Heavy metals in food always spark concern within communities. We all know that heavy metals and edibles do not mix, whether in agriculture or seafood. Like microplastics, heavy metals accumulate slowly in the body until the toxicity levels are high enough to cause problems. For example, long-term exposure to cadmium - our heavy metal of concern - through food leads to liver damage and toxicity in organ systems, including the skeletal, urinary, and cardiovascular systems (Rahimzadeh et al., 2017).

In developed countries, the risks of heavy metal pollution are understood and strict regulations are often imposed to ensure food safety. So why, even with these efforts, do we ever so often hear that random samples have exceeding rates of arsenic, cadmium, lead, and other metals?

To answer this, we need to look at food-exporting countries. What is their side of the story?

A study comparing 796,084 datapoints on soil concentrations of key toxic metals from 1,493 regional studies found that cadmium has the highest global exceedance rate at 9.0% (−1.9%/+1.5%). Exceedance of cadmium in agricultural soil is most notable in northern and central India, Pakistan, Bangladesh, southern China, and parts along the belt highlighted below (Hou et al., 2025).

Figure 1. Global soil pollution by toxic metals exceeding agricultural thresholds (AT). Figure from Global soil pollution by toxic metals threatens agriculture and human health. Science, 388(6744), 316–321. (A) Aggregate distribution of exceedance of arsenic, cadmium, cobalt, chromium, copper, nickel, and lead; color code shows the maximum probability of exceedance among the seven metals. (B and C) Zoomed-in sections of globally important food production areas. (D) Predicted Cd exceedance rates and average soil pH indicative of Cd mobility in the major rice export countries. Country abbreviation: IN, India; TH, Thailand; VN, Vietnam; PK, Pakistan; CN, China; US, United States; BR, Brazil; PY, Paraguay; EU, European Union; AR, Argentina.

Our team confirmed the severity of cadmium pollution through our research. But what is cadmium exactly?

Cadmium is a heavy metal used in various fields, including nickel-cadmium batteries, pigment, and corrosion-resistant coating (Zhao et al., 2024). Its high bioaccumulation factor makes it particularly dangerous in agriculture, even at low concentrations, marking the severity of cadmium pollution (Tytła, 2019).

In China alone, cadmium also has the highest exceedance rate among heavy metals (7.0%), with approximately 7.6 million hectares of farmland contaminated. Contaminations in grains result in an estimated annual economic loss of about 20 billion RMB (Zhao et al., 2024). Research further shows that the exceedance rate of cadmium is as high as 17.57% in rice and 21.76% in vegetables, with the problem being particularly severe in southern provinces such as Hunan, Yunnan, and Guangxi (SHI et al., 2022).

During our ideation, we saw the problems caused by cadmium pollution and decided it was worth investigating. To do that, we backtracked to the source.

Sludge is a major source of cadmium release (UN Environment Programme, n.d.). It is done during the production of cadmium products or the mining of zinc (Werner et al., 2024). When heavy metal pollutants and waste are not disposed of properly, they runoff into water and enter the sewage system (Briffa et al., 2020). During water treatment, sludge is produced as a byproduct. While the treated water is clean and safe for consumption, pollutants, including heavy metals, remain in the sludge, which is then disposed of or applied to farmlands (Nguyen et al., 2022). Heavy metals like cadmium seep from the sludge into the soil and are taken up by crops, contaminating grains and vegetation.

The potential use of sludge as fertilizer has long been explored due to its rich organic content, which is why it is sometimes applied directly to farmlands (Van Den Broek et al., 2024). However, one major obstacle to the upcycling of sludge is its heavy metal content (Janaszek & Kowalik, 2023; Suanon et al., 2016), with cadmium posing the most significant risk (Janaszek & Kowalik, 2023). There have been past cases of heavy metal poisoning due to the application of contaminated sludge on farmlands. As a result, the use of raw sludge as fertilizer has been banned in some countries.

We are certainly not the only ones aware of the benefits of sludge remediation. Many efforts have proposed different methods to remove heavy metals from sludge for safe disposal or reuse. However, our research found little data focused specifically on cadmium removal. Furthermore, existing solutions play out to be less efficient at remediating cadmium compared to other heavy metals (Geng et al., 2020). This may be because industries have low awareness of cadmium pollution due to its very low concentrations relative to other metals, as indicated in our consultation with Dr. Xiyue JIA.

Therefore, we realized that to solve the problem, we need to create a solution specifically designed to remove cadmium.

The conventional method of heavy metal remediation from sludge consists of two general steps as suggested by research (Geng et al., 2020) and our consultation with Prof. Zhiguo YUAN AM:

1. Leaching heavy metal ions out of sludge into the liquid phase
2. Precipitating heavy metal ions into solids for removal

Our project replaces the second step with engineered P. putida that captures cadmium to achieve remediation. As mentioned, cadmium removal rates using general methods are less effective. Our biological application emphasizes the ability to selectively capture cadmium with live cells, even in a competitive environment with high concentrations of zinc or other heavy metals.

