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Soil, one of the most common and valuable resources on this planet, is the great parent of all. It gives birth to all plants and carries the hope of a bumper harvest. In Chinese culture particularly, soil stands for the origin of human beings and a paean to labor.
As students from China Agricultural University, we always pay special attention to soil, the mother of agriculture and Chinese cultivation culture. However, we must accept the fact that our soil has been going through serious degradation for decades, and soil salinization is one of the major threats.
How serious can the saline-alkaline soil problem be? We have been continuously doing surveys since we noticed this topic, and below are some results:
To get a full view of this problem and what we can do further, we also did research in what have been done before by participating in related seminars.
The most valuable one was an online plant nutrition seminar organized by the Key Laboratory of Plant-Soil Interactions, MOE, National Academy of Agriculture Green Development. Prof. Liu Xiaojing from Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, gave a presentation on improvement and utilization of saline-Alkaline soil. Prof. Tibor Toth, from Institute for Soil Sciences and Agricultural Chemistry, Centre for Agricultural Research, Hungarian Academy of Sciences, gave a presentation on management of salt-affected soils.
Thanks to the report, we have a deeper understanding of soil problems. We learned that irrigative, agricultural, chemical and biological measures are four main approaches to this problem nowadays. According to professors, agricultural and chemical measures are often not effective enough in the long run, and irrigative measure leads to high costs, apparently not suitable for developing countries. Besides, these three measures have bad performance on dealing with secondary salinization and seawater intrusion.
Both Prof. Liu Xiaojing and Prof. Tibor Toth recommended biological measures as the final solution, for it is eco-friendly, safe and efficient. Particularly, Prof. Tibor Toth attached great importance to microbial engineering in soil restoration, naming some previous research in bacteria methods that inspired us to try synthetic biology later.
Fig. 1 Prof. Tibor Toth sharing his idea of microbial engineering in the seminar.
To better understand the soil issue and grasp the core values of our project, we get in touch with people involved in soil salinization to absorb their opinions and expectations.
Fig. 2 Visit the History Museum of CAU
Fig. 3 Statues Related to the Spirit of Qu Zhou(Mr.Xin Dehui, a respectable professor from CAU who devoted himself into soil restoration project)
Synthetic biology project is not just a party for the minority with scientific background. Instead, as an environment project directly interacting with the society, we need to know how the public, especially the citizens, recognize the threat of saline-alkaline soil and how they feel about synthetic biology as a solution to environmental problems.
We sent out questionnaire and collected 60 valid results, 24 results from men, 33 results from women and 2 results from other genders.
Fig. 4 Questionnaire survey results
From the results we can know that 67% of the respondents agree to the urgency of improving the saline-alkaline land. However, only a few respondents say they know the problem of saline-alkaline soil well and thoroughly. Approximately 75% of the respondents say they’ve heard of soil salinization more or less before but never get to know the details. These results suggest us that the ultimate goal of our project is accepted and approved of by the public. Meanwhile, we should also take actions in public education on details about soil salinization.
We also asked the respondents on attitudes towards synthetic biology implementation. 81% say they know clearly or at least have heard of synthetic biology. However, only 57% show approval of its implementation in Agriculture & Food industry. If synthetic biology is used to improve the environment, people showed more tolerance, with 78% respondents approve of such an application. Nevertheless, they still express concerns on its influence in ecosystems and food chain.
As a project actually between the agriculture track and the environment track, CAU_China values these objections and approvals seriously. As a matter of fact, these worries warn us about the necessity of an effective kill switch and an overall risk management method, which has been fully described in Design and Safety.
Based on background research, we concluded and prioritized values for our project to achieve:
You can go to Project, Implementation, and Education wiki pages to see our efforts to achieve those values.
As undergraduates, we knew clearly that the first version of our project design definitely had flaws and unrealistic imagination. To polish our project design 1.0 into a simple and realistic one, we have consulted many experts in different fields to exchange thoughts and actively seek their help in design and experiments.
Special thanks to all the experts who had offered suggestions:
Professor Yang Jinshui informed us that transforming too many genes into the bacteria may put burden on their growth, which is not a good thing particularly when salt and alkali stress already exist. There would be plasmid incompatibility as well. Also, he pointed out that the symbiotic system in the first version of our design didn’t seem practical, because Bacillus subtilis and Corynebacterium glutamicum are not known mutually beneficial symbiotic relationship in soil, and if they are isolated in space, the treatment efficiency will be greatly reduced.
Professor Tian Changfu, an expert in rhizobia , suggested conducting relevant research in interaction between bacteria and plants’ roots in the soil, in order to help our bacteria proliferate. Secondly, we learned from him that using our bacteria through drip irrigation or spraying may lead to better restoration efficiency. More importantly, Professor Tian pointed out that if the bacteria will be put into the soil, its plasmid will soon be lost since there is no resistance stress for screening.
Professor Guo Yan told us that the restoration process of saline-alkaline land is very complicated and it is hard to finish the restoration with just one method. Therefore, he suggested using a series of comprehensive treatments. Besides, Professor Guo emphasized the importance of biosafety and suggested that we should test the efficiency of our toxin gene and kill switch periodically.
Professor Feng Gu introduced that the lack of water resources is the bottleneck of restoring saline-alkaline land and explained the toxic mechanism of saline-alkaline soil to plants. In addition, he suggested that in order to improve the survival rate of bacterial products in soil, we can consider their interaction with plants and colonize them in plant roots.
In the first version of our design, we planned to build a symbiotic system, in which B. subtilis can produce γ-PGA with the glutamic acid provided by C. glutamicum. After discussing with Professor Yang, we greatly changed our design into the version you see now, in which C. glutamicum has the ability to synthesize γ-PGA itself, in order to avoid inefficiency of the symbiotic system. What’s more, with advice from Professor Tian, we decided to insert every final genetic circuit into the genome of the bacteria so that they won’t be lost due to a lack of screening in natural environment. In addition, with advice from Professor Guo, we added a composite kill switch to our bacteria that can respond to salinity and pH changes. Finally, since professors all mentioned a combination with other soil restoring methods, we decided to focus on the interaction between bacteria and plants’ root system in the second stage of our project in the future.
Having decided on the final design, we went on to have in-depth discussions with experts about our end products and future plans after iGEM. To ensure that our bacteria is applicable in the real environment, we need to find a carrier for bacteria and a possible way to turn into products. Our preliminary choice was the bacterial agent, so we focused on the advantages and feasibility of transforming our engineered bacteria into bacterial agents in this stage of interviews.
Fig. 5 Discussion with Professor Feng Gu
Fig. 6 Interview with Professor Zhou Wenkun
Hearing that we planned to further include interaction with plants in our project, Professor Zhou Wenkun recommended Thellungiella salsuginea , which is a well-used model plant in salt stress research. It grows fast and can handle some basic environmental stress well, which is suitable for the comprehensive treatment of saline-alkaline soil.
Professor Xu Li let us pay attention to some details during the actual implementation of bacterial agents. It mainly lies in the secondary salinization of soil and the setbacks that viscosity of polyglutamic acid brings in the treatment of saline alkali soil.
On inspirations from Professor Xu, we further contacted Professor Li Huixin for more information on bacterial agents. He explained that using the bacterial agents instead of γ-polyglutamic acid is definitely a better choice, since the living bