Abstract

Heavy metals in water pose serious health risks. Traditional methods like chemical precipitation and ion exchange have drawbacks, including extra pollutant introduction and high consumable use. Bioremediation offers a more sustainable solution. Our team engineered E. coli to express rice metallothionein (OsMTI-1b) for metal binding and a urease gene cluster from Sporosarcina pasteurii for Microbiologically Induced Calcite Precipitation (MICP). Our bacteria bind heavy metals and form carbonate precipitates, allowing easy separation from treated water. This scalable, eco-friendly method is ideal for industrial wastewater treatment plants.

 

Part 1. Heavy Metals

Heavy metals, such as lead, mercury, cadmium, and chromium, are metallic elements with high atomic weights and densities (Ali & Khan, 2018; Mir Mohammad et al., 2021). These metals are naturally present but are released into the environment through industrial activities, mining, and waste disposal. Heavy metals pose serious health risks, including neurotoxicity, nephrotoxicity, and carcinogenicity (Abd Elnabi et al., 2023; Balali-Mood et al., 2021; Mitra et al., 2022). Heavy metals are toxic not only to humans but to almost any organism from bacteria, to plants and wild animals. As a result, heavy metal pollution can disrupt the ecosystem and endanger biodiversity (Tchounwou et al., 2012).

 

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

 

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).

 

Various methods are used to remove heavy metals from water. Chemical precipitation forms insoluble metal precipitates but is costly, produces toxic sludge, and introduces other pollutants like aluminum (Atari et al., 2019; Bakar & Halim, 2013). Ion exchange and adsorption use materials like ion exchange resin and activated carbon to capture heavy metals, but they have limited capacity and require large amounts of consumables (Ayach et al., 2024; Monir et al., 2024). Electrochemical methods induce redox reactions to precipitate heavy metals but are energy-intensive (Guo et al., 2024; Liu et al., 2019). Bioremediation utilizes microorganisms or plants, though it may be less effective for certain contaminants and depends on environmental factors (Kapahi & Sachdeva, 2019; Qasem et al., 2021).

 

With all the pros and cons, no method is perfect. As of now, chemical precipitation is the most used strategy in the real world due to its high efficiency. However, we believe that bioremediation is the future because it offers a more environmentally friendly and sustainable approach to heavy metal removal. As an iGEM team, our goal is to engineer bacteria to overcome the low efficiency of bioremediation.

 

Part 2. Our solution: MT and MICP

Part 2.1 Metallothionein (MT)

Metallothioneins (MTs) are small, cysteine-rich proteins found in various organisms, including mammals, plants, and microorganisms, that bind and sequester metal ions (Coyle et al., 2002). Due to their high cysteine content, MTs have a strong affinity for metals, playing roles in metal ion transport, antioxidant defense, and protection against metal toxicity (Carpene et al., 2007; Klaassen et al., 1999). Introducing MTs into our engineered cells offers two benefits for heavy metal removal: directly binding and lowering metal ion concentrations, and enhancing the cells’ tolerance to toxic metal ions (He et al., 2024; Lu et al., 2023).

 

Figure 2. The general structure of metallothionein. Four and three atoms of metal ions are coordinated in α- and β-domains, respectively (Klaassen et al., 1999).

 

Part 2.2 Microbiologically Induced Calcite Precipitation (MICP)

Microbiologically Induced Calcite Precipitation (MICP) is a biomineralization process driven by bacteria, primarily ureolytic bacteria, that catalyze the formation of calcium carbonate minerals. Urease enzymes in these bacteria hydrolyze urea into ammonia and carbonate ions, raising the pH and allowing carbonate ions to react with calcium (or other cations) to form calcium carbonate precipitates (Bachmeier et al., 2002; Sarayu et al., 2014). These precipitates accumulate around bacterial cells, forming solid mineral deposits (Wu et al., 2021).

 

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

 

MICP shows promise for heavy metal removal as the precipitation process can occur with various metal ions. For instance, in the presence of cadmium ions, cadmium carbonate forms, reducing cadmium concentration and toxicity (Qasem et al., 2021; Zeng et al., 2021). Thus, MICP is an effective method for removing a wide range of heavy metals, making it ideal for our project.

