Team:NAU-CHINA/Implementation
IMPLEMENTATION
OVERVIEW
The Application
Concerning Safety
Challenges to Conquer
OVERVIEW
Our Project
Our project is aiming to solve the problem of uneven special distribution of selenium. We engineered E.coli BL21 which expressed sefA and ssuE in a low intensity by virtue of synthetic biology strategies. We have validated that selenite can be metabolized into SeNPs, indicating that our strategy could work well in lab-scale condition.
Our Final Product
We lysed the bacteria and centrifugated to purify SeNPs. The purified SeNPs will be lyophilized into powder, which became the main body of our final product.
Apart from being used as fertilizer to promote agricultural industries, SeNPs are gaining importance in the electronics and optics industries because of their unique properties in photoelectricity, semiconductive theory, catalytic activities etc. SeNPs also possess excellent antioxidant, anticancer, and therapeutic properties, making them particularly valuable in pharmaceutical and biomedical applications.
End Users
Therefore, from a biotechnological standpoint, the microbial biotransformation of selenium could be used for:
(1) the remediation of industrial effluents rich in Se.
(2) the synthesis of biomaterials with industrial applications such as fertilizers and health products.
So we imagined that our project end users are industrial producers and farmers.
The Application of Our Project in the Real World
For Industry
It is suggested that microbiological selenium detoxification may be the simplest and most economical approach. In order to put our engineering bacteria into use, according to the literature, we had also designed a supporting pilot-scale biological reactor planned to be established downstream of mines or smelting plants.
Fig.1 the pilot-scale bioreactor for selenium recycle along rivers
The anatomy of the bioreactor is shown in the Figure 1. The reactor consisted of four medium-packed tanks connected by grade SDR 41 polyvinyl chloride (PVC) pipe. Each tank was sealed with two PVC caps where the gas vent valves were installed. Tanks 1 and 2 were filled with Tri-Packs produced by Kelong Petrochemical Equipment Packing Co., LTD in Pingxiang Jiangxi province. Tanks 3 and 4 also contained silica sand to provide as much surface area as possible for the growth of bacteria.
Tri-Packs could provide maximum surface contact between gas and the scrubbing liquid by facilitating continuous formation of droplets throughout the packed bed which results in high scrubbing efficiency, and minimizes packing depth. Flow through the system would be determined according to the real situation. The upstream feed pump is connected to selenium-enriched sewage in which the heavy metals have been pre-adsorbed and glacial acetic acid as carbon source & electron donor, the two would pass through a static inline mixing valve and then blended into the reaction tank for the production of SeNps.
To inoculate our engineered bacteria into this system, initially, our engineered bacteria would be harvested, resuspended in 50% glycerol and refrigerated in -20℃ until they were used. Then, the cells were thawed and divided into four equal portions that were poured into tanks 1 to 4 through inoculation ports located at the top of each tank.
In our envisagement, every monthly incubation, we will shut down the system temporally and extract the SeNPs produced our engineered bacteria from Tri-Packs. After cleaning and refilling the tanks, we will reboot the system and put into the production again.
After the extraction and lyophilization of SeNPs, we will conduct characteristic measurement and live bacteria detection. After confirming that there is no risk of live bacteria contamination and genetic drift, we will mix with the fertilizer produced developed by our University to make Se fertilizer applied to promote soil improvement and plant growth in selenium-deficient areas.
For Farmers
The SeNPs fertilizer is the easiest application in the real world. SeNPs fertilizer can be used into foliar fertilizer and soil fertilizer. To test SeNPs as a fertilizer in the real world, we tested our hypothesis by cultivating alfalfa in the lab. We added different concentrations of SeNPs into the nutrient solution of alfalfa culture, and found that the addition of 0.5g/L of SeNPs has excellent promoting effect on the growth of alfalfa (more details in Proof of Concept). Our experiment proves that SeNPs can be used as fertilizer in the real world.
Concerning Safety
We fully complied with iGEM safety policy during the whole experiment period. We didn't release any of our engineered bacteria into the environment. All validation was done in the laboratory.
In the implementation section, we firstly looked back on the protocols during our experiments. In our plan, the pilot-scale bioreactor will be just like the prototype in the lab. The majority of our germs will be killed after having accomplished their mission due to the extraction of SeNPs. As to the leak of the minorities, evidence founded that if they leak into the water without pre-treatment, the heavy metal contents in the area will adversely damage the reproduction of our germ and leads to the extinction.
For the final product extraction, lysozyme was added and UV sterilization was carried out. But small amounts of DNA remain, which is harmless compare with genetic drift on a natural scale.
Challenges to Conquer
The main challenges are on the application of pilot-scale bioreactor:
1. How to ensure the heavy metal content of the water entering the system is within the range where bacteria can survive?
We cannot let the polluted water enter the system without any process, otherwise, our engineered bacteria certainly will die. Luckily, Some new polymer materials have the property of adsorbing heavy metal ions. We are planning to build another tank for ions absorption before the contaminated water directly enter the main body. With the simple pretreatment procedure, the system will be more vital and efficient.
2. How to determine the calibre of connection pipes and the suitable lift of pumps?
To build such a bioreactor, data about the flow rate of the Fuyang River, the height between land and water etc. are also required. These all involve the application of chemical principles, requiring some help from professionals.
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