Team:NAU-CHINA/Implementation
End Users：Agricultural Producer
In the process of agricultural production, lead pollution in the soil enters the human body through the way of "soil → crop → human", posing a threat to human health. And lead pollution also causes damage to the agricultural economy. In this process, agricultural producers are closely related to the cultivation of soil and the production of crops. The main reasons we choose agricultural producers as end users are as follows.
Firstly, remediation of heavy metal contaminated soil may provide direct benefits to agricultural producers. Agricultural producers are users of soil and producers of crops. Lead pollution in cultivated soil enters the human body through the enrichment of crops and poses a serious threat to health. It is known that lead pollution is one of the important causes of disease. According to a large domestic epidemiological survey in 2015, the average blood lead level(BLL) of urban children under the age of 6 in China was 35.35μg/L, higher than that of 20μg/L～25μg/L in the US in 2000 and less than that of 30μg/L in Europe. If we can repair the lead pollution, this will directly benefit farmers.
Secondly, remediation of heavy metal contaminated soil may help farmers in China recover their economic loss caused by heavy metal pollution. It is reported that in China, 12 million tons of vegetables and crops are polluted by heavy metals annually, causing an economical loss of 3 billion dollars. The solution of soil problems would save the vegetables and crops, and in turn bring income to farmers.
Therefore, for the sake of the general public and agricultural economy, we choose agricultural producers as our end users.
How to use
We envision that others can form a closed loop between laboratories, biotechnology companies, and farms when using our approach devised in this project.
After obtaining the engineered bacteria in the laboratory, we will register them for patents and sell them to biotechnology companies and provide certain technical support.
The biotechnology companies will carry out further screening, expansion and freeze-drying preservation. They will wash the artificially cultivated earthworms and put them in a turnover box with moist absorbent paper, followed by adding the freeze-dried powder of engineered bacteria. After one day, they will wash and sell the earthworms to the farmers, while providing a certain assessment of heavy metal pollution in farmland.
Before planting, farmers inoculate earthworms in the cultivated land and supplement the inoculation according to the changes in the number of earthworms. At the same time, farmers will provide some real application feedback for the laboratory to make technical improvements.
As for the number of inoculated earthworms, we simulated the relationship between different inoculation amounts and effects through Cellular Automata model. So far, 7500 earthworms were put into each pile at an interval of 10 meters.
Further Development
In addition to traditional applications, we also envision the further development of the project. We expect that our project can be combined with vermicomposting and develop a multi-functional and sustainable application to better solve certain environmental problems.
- Used in vermicomposting
- Multi-functional application
- Sustainable application
Vermicompost is the product of the decomposition process using various species of worms to create a mixture of decomposing vegetable or food waste, bedding materials, and vermicast.
Earthworms eat a lot of manure and take advantage of the synergy of intestinal microbes. They can efficiently transform organic matter and accelerate fertilizer maturity. However, the lead in “farm manure” is also an important source of lead pollution on farmland. If earthworms inoculated with engineered bacteria are applied to vermicomposting, they will greatly reduce the lead pollution in fertilizers entering the soil and avoid the use of chemical fertilizers, which will promote the development of vermicomposting and contribute to the sustainable agriculture.
After the engineered bacteria have made progress in the treatment of lead pollution, we will design other recombinase to obtain free dissociative sulfur and phosphate groups to deal with other heavy metal pollution.
After fully verifying the safety and security of the project, we plan to release the engineered bacteria directly into the soil environment to ensure their roles in every generation, which will greatly reduce the cost of project implementation.
Applications in the Real World
When it comes to the applications in the real world, we believe that the cultivated land in southern China is the most suitable agricultural land that meets our expectations.
The lead concentration in polluted southern cultivated land is mostly in the range between the screened value and the controlled value, according to the soil risk control standards for agricultural land issued by the Chinese government in 2018. In the national soil risk control standards, the quality and safety of edible agricultural products were taken to set the screening value and control value of soil pollution risk in agricultural land. When the lead content of cultivated land exceeds the screening value, the quality and safety of agricultural products may be at risk, while the risk is higher when the lead content exceeds the control value.
Pb Concentration
The lead content of the cultivated land we selected is between the risk screening value and the risk control value, which determines that the target land still has production efficiency and will not harm the engineered bacteria carrier Eisenia fetida.
The traditional method of physical and chemical lead fixation combined with phytoremediation will adversely affect the production efficiency. In contrast, our project hopes to treat the lead pollution while maintaining the production efficiency of cultivated land.
