Team:CSU CHINA/Description

Description

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1.Background and Significance

1.1 The harm of cadmium

1) Cadmium is a non-essential element for the human body.

2) Enrichment: Cadmium can accumulate in organisms and enter the human body through the food chain to cause chronic poisoning. The cadmium in water and soil would be absorbed by crops such as rice. Then humans take the rice contaminated with cadmium, causing the cadmium to accumulate in the human body.

3) Accumulation: After cadmium is uptaken by the human body, the cadmium thioprotein would be formed in the body. It is selectively accumulated in the liver and kidney. The kidney can absorb nearly one-third of the cadmium that enters the body, which is the “target organ” of cadmium poisoning. Other organs such as the spleen, pancreas, thyroid, and hair also have a specific accumulation amount.

4) Symptoms: Cadmium damages the renal tubules, which would cause diabetes, proteinuria and aminoaciduria. Dr. Luo from Xiangya Hospital told us, the long-term exposure to cadmium-polluted water and rice will lead to chronic cadmium poisoning, and further caused itai-itai disease and loss of renal function, which poses significant threats to the public health.

Figure1 Interview with Dr. Luo

1.2 Current status of cadmium pollution

1) Agricultural production: The impact on crops makes reducing cadmium content in water urgently needed to be solved. According to the Bulletin of the National Survey of Soil Pollution, China’s overall soil environment is not optimistic. Soil pollution is severe in some areas, and cultivated land’s soil environmental quality is also worrying. The exceeding rate of cadmium pollutants reached 7.0%, showing a gradual increase from northwest to southeast and from northeast to southwest. It is one of the primary contaminants in cultivated land, woodland, grassland and unused land. There are 20 million h㎡ of land contaminated by heavy metals in China, accounting for 1/5 of China’s arable land. Among them, the cadmium pollution of farmland is as high as 1.3 x10 ⁴ h ㎡, involving 25 provinces and cities in China. The annual production of “cadmium rice” is as many as hundreds of millions of kilograms.

Through the interviews with SINOGRAIN Company and farmers, in China, the main way of soil cadmium pollution is the cadmium polluted-water irrigation, the heavy metal polluted rice result in 12 million tons grain discarded and more than 20 billion economic loss, and there is currently no effective way for farmers to absorb the cadmium for agricultural irrigation water they use.

Figure 2 The vast farmland

2) The Source of Pollution: The pollution of cadmium in water bodies mainly comes from surface runoff and industrial wastewater. The cadmium content in the wastewater discharged from the production of sulfuric acid from pyrite ore, and phosphate fertilizer production from phosphate rock is relatively high. Cadmium particles from atmospheric lead-zinc ore and non-ferrous metal smelting, combustion and incineration of plastic products all of these may enter the water. Cadmium is discharged from catalysts, pigments, plastic stabilizers, synthetic rubber vulcanizers, and fungicides that use cadmium as raw materials can also cause water pollution. As for urban water, the cadmium content in drinking water is often increased due to the corruption of containers and pipelines. The discharge of industrial wastewater makes the cadmium content in offshore seawater and plankton higher than that in the distant sea. The cadmium content in the surface water of industrial areas is higher than that in non-industrial areas.

Figure 3 Industrial waste water being discharged

1.3 the significance of our project

If our modified Synechocystis sp. PCC6803 can be successfully applied to cadmium-polluted water bodies. It will

• 1) Reduce cadmium ions in water, significantly reduce cadmium in industrial wastewater.
• 2) Reduce the cadmium in farmland and food
• 3) Reduce the incidence of cadmium poisoning in the population.
• 4) Reduce the diseases related to cadmium pollution
• 5) Contribute to public health and environmental governance.

2.Aims

2.1 Use the modified Synechocystis to reduce the cadmium content in the water.

We, CSU_CHINA, is from Hunan Province. It is a sizeable rice-growing province and a significant producer of non-ferrous metals in central China, serious with cadmium pollution. How to use Synechocystis. To reduce the cadmium content in water through synthetic biology methods has become a problem for our team. The ultimate goal of our project is to use the transformed Synechocystis sp. PCC6803 in water contaminated by cadmium.

2.2 Improve the growth rate of Synechocystis in a high cadmium concentration environment.

The high cadmium concentration is extremely toxic to cells. It may cause the photosynthesis failure of Synechocystis, protein inactivation, or the production of high concentrations of ROS to induce cell death through cellular oxidative stress. The problem of growth in the cadmium concentration environment increases the growth rate of algae under cadmium ion stress.

2.3 Avoid environmental pollution caused by the escape of Synechocystis.

Since the flight of genetically engineered Synechocystis may cause pollution, we need to fix the Synechocystis in the calcium-alginate microbeads. To be environmentally friendly, the microorganisms are supposed to automatically die instead of multiplying and destroying the environment.

3.Achieve

3.1 Increase the absorption and tolerance rate of Synechocystis.

To achieve the principle of pollution-free and high adsorption rate, we considered using common cyanobacteria model algae—Synechocystis to meet our requirements. For ordinary single-celled organisms, cadmium is concentrated on the cell wall and absorbed into the cell via transporters. However, the excessive absorption of Cd leads to a series of biochemical imbalances in the cell. It also inhibits cyanobacteria’s photosynthesis, inactivates proteins or produces large amounts of ROS to induce cell death. It is difficult to achieve high absorption and high tolerance rate conditions, so we hope to design a cadmium removal engineering bacteria from the three functions of improving the transportation, chelation and redox function of Synechocystis. Select the transporter that is mainly responsible for absorbing cadmium ions in higher plants or yeasts. After transporting many cadmium ions into the cell, the chelating protein accumulates heavy metal ions. It prevents them from being transported out of the cell. Simultaneously, it increases the expression of oxidoreductase and eliminates reactive oxygen clusters in cells increases the growth rate of algae under cadmium ion stress.

