Section 1 : Set the project content

1.1 Determine the orientation of our project

Rare earth elements are one of the most important resources in the world. They can be widely used in civil and military fields. Rare earth elements are also indispensable in high-tech industries such as equipment manufacturing, new energy and emerging industries. As strategic resources, they are valuable and crucial. In 2017, our team designed REEBOT to treat acidic industrial wastewater using methods of synthetic biology and extract rare earth elements from it. Since then, we have continued to focus on wastewater treatment technologies in the rare earth industry and do more research in this area.

After further study, we learned that the global total proven rare earth quantity is about 120 million tons, and the annual production is about 210,000 tons. According to statistics, in 2012, China mined 76,000 tons of rare earth and produced more than 20 million tons of drainage. Based on this, it is estimated that it takes an average of 263 tons of water to mine one ton of rare earths. According to relevant laws and actual sample data, we estimated that the total amount of rare earth elements in the world's annual rare earth wastewater is 21×263×10⁷×0.05g=2760T (in which the rare earth mining wastewater contains about 0.05g of rare earth elements per liter). This will undoubtedly lead to the impact on the environment and the waste of resources. Through the questionnaire survey, we collected the public's understanding of rare earth wastewater, and further confirmed the importance of our project. In order to make the technology more environmental friendly and efficient, our team designed REE Miner.

Investigate the specific problems of diposing industrial drainage

After online survey and enterprise visiting, we learned that the mainstream method of drainage treatment is method of chemical precipitation: the rare earth elements can be collected in the form of compounds by adding chemical reagents in drainage through cooling and precipitation. At present, the mature processing technologies in the industry mainly include: neutralization precipitation method, sulfide precipitation method, and heavy metal capture (chelating) agent treatment method. However, the current methods still need to be improved. First of all, rare earth elements in wastewater cannot be fully recycled, resulting in economic and environmental loss. Secondly, the current treatment methods need to add a large number of chemical reagents, which cannot achieve zero emission under the existing standards. Prolonged exposure to such environments may affect the health status of staff members.

Section 2 : Synthetic Biology Design

In order to make the engineering bacteria solve the problems effectively, we would focus on the following three aspects.(1) How to make engineering bacteria adsorb REEs with high selectivity in acidic wastewater; (2) How to increase the efficiency of engineering bacteria adsorbing REEs; (3) How to fix cells in a piece of area to facilitate subsequent recovery.

2.1 Consult the authoritative experts

On the basis of field research, we collected a large number of documents and visited scholars in relevant fields to get some guidance.

According to the project of our team in 2017, it was confirmed that LBT protein expressed in E.coli had REEs binding properties.It is known from literature review that dLBT protein has two REEs binding sites, so dLBT is also used to adsorb REEs in this year's project. In addition, we checked the current paper in related fields. Recent work has demonstrated the presence of specifically Ln(III)-dependent MDHs (Ln-MDHs) in several organisms, from which they identified a previously uncharacterized Ln(III)-binding protein——Lanmodulin（LanM）. It contains four metal coordination motifs (EF hands), three among which have Ln(III) binding properties. Moreover, the gene of this protein has been successfully introduced into E. coli for expression. So in this year's project, we will design a fusion protein containing LanM to improve the adsorption efficiency of REEs.

Yunjun Yan, professor of College of life science and technology in HUST, having high attainments in broad-spectrum biosorption technology. He affirmed our preliminary idea and said that microorganisms have been well acknowledged as biosorbents for the adsorption of heavy metals. He has also studied both dLBT and LanM. He mentioned that LanM possesses not only a high selectivity for REEs over other common metal cations, but excellent suitability under acidic conditions(pH=3). When it comes to the methods of microorganism incubation, he pointed out that among various immobilization techniques, packed-bed column system is a suitable option. By employing this technique, continuous adsorption and recovery can be automatically performed. He also reminded us what details should be taken into consideration the cells which need to be fixed should be those that have absorbed REEs. Not all the cells should be fixed. A suitable induction system can be designed to solve this problem.

Guofan Zhang, professor of School of Resource Processing and Bioengineering in Central South University, having high attainments in rare earth mineral processing. We learned from him that the rare earth mineral processing process, as well as the causes of drainage. The drainage is produced during the chemical precipitation of rare earth ions, which is weakly acidic and contains many ammonia and nitrogen compounds. Some rare earth mines use microorganisms to remove ammonia nitrogen from the drainage and then discharge it after sterilization. This confirms the feasibility and safety of our project.

2.2 Communicate with the enterprise to substantiate the necessity of our project

In order to make our project more applicable in actual production, the team members of HUST-China visited several enterprises to conduct research. We went to Ganzhou, Jiangxi Province, known as the kingdom of rare earth, and visited the China South Rare Earth Group to learn how rare earth elements are recycled in practice. Team members also visited Lincang city, Yunnan province, visited LinXiang huazhong university of science and technology institute of science and technology innovation, to get hold of rare earth condition in Yunnan province.

Section 3 : Evaluation,verification,and optimization of our project

3.1 Make a return visit to the relevant enterprise

We took our design to Ganzhou Rare Earth Youli Technology Co. Ltd which is mainly responsible for treating the wastewater generated from the rare earth production process, and communicated with the engineers there to improve our solution. We conducted on-site inspections to further optimize and improve the hardware design.

The technicians put forward a new problem. Rare earth ions need to be combined with PmrB protein during induction, and these REEs cannot be trapped by LanM or dLBT, which reduces the recovery efficiency. Perhaps additional ion activation could be added to increase the enrichment of REEs. We further learned that mining wastewater often contains Cu²⁺, so we thought that we could design a pathway activated by Cu²⁺.

Then, we continued to search for the information and found the GolS induction system, which is a negative control induction system. In the absence of copper ions or gold ions, the repressor protein GolS represses the expression of downstream genes. When copper ions or gold ions bind to the repressor protein, it interferes with its repression effect, and the downstream genes can be expressed smoothly. And this system also has a precedent for successful expression in E. coli. So we replaced the pmrCAB system with the GolS system, and designed two additional pathways driven by copper ions.

4.2 Determine the hardware design

Engineer proposed that in order to achieve a high adsorption rate of metal ions, we could increase the contact times between the engineering bacteria and the wastewater. Considering the fact that the engineering bacteria can be adsorbed on SiO₂ , the hardware part of our design is shown as follows:

We constructed a quartz sand adsorption column, mixed the quartz sand with sodium carbonate, and put them in layers. This allows us to distribute the quartz sand density flexibly while adjusting pH. By stratifying the adsorption, we can also validate our flow absorption model.

Return visit after preliminary experimental results

We also came to a rare earth company in Changting County, Longyan, Fujian Province, to conduct enterprise research. In addition to figuring out the existing rare earth element mining process and future development trends, we also collected 5L mother liquor and 10L waste liquor after industrial treatment as raw materials for the team experiment.