In southern China the engineered bacteria carrier—Eisenia fetida is commonly used as one of vermicomposting species in agricultural production. It shows good heavy metal tolerance in heavy metal contaminated sites. With carefully selected lead content, our earthworms will be able to ensure their normal growth while tackling with lead contamination at the same time.
Safety
Due to the isolation during the epidemic, we cannot enter the laboratory for experiments this year, but safety work is always of first priority. We studied lectures and regulations related to safety and ethics to improve the safety awareness of our team members.
Security of project design
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Overall biosafety
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Gene safety
Our project envisages using Bacillus subtilis WB800N as the chassis organism, Bacillus subtilis YCD as the source of phytase, and Eisenia fetida as the engineering bacteria carrier. Bacillus subtilis is a safe and commonly used strain in laboratory, and usually does not cause obvious risks to human health, the community or the environment. Eisenia fetida is a widespread species in China and is widely used as a compost species in southern China. The common strain and widespread species can guarantee the safety of our project.
We designed a kill switch in the gene pathway to trigger suicide when the engineered bacteria enter the natural environment, avoiding the risk of gene drift to the greatest extent.
Implement security
When we envision the project to function in the real soil environment, we will follow the principle of gradual evaluation, and gradually increase the release scale.
After obtaining the earthworms containing engineered bacteria, we envisage building an ecological box similar to the soil environment of the target cultivated land. We will apply earthworms in the ecological box, and conduct ecological evaluation after a period of time, mainly considering the quantity of soil organisms and whether there are living engineered bacteria in the soil, etc.
After ensuring the safety of the application in the ecological box, we will conduct a release evaluation in the test field of Nanjing Agricultural University located in the Baima Experimental Base of Nanjing. We will design three different release scales, namely, 100 bars/m2, 130 bars/m2, and 160 bars/m2 to gradually expand the release scale. If other soil biomass is found being abnormally reduced or the number of engineered bacteria detected in the soil environment exceeds the error range, we will immediately stop the release and improve the pathway.
In short, we will evaluate each step of the implementation and rationally optimize the implementation plan.
Laboratory safety
The isolation during the epidemic prevent us from entering the laboratory for experiments this year, but we still have received laboratory safety training and passed the laboratory safety exam. We are familiar with laboratory safety operations and emergency measures.
The followings are our supplementary materials to ensure laboratory safety：
Supplementary MaterialsChallenge
How to ensure the vitality of earthworms and engineered bacteria?
Soil animals are sensitive to the toxicity of pesticides. Long-term use of pesticides has a great impact on the types, quantity, flora and respiratory intensity of soil animals. Relevant studies have shown that pesticide pollution will lead to the decline of soil microbial functional diversity, and also reduce the use of carbon substrates by microorganisms. Therefore, the use of pesticides and chemical fertilizers on farmland may cause problems, for example, low vitality of earthworms or engineered bacteria.
However, the development of vermicomposting technology can improve the utilization efficiency of nutrients in organic fertilizer by plants. Therefore, if follow-up research can give full play to the synergistic effects of vermicomposting and beneficial microorganisms, the use of pesticides and chemical fertilizers will be greatly reduced.
Wrong suicide
When freeze-dried powder of our engineered bacteria are fed to earthworms in incubator, they may recover without entering the intestine of earthworm, which will lead to unexpected suicide of the engineered bacteria. This kind of death of bacteria will cause some economical loss.
There are two aspects we can explore. One is improving earthworms’ feeding strategy, which is suitable for growth of earthworms as well as not suitable for freeze-dried powder of bacteria to recover. Another is looking for one kind of anaerobionts as our engineered bacteria which cannot survive outside the intestine of earthworm.
With more and more efforts, we can solve this problem and realize our ideal function in the future.
References
- [1] Zhang Weixin, Shen Zhifeng, Shao Yuanhu, et al. Research progress in soil biology and sustainable agriculture[J]. Acta Ecologica Sinica, 2020, v.40(10):33-56.
- [2] Xu Qin. Overview of the geographical distribution of terrestrial earthworms in China[J]. Journal of Beijing Institute of Education, 1996(03):54-61.
- [3] Zhang Xiuying, Chen Dongmei, Zhong Taiyang, et al. Evaluation of Lead in Arable Soils, China[J]. CLEAN - Soil, Air, Water, 2015, 43(8):1232-1240.