Abstract

Heavy metals pose significant health risks, including neurotoxicity, kidney damage, and cancer. To enhance bioremediation, our team engineered E. coli to express rice metallothionein for efficient metal binding, along with genes from Sporosarcina pasteurii to enable Microbiologically Induced Calcite Precipitation, transforming heavy metal ions into easily removable precipitates. We integrated feedback from a range of stakeholders, including the general public, experts in heavy metal treatment, aquaculture specialists, microbiologists, and wastewater treatment engineers. These insights helped us refine our project, leading to the development of an effective heavy metal-removing E. coli strain and a hardware solution compatible with wastewater treatment plants.

 

Highlights

Interviewee/ Respondent	Institution	Suggestions & Takeaways
1163 people	The public	The public recognizes the significance of heavy metal pollution and is eager to see heavy metal pollution being handled.
Dr. Siqing Chen	Yellow Sea Fisheries Research Institute	Heavy metals in water systems can harm aquatic animals and potentially accumulate in the human body through the food chain.
Dr. Jinlong Jiang	Third Institute of Oceanography	Prevention at the source is crucial in addressing heavy metal pollution. Industrial enterprises should improve wastewater treatment.
Ms. Sha Yu	Xianyang No. 1 Water Treatment Plant	Heavy metals mainly come from industry and should be managed there, as standard water treatment plants lack facilities for their removal.
Mr. Jining Cheng	China Coal Xi'an Design Engineering Co., Ltd.	Chemical precipitation is preferred for its cost and efficiency, while current biological methods are less efficient.
Prof. Jun Zhang	Northwest University	Microorganism-based heavy metal removal is promising with advances in synthetic biology, especially with MICP.
Prof. Xufen Zhu	Zhejiang University	BL21's high expression caused misfolded proteins to be non-functional, while DH5α's lower levels enabled proper function.
Mr. Shuicai Xu	Deqing County Water Treatment Office	Introduced us to wastewater treatment facilities, which inspired the development of a device for our engineered bacteria.
Mr. Hangshuai Liu	Dawang Wastewater Treatment Plant	Control the amount of engineered bacteria entering the next steps. Heavy metal precipitates must be carefully managed.

 

1. Research on heavy metal pollution in China

Heavy metals like lead, mercury, cadmium, and chromium are toxic elements with high atomic weights and densities (Ali & Khan, 2018; Mir Mohammad et al., 2021). They are released into the environment through industrial activities, mining, and waste disposal, posing serious health risks such as neurotoxicity, nephrotoxicity, and carcinogenicity (Abd Elnabi et al., 2023). These metals also harm other organisms and disrupt ecosystems, threatening biodiversity (Tchounwou et al., 2012).

 

Figure 1. Position of heavy metals in the periodic table (Mir Mohammad et al., 2021).

 

Our literature research found that heavy metal pollution is a significant issue in China due to industrialization, urbanization, and agriculture. In our hometown, the Yangtze River Delta, studies conducted in 2018 identified significant ecological risks from mercury, cadmium, and chromium in three major cities in this region, Hangzhou (Qinxuan et al., 2018), Ningbo (Mei-juan et al., 2018), and Haiyan (Zhan-jun et al., 2018), with carcinogenic risk indices reported to be up to 24,800 times higher than safe levels. The pollution level in the Yangtze River Delta is not the worst. Reports indicate that other provinces in China, particularly Guangxi, Fujian, and Liaoning, suffer from more severe cadmium and lead pollution than the Yangtze River Delta (Wu et al., 2022; Yu et al., 2017).

 

To assess public awareness of heavy metal pollution and its causes, we conducted a survey. The results showed that though some uncertainty exists (9%-10% are unsure), most people still acknowledge the issue’s importance (Figure 2A). Also, most people believe heavy metal pollution is closely related to human life, with 86% recognizing its relevance (Figure 2B). To conclude, the public is eager to see heavy metal pollution being handled.

 

Public Survey

Figure 2. Public awareness and perception of heavy metal pollution.

