better insight into sludge treatment. Their representatives illustrated the importance of the type of sludge we were targeting, which turned out to be a much more complicated target than we first anticipated. The key recommendation being to target industrial sludge rather than sewage sludge, as the major source of cadmium is from industrial activities, which pollutants will land in industrial sludge more.
The next time we carried out a meeting with BioMetallica, we suggested the above and was met with the concern that even though other heavy metals have more value than cadmium, the general market of metals does not have a large demand for toxic and harmful ones. It is still hard to justify the rationale for implementing this project on the end of the treatment facility. They further suggested that if our main focus is to remediate cadmium, we should focus on the removal process, urging us to define a clear goal instead of having these additions.
The problem we had to address was our definite target of addressing cadmium pollution. Through these consultations, we were constantly being challenged as to why we targeted cadmium, unable to find the key evidence to convince our industrial stakeholders as to why our project was necessary. We contacted Dr. Xiyue JIA, a co-author of a paper that heavily impacted our decision in changing the project direction to cadmium pollution in search of that evidence. To which we were validated that cadmium did in fact pose the most significant threat to soil pollution for multiple reasons, and that industrial awareness was not high due to the challenges that were pointed at us (low concentration, low cost effectiveness) .
We compared our previous research on current methods of sludge remediation, finding little data recorded on the effectiveness of cadmium removal. Those methods which cadmium was documented showed lowered efficiency towards remediated cadmium compared to other heavy metals. (Geng et al., 2020). This added to Dr. JIA’s consultation led us to believe that our project may target this gap and provide an effective remediation method that is specific to cadmium.
With this in mind, we further researched the implication methods of our project to reduce the processes needed and focused on the specific removal of cadmium rather than including additional steps for recovery.
Fourth design
Fourth design
Packed bed column was chosen to be the simplest and effective way of application posing an easy separation method of our chassis from the sludge
This design was chosen after throughout research on cadmium recovery methods and comparison with application methods such as direct application plus filtration. We implemented the suggestion of focusing on only our remediation method and excluded the idea of metal recovery.
In our final meeting with BioMetallica, we presented the full project goal and pipeline to them, which we received positive feedback towards. The only suggestions left were for the diagram to be more specific, thus leading to our final pipeline design.
Fifth Design
Fifth design
Evaluation of our solution
As part of iHP feedback stakeholders, they implored us to adopt a more entrepreneurial and application-based approach to the project. Thus, we extrapolated our data to existing data and literature in order to demonstrate the current feasibility of the project.
Firstly, we have analysed the data of our ICP-OES analysis and converted the values for real-world comparison. In the figure below, the first two bars plot the decrease caused by ECMT and EC but converted into mg and scaled up. In the following third bar plot coloured red, it subsequently shows the difference between the two groups. In other words, it represents the estimated decrease in cadmium caused by MT activity. Based on this, it is expected that 1 litre of ECMT sample expressing the metallothionein at a density of OD = 1 can remove an estimated 70 mg of Cadmium.
Approximation of cadmium removal efficiency of PflQ2MT
To calculate this value, we assumed that OD and subsequent protein expression can be linearly scaled up to OD = 1. This is supported by the data found in Week 10 of our Notebook page, which shows that the bacteria grow in a relatively linear relationship between OD 0.5 and OD 1.0. As mentioned in our Experiments page, we subcultured our bacteria to around 0.6 before inducing with IPTG. Thus, we believe this is a reasonable assumption to make when scaling up the density of our samples.
As mentioned prior, we subsequently calculated the difference between ECMT and EC groups to account for any potential reduction of cadmium caused by dilution or native mechanism within unengineered E. coli. Comparing previous literature suggesting that sludge has a concentration of around 4 mg of cadmium/L of sludge, we therefore estimate that 1 L of our bacteria at OD = 1 can approximately treat 18 L of sludge at its current rate of expression. While time restrictions prevented us from testing the MT function in P. putida, the western blot confirming its expression in the chassis, similar to E. coli, provides grounds to suggest that it would possess the same functionality. Further characterisation of the protein and its expression could be done to further optimise the amount of cadmium removal that could be achieved.
Regrettably, past stakeholders were unable to provide estimated values for our cost-approximation and characterization of our project would require the preestablishment of a specific sludge source. As described in our Description Page, we aim to replace the non-specific precipitation of heavy metal ions with our bacterial solution. Through this, we present an advantage over traditional methods as we prevent the need for chemical precipitation. These methods have been cited to potentially cause secondary pollution, or alkaline residues which are damaging to the environment, especially if executed at an industrial scale (Gomes et al., 2016; Zhang et al., 2024). Therefore, while we understand the actual efficiency of our solution is still ambiguous and at its current stage is likely to be not as cost-effective, our solution is still able to be effective at cadmium removal while having added advantages of being specific and more sustainable which can justify its implementation in real-world situations.
Therefore, we believe our solution proposes promising advantages and shows potential for cadmium-specific remediation at a larger scale.