3.2 Ensure algae will not pollute the environment.

We designed a “toxin-antitoxin” suicide system that can inhibit the expression of toxins under blue light. In other words, when the engineered algae are irradiated by blue light, it will usually survive; once the cyanobacteria escape from the preset environment and out of the blue light, they will begin to secrete toxins and die. Thus, the biosafety of algae can be guaranteed under the light-controlled “toxin-antitoxin” system.

3.3 Recycle the cadmium in the calcium-alginate microbeads.

We can deliver the calcium-alginate microbeads enriched with cadmium ions to the chemical plant processing and achieve the recycling of cadmium, thus turning the toxic heavy metal pollutants into precious resources.

4.Inspiration and Reference

4.1 How did we think of using Synechocystis to adsorb cadmium ions:

Biosorption is currently one of the most promising methods in treating heavy metal wastewater. As a biosorbent, cyanobacteria have broad development and application prospects. It can enrich metals, has high adsorption performance, is easy to survive in natural environments, can grow in extreme environments, and has strong stress resistance. The algae cell wall has a large surface area, viscous and negative charge. It can provide many functional groups such as hydroxyl, carboxyl, amino, amide, phosphate, etc. to bind to metal ions. The main component of the viscous glial sheath on cyanobacteria’s cell wall is acid mucopolysaccharide or pectin, which has a strong ability to adsorb small particles and metal ions. There are also metal ion binding sites in cyanobacteria cells, such as metallothionein and polyphosphates. This intracellular binding is usually related to the detoxification mechanism of algal cells. For example, cyanobacteria can induce metallothionein’s production to bind metals in a heavy metal environment. The number of polyphosphates will increase with a large number of negative charges on the surface. N. Rangsayatorn and other studies have shown that Spirulina platensis has strong tolerance and adsorption capacity to \({Cd}^{2+}\). It has high feasibility of treating wastewater containing a low concentration of \({Cd}^{2+}\). These studies have shown that cyanobacteria have good application potential in the recovery of precious metals. Synechocystis, one of the cyanobacteria, can accumulate cadmium ions well, is characteristic in good adsorption and stress resistance, and can produce metallothionein detoxification. Therefore we chose it as the bioadsorbent for cadmium ions. The entire sequence of Synechocystis sp. genome and plasmid has been tested. It can naturally transform foreign DNA and is easy to operate.

4.2 Why should we embed Synechocystis in calcium-alginate microbeads:

Studies have shown that immobilized algae adsorb heavy metals faster and more efficiently than suspended algae. Immobilization technology can make the biosorbent have ideal physical, chemical and mechanical properties (including particle size, porosity, impact resistance, etc.), making it suitable for filling various reactors and meeting the requirements of industrial applications. Carriers used for immobilization include calcium alginate, polysulfone, sodium polysilicate, epoxy resin, silica gel and agar. The choice of carrier has a more significant impact on the adsorption performance of immobilized cells. We used calcium alginate embedding for two purposes: the first is for fixation, another is for easy retrieval, easy placement, and relatively centralized placement.

4.3 Why do we use genetic engineering to transform Synechocystis:

Induce and extract heavy metal resistance genes through genetic engineering technology, fuse them into strains with short generations and strong adaptability to construct a project with high selectivity and high adsorption capacity. Strains are a new research topic in the field of biosorption. These genetically engineered bacteria have significantly enhanced tolerance to heavy metals. Their adsorption capacity and selective adsorption capacity have also been improved considerably. Cyanobacteria is an ideal receptor system for expressing foreign target genes. Since its cell structure and genetic characters are similar to Gram-negative bacteria, various molecular biological operations applicable to Escherichia coli are also relevant to cyanobacteria cells.

4.4 Advantages of using Synechocystis to recover cadmium ions:

In the presence of multiple heavy metal ions, the research and development of biosorbents that specifically absorb heavy metals in wastewater will directly lead to the significant recovery and reuse of heavy metal resources. Promoting role. With the development of genetic engineering, genetic engineering technology to construct microorganisms with high selectivity, high adsorption capacity, and increased tolerance will be a new development direction for biosorbents. Sewage plants usually use chemical and physical sedimentation, which will cause secondary pollution. Suspended particles in the water body have good adsorption performance for trace cadmium, which is also the primary mechanism of river water self-purification. Still, when the river water’s pH value is 7~8, the cadmium sediment may be resolved, causing secondary pollution. Therefore, we adopt a biological treatment method to completely recover the cadmium ions adsorbed by the alginate ball to prevent secondary decay.

4.5 How do we think of establishing a “toxin-antitoxin” suicide system:

When our team communicated with the XMU-China team, they said that their project AnTEA-Glyphosate designed a biological containment module. To prevent engineered bacteria from escaping affecting the environment, their team also developed a series of “suicide switches.” So our team was much inspired and thought the toxin-antitoxin system was fascinating. After consulting a lot of literature, we decided to use RelB as an antitoxin and RelE as a toxin. And we use the blue light control system because it is prevalent, and there is a lot of research to refer to. It is relatively easy to implement and build a similar system to solve the problem of Synechocystis escape.