To learn more about more about the development of our real-world solution and how it compares to existing solutions, please take a look at our iHP Page under Application Development.

HKUST iGEM 2025 Slupeer team aims to design novel bio-engineered bacteria for cadmium bioremediation and its corresponding application workflow. Using synthetic biology techniques, we are engineering Pseudomonas putida KT2440 to achieve cadmium extraction and detection, with high-level biosafety control.

Main Circuit for Cadmium Binding

The main circuit achieves cadmium-specific binding and extraction by surface-expressing PflQ2 Metallothionein (PflQ2MT, BBa_25VCEOE5). PflQ2MT is displayed on the outer membrane of Pseudomonas putida KT2440 via C-terminal fusion to Lpp-OmpA (BBa_K1694010), an outer membrane anchor protein. This allows PfIQ2MT to interact with cadmium ions in liquid solutions. A TEV cleavage site (BBa_J18918) is introduced between Lpp-OmpA and PfIQ2MT to verify its surface display. The concept of surface binding could be further extended to other heavy metals, and we discovered binding protein EC20 (BBa_25WWG3IU) as a candidate with higher efficiency and selectivity.

Figure 2. Demonstration of our Main Circuit.

Biosensor

To enable real-time visualization and quantification of remaining cadmium ions and monitor the remediation rate, we engineered a toehold switch-based biosensor in P. putida . Utilizing the Cd2+ concentration-dependently activated Pcad promoter (BBa_25CAR3OY) regulated by CadR (BBa_251WLXJ9), the presence of cadmium leads to the expression of a specific trigger RNA (BBa_K2621015). As described by Soudier et al. (2022), toehold switches are RNA sensing devices that rely on a conformational switch in secondary structure. The trigger RNA hybridizes with the synthetic sensing mRNA molecule (BBa_25S6BMR8), unfolding the hairpin structure that normally sequesters the Ribosome Binding Site (RBS) and the start codon. This conformational change exposes the RBS, allowing translation initiation of the reporter gene, mScarlet-I3-NCwt (BBa_25IVGUN2). Consequently, the resulting red fluorescence intensity directly correlates with the concentration of Cd2+ present in the environment. To ensure rapid signal turnover for real-time detection of Cd2+ concentrations, the reporter protein is fused to the LVA degradation tag (BBa_K3257071), which targets the protein for degradation.

Figure 3. Demonstration of the Biosensor.

Kill Switch

A kill switch prevents the persistence of the genetically modified bacteria in case of leakage into the natural environment. Our chassis, Pseudomonas putida KT2440, operates in a high-citrate environment at a pH around 4, to dissolve and stabilize cadmium ions in liquid solutions. If the bacteria are accidentally released into a natural environment with lower citrate concentration and higher pH, the opdH-tctCBA-tctDE citrate-sensing system (BBa_25CTXIKQ) would induce the expression of the E lysis protein (BBa_K2152003) which originate from bacteriophage Phi X 174. This initiates cell lysis and death by inhibiting bacterial cell wall biosynthesis.

Figure 4. Demonstration of the Kill Switch.

Our proposed application pipeline is based on five steps:

Figure 5. Demonstration of our sludge processing pipeline.

1. The chassis is bound to a packed bed column. The reactor contains a minimum amount of citric acid to prevent the kill switch from activating.
2. Sludge is mixed with citric acid to leach cadmium.
3. The sludge solution passes through the reactor, where the chassis captures the cadmium.
4. The column is removed for proper disposal once saturated.
5. Citrate may be recycled, and treated sludge proceeds to downstream processes.

Column Setup Definition: A column reactor for bacterial sludge treatment is typically a vertical vessel that retains a high-density biomass zone, allowing liquid (containing sludge and cadmium) to pass through downward (Price et al., 2015). We propose to include packed bed columns (using inert materials for bacterial attachment) in this design. The column can include packing to increase surface area for biofilm formation and biomass retention (Zune, 2015).

Retention of bacteria can be achieved by immobilizing them on beads inside the column to promote biofilm formation. A packed column design, or the introduction of internal structures, maximizes contact area while reducing cell washout by decoupling solids and hydraulic retention time (Keet et al., 2024).

With respect to the timeframe of iGEM, the following is a development plan for the future engineering cycles of the project.

1. Validation of proposed column pipeline through characterization and small scale experiments.
   1. Testing with different sludge samples
   2. Characterization of bacterial efficiency and industrial application cost
   3. Establishing parameters for column application
2. Establish collaborations with potential end-users.
   1. Fine tune project according to components of the local sludge
3. Extension of application
   1. Extend project application to different heavy metals while maintaining effectiveness against cadmium, ultimately achieving a circular economy by upcycling sludge into fertilizer.

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