 

Respondents:

1163 people

Results and takeaways:

1) Some uncertainty exists (36%), but most people recognize the importance of heavy metal pollution.

2) Many people have a basic understanding of heavy metals, indicating widespread interest among the respondents.

2) Most people (86%) believe heavy metal pollution is closely related to human life.

 

To assess the impacts of heavy metals on animals, we interviewed Dr. Chen, an aquaculture expert. Dr. Chen emphasized that in marine aquaculture, heavy metals such as manganese and iron in groundwater can impair fish growth, weaken immunity, and even cause disease or death. Furthermore, removing these metals is costly, as materials like gravel, quartz sand, and activated carbon require frequent replacement. He also stressed that if heavy metals in water systems are not properly managed, they can harm aquatic animals and potentially accumulate in the human body through the food chain.

 

Interview

Interviewee:

Dr. Siqing Chen

Institution:

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Science

Field of research:

Marine aquaculture

Suggestions and takeaways:

1) In aquaculture, heavy metals affect water quality, and EDTA or other chelating agents are commonly used to treat them.

2) Heavy metals like manganese and iron in groundwater can affect fish growth, lower immunity, and even cause disease or death. 

3) Removing heavy metals is costly, as materials like gravel, quartz sand, and activated carbon must be replaced regularly. 

4) Heavy metals in aquaculture not only harm marine life but also pose risks to human health.

Given the severe heavy metal pollution in the Yangtze River Delta and its serious threats to humans and animals, we believe that action is needed. Therefore, we decided to develop a method to reduce heavy metal levels in the environment, aiming to protect both people and the ecosystem.

 

2. Research on sources of heavy metals

To identify the most effective and suitable method for heavy metal removal in the Yangtze River Delta, we need to focus on two key aspects. First, determining the sources of heavy metals, as this will guide where efforts should be concentrated. Second, assessing the available heavy metal removal methods and evaluating which are feasible for real-world application.

 

To understand where efforts to reduce heavy metal pollution should begin, specifically identifying the main sources, we interviewed Dr. Jinlong Jiang, a marine environmental specialist. Dr. Jiang noted that while China's marine ecological environment has improved in recent years, heavy metal pollution remains a serious issue, with industry being the primary source. He emphasized that prevention at the source is critical in addressing heavy metal pollution. He stressed the importance of industrial enterprises strengthening wastewater collection and treatment to mitigate the spread of pollution. This has given us a clear direction for our efforts.

 

Interview

Interviewee:

Dr. Jinlong Jiang

Institution:

Third Institute of Oceanography, Ministry of Natural Resource

Field of research:

Marine ecological protection and resource utilization

Suggestions and takeaways:

1) The main source of heavy metal pollution is land-based, primarily entering the ocean through rivers and agricultural runoff, accounting for 70% to 80% of the total pollution. 

2) Reducing heavy metal pollution in freshwater rivers can significantly improve the marine environment. 

3) The extent of heavy metal pollution varies by region, with industrial and agricultural activities being the main sources in certain areas. 

4) Industrial enterprises must strengthen wastewater collection and treatment to reduce the spread of pollution.

 

We visited a water treatment plant and spoke with the general manager, Ms. Yu, who confirmed that heavy metal pollution is a serious issue they regularly test for. She explained that heavy metals primarily come from industrial sources and should be addressed within the industrial system, as she noted that in standard water treatment plants providing tap water, there are no specialized facilities for heavy metal removal.

 

Interview

Interviewee:

Ms. Sha Yu

Institution:

No. 1 Water Treatment Plant, Xianyang Water Group Co., Ltd

Position:

General Manager

Suggestions and takeaways:

1) Water quality monitoring follows a three-tier system, testing at the plant, in the distribution network, and at the user endpoint, with routine and heavy metal tests conducted monthly. 

2) Tap water treatment includes mixing, flocculation, sedimentation, filtration, and disinfection, then distributed through pipelines to the city and homes. 