 

Part 3. Modification of Escherichia coli

Part 3.1 Chassis: Escherichia coli DH5α & BL21(DE3)

Although ureolytic bacteria, such as S. pasteurii, have the ability to induce MICP naturally, most of them are not well studied and are difficult to perform genetic modifications. E. coli is a safe and well-known platform for expressing exogenous genes. We chose it for its ease of use and potential for future improvements. We hope future iGEM teams can easily build on our work.

 

In this project, we used E. coli strains DH5α and BL21(DE3). DH5α is commonly used for molecular cloning and plasmid amplification because of its efficiency and DNA stability, though its protein expression levels are lower than other strains. In contrast, BL21(DE3) is designed for high-level protein expression, especially with T7 promoter plasmids. The absence of proteases like OmpT and Lon helps minimize its protein degradation, leading to higher yields of intact proteins (Jeong et al., 2015).

 

Part 3.2 OsMTI-1b

Rice (Oryza sativa) contains metallothioneins (OsMTs) known for efficient heavy metal absorption. Of the eleven OsMT isoforms, OsMTI-1b (noted as OsMT1 in this project) has demonstrated the highest metal-binding capacity (Ansarypour & Shahpiri, 2017; Shahpiri & Mohammadzadeh, 2018). To maximize OsMTI-1b expression and function in E. coli, we fused a signal-like sequence (Non) and part of the outer membrane protein A (OmpA) to the N-terminus of OsMTI-1b, creating a 20.67 kDa fusion protein for surface display on engineered bacteria.

 

Part 3.3 Urease gene cluster

Sporosarcina pasteurii, formerly known as Bacillus pasteurii, is a Gram-positive bacterium recognized for its strong ureolytic capabilities (Wu et al., 2021; Yoon et al., 2001). Commonly found in soil and natural environments, S. pasteurii alters its microenvironment, contributing to important ecological processes. It is safe for humans, animals, and plants, making it an excellent gene donor for our project (Omoregie et al., 2019). The model strain DSM 33 has had its entire genome sequenced, allowing us to identify and amplify its urease genes, which are responsible for microbially induced calcium precipitation (MICP). Fortunately, these urease genes are located close together in a gene cluster, allowing us to amplify the entire 5300 bp cluster, which consists of seven genes: ureA, ureB, ureC, ureE, ureF, ureG, and ureD (Pei et al., 2023; You et al., 1995).

 

Part 3.4 Recombinant vector

For exogenous gene expression, we find pET-28a an effective vector. pET-28a contains a high-copy origin of replication (high levels of expression and easy extraction), a kanamycin-resistant selection marker (superior to ampicillin due to higher stability of kanamycin), and a T7 promoter system. The T7 promoter can be very strong but requires inducers to function, which is not suitable for our project since our proposed application is in the water treatment plants.

 

We found a strong constitutive promoter, BBa_J23100, from the iGEM Registry. It is reported as the strongest in its family, with effectiveness being confirmed by multiple iGEM teams (Anderson, 2006). Other advantage includes short length (35 bp) and compatibility (compatible with RCF[10], [21], [23], [25], and [1000]). As a result, we planned to incorporate BBa_J23100 into pET-28a and put OsMTI-1b and the urease gene cluster under its control (Figure 4B-D).

Figure 4. A. Carbonate precipitates induced by E. coli harboring pET28a-Ure. The dark grains were calcium carbonate precipitates; B. Plasmid map of pET28a-OsMT1; C. Plasmid map of ET28a-Ure; D. Plasmid map of pET28a-OsMT1-Ure.

 

Summary

Heavy metals like lead, mercury, cadmium, and chromium pose serious health risks. Traditional methods, such as chemical precipitation and ion exchange, have limitations, including introducing other pollutants and the high cost of consumables. Bioremediation offers a more eco-friendly alternative. Our team aimed to enhance bioremediation by engineering E. coli to express rice metallothionein (OsMTI-1b) for metal binding, along with a urease gene cluster from Sporosarcina pasteurii to enable Microbiologically Induced Calcite Precipitation (MICP). Our engineered bacteria will bind heavy metals in water and induce the formation of carbonate precipitates, allowing easy separation of the bacteria and precipitates from the treated water. These engineered bacteria are designed for use in areas needing heavy metal remediation, such as industrial wastewater treatment plants. This approach provides a scalable, greener alternative to conventional methods, helping ensure safer water supplies.

 

 

References