3) In typical water treatment plants that provide tap water, there are no specialized facilities for heavy metal removal because only water sources that test free of heavy metals are used.

4) Emergency groundwater sources are monitored every six months to ensure they meet safety standards for use when needed.

 

3. Research on current heavy metal removal methods and find our solution

After concluding that heavy metals should be neutralized in industrial settings, we began exploring the current options for heavy metal removal. We believe a thorough evaluation of all available methods is essential before making any decisions. We interviewed Mr. Cheng, the technical director of a coal company with expertise in heavy metal treatment. He mentioned four primary methods: chemical precipitation and coagulation, reverse osmosis, electrochemical techniques, and biological treatment. According to him, chemical precipitation is the most commonly used method due to its high efficiency. In contrast, current biological treatment methods have drawbacks, such as the need for additional nutrients, low efficiency, and the potential introduction of organic pollutants.

 

Interview

Interviewee:

Mr. Jining Cheng

Institution:

China Coal Xi'an Design Engineering Co., Ltd.

Position:

Technical Director

Suggestions and takeaways:

1) Common methods for heavy metal treatment include chemical precipitation and coagulation, reverse osmosis, and electrochemical techniques.

2) Treatment costs and effectiveness vary. Chemical precipitation and coagulation are low-cost and simple, while reverse osmosis and electrochemical techniques are more effective but more expensive. 

3) Economic issues are the main barriers in heavy metal treatment, with significant cost differences between methods. 

4) Biological treatment methods have limitations such as the need for nutrients and low efficiency that require improvements.

 

Following Mr. Cheng's insights, we did further research on current heavy metal removal methods. Chemical precipitation forms insoluble metal precipitates but produces toxic sludge, and may introduce pollutants like aluminum (Atari et al., 2019). Ion exchange and adsorption rely on materials like resin and activated carbon but have limited capacity and high consumable use (Ayach et al., 2024). Electrochemical methods trigger redox reactions to precipitate metals but are energy-intensive (Guo et al., 2024). Bioremediation uses microorganisms or plants, but its effectiveness depends on environmental factors (Fu & Wang, 2011; Qasem et al., 2021).

 

In conclusion, no method is perfect. Although Mr. Cheng does not view bioremediation as an effective solution for heavy metal removal, we see its challenges as opportunities for improvement and innovation. Moreover, bioremediation offers a significant advantage over other methods: it is more environmentally friendly and sustainable. As an iGEM team, our goal is to engineer bacteria to improve bioremediation efficiency.

 

4. Research on engineered bacteria design and build

After defining our goal of improving microorganisms for heavy metal removal, we consulted Prof. Jun Zhang, an expert in this field. She affirmed that using microorganisms for heavy metal removal is both feasible and promising, especially with the rapid advancements in synthetic biology. She also introduced us to Microbiologically Induced Calcium Carbonate Precipitation (MICP), a process where bacterial activity catalyzes the formation of calcium carbonate minerals, which accumulate around bacterial cells as solid deposits.

 

Figure 3. Schematic of microbially induced calcite precipitation (MICP) catalyzed by Sporosarcina pasteurii, a ureolytic bacteria (Wu et al., 2021).

 

MICP is ideal for our project because heavy metal ions can solidify and be removed from the treated water. The process is effective with various metal ions; for example, cadmium ions can form cadmium carbonate, reducing both concentration and toxicity (Qasem et al., 2021). Therefore, MICP holds great potential for the removal of a wide range of heavy metals, making it a perfect fit for our project. Prof. Zhang also emphasized the importance of developing a lab-scale bioreactor.

 

Interview

Interviewee:

Prof. Jun Zhang

Institution:

The College of Life Sciences, Northwest University

Field of research:

Environmental microbial community diversity and wastewater treatment

Suggestions and takeaways:

1) Microbial wastewater treatment includes precipitation, adsorption, and final discharge.

2) Bioprecipitation, such as Microbiologically Induced Calcium Carbonate Precipitation (MICP), shows strong potential in synthetic biology.

3) Bioreactor design should align with bacterial requirements, and lab-scale models are key for hardware development.

 

We discovered another approach for heavy metal removal through enhanced absorption using metallothioneins (MTs). MTs are mall, cysteine-rich proteins that bind and sequester metal ions (Coyle et al., 2002). By introducing MTs into our engineered cells, the proteins are expected to directly bind heavy metal ions and enhance the cells' tolerance to toxic metals (He et al., 2024; Lu et al., 2023). We chose to engineer the most commonly used Escherichia coli BL21 as our chassis, incorporating a rice MT and the MICP-inducing genes from Sporosarcina pasteurii.

 

As our design was finalized, we began our experiments. We successfully assembled recombinant vectors carrying our genes of interest and transformed them into E. coli BL21. However, when testing the MICP-related genes, BL21 showed no function, while the DH5αdemonstrated clear functionality. This result was unexpected, as we had anticipated higher gene expression in BL21 compared to DH5α.

 

To understand this unusual outcome, we consulted Prof. Zhu from Zhejiang University. After reviewing our vector design and confirming there were no errors, she suggested that the high protein expression rate in BL21 might have overwhelmed the cell's folding machinery, leading to misfolded proteins or inclusion bodies, rendering them non-functional. In contrast, DH5α’s naturally lower protein expression levels likely allowed for proper protein folding. We performed SDS-PAGE as she suggested, and confirmed her hypothesis.

 

In the end, we decided to abandon BL21-based strains in favor of stability, as our engineered DH5α strains demonstrated reliable heavy metal removal abilities in subsequent tests (see Engineering Success https://2024.igem.wiki/hangzhou-sdg/engineering for more details).

 

Interview

Interviewee:

Prof. Xufen Zhu

Institution:

Life Sciences Institute, Zhejiang University

Field of research:

Microbiology and enzymology

Suggestions and takeaways:

1) Our vector design was correct.

2) The high protein expression rate in BL21 might have overwhelmed the cell's folding machinery, leading to misfolded proteins or inclusion bodies

3) DH5α's naturally lower protein expression levels likely allowed for proper protein folding.

4) The issue of BL21 could potentially be mitigated by adjusting culture conditions, such as lowering the temperature.

 

5. Research on hardware development and real-world implementation

Our engineered E. coli demonstrated strong heavy metal removal capabilities in lab tests, particularly for Cd²⁺ and Pb²⁺, with maximum removal rates of 86% and 99% respectively. We also determined the applicable concentration ranges: 0-0.1 mM for Cd²⁺ (0-18.33 mg/L of CdCl₂) and 0-1 mM for Pb²⁺ (0-331.21 mg/L of Pb(NO₃)₂). The actual upper limit could be higher, as we only tested 10-fold dilutions, which means concentrations smaller than these intervals might have been missed (see Engineering Success https://2024.igem.wiki/hangzhou-sdg/engineering for more details). To better understand real-world conditions and assess the competitiveness of our engineered E. coli, we researched the applicable concentration ranges of other heavy metal removal methods. We found that these methods typically handle cadmium concentrations of 25-50 mg/L and lead concentrations of 100-1000 mg/L, which are comparable to the capacity of our engineered E. coli. (Fu & Wang, 2011). This suggests our bacteria have promising potential for practical application.

 

Following Prof. Zhang's advice, we decided to build lab-scale bioreactor models. Our goal is to develop a bioreactor with supporting devices that can seamlessly integrate into existing wastewater treatment systems, ensuring that our engineered bacterium and hardware can be easily accepted for real-world applications. To begin with, we visited a wastewater treatment plant in Deqing and spoke with Mr. Shuicai Xu, the Inspection and Evaluation Team Leader, who introduced us to the plant’s current facilities. Inspired by their setup, we designed a device consisting of a reaction tank where bacteria bind to heavy metal ions and MICP occurs, and a sedimentation tank where the bacteria and heavy metal precipitates coagulate and are removed from the water (see Hardware https://2024.igem.wiki/hangzhou-sdg/hardware for more details).

 

Interview

Interviewee:

Mr. Shuicai Xu

Institution:

Deqing County Water Treatment Office

Position:

Inspection and Evaluation Team Leader

Suggestions and takeaways:

1) Wastewater treatment plants in Deqing use the A²O process, treating 60% domestic and 40% industrial wastewater.

2) Automated online monitoring collects samples every two hours for water quality testing, and authorities regularly inspect to ensure compliance.

3) Third-party agencies test heavy metal levels, ensuring no secondary pollution into the environment.

4) Wastewater treatment benefits the environment and boosts local economies by attracting tourists with a clean water environment.

 

We then visited Dawang Wastewater Treatment Plant and spoke with technician Mr. Liu. He appreciated our solution and believed that our engineered bacterium could be useful in the pre-treatment stage. He emphasized the importance of eliminating any remaining bacteria. Even though sterilization is part of the later process, Mr. Liu highlighted the need to control the amount of engineered bacteria entering the subsequent steps to avoid adding stress to the system. To address this, we added a UV lamp after the sedimentation tank to kill most of the bacteria (see Hardware https://2024.igem.wiki/hangzhou-sdg/hardware for more details). We will continue testing and improving our hardware.

 

Interview

Interviewee:

Mr. Hangshuai Liu

Institution:

Dawang Wastewater Treatment Plant, Xixian New Area, Shanxi

Position:

Technician

Suggestions and takeaways:

1) Wastewater undergoes screening, sedimentation, biological treatment, advanced treatment, and disinfection. 

2) Solid-liquid separation occurs in the secondary sedimentation tank using coagulants, followed by sterilization with sodium hypochlorite that targets E. coli. 

3) Improving bacterial adsorption requires further research on environmental factors like pH and nutrient needs.

4) The heavy metal precipitates should be handled separately and should not be mixed with sludge from domestic wastewater.

 

As Mr. Liu mentioned, removing heavy metals from polluted water is not the final step. The heavy metal precipitates must also be carefully managed. The heavy metal precipitates, once separated from the treated water, should first be dried to reduce their weight and volume, facilitating easier handling (Fytili & Zabaniotou, 2008). If the dried precipitates contain high percentages of valuable elements like mercury or cadmium, they should undergo specialized recycling processes (Du et al., 2023). Otherwise, the dried precipitates should be solidified with stabilizers such as cement or other immobilizing agents to prevent the release of heavy metals. The cement-stabilized precipitates can then be safely disposed of in landfills or, after rigorous evaluation, potentially repurposed as building materials (Chen et al., 2009; Liu et al., 2022).

 

6. Regulations of GMO

We are working on a genetically engineered bacterial product, and given China's strict regulations, we wanted to ensure it could eventually reach the market. After reviewing the regulations, we found that although genetically modified bacteria must pass a series of rigorous tests, market approval is possible. In 2023, 113 genetically modified organisms were granted Safety Certificates, which suggests our engineered E. coli could follow the same path in the future.

 

The route to approval is as follows:

1) According to the Biosecurity Law of the People's Republic of China and

Measures for the Administration of the Safety Evaluation of Agricultural Genetically Modified Organisms (2016 Revision, Article 13), our engineered bacteria must go through three stages of trials: intermediate test, environmental release test, and production test.

 

2) During each stage, factors including genetic stability, potential toxicity, spreading capacity, impacts on non-target organisms, etc. will be evaluated (Appendix III. 1).

 

3) Submit the Application for the Safety Evaluation of Agricultural Genetically Modified Organisms to the State Commission for the Safety of Agricultural Genetically Modified Organisms and the Office for the Safety Management of Agricultural Genetically Modified Organisms to start the evaluation process.

 

4) If approved, the Ministry of Agriculture and Rural Affairs of China will issue the Agricultural Genetically Modified Organisms Safety Certificate.

 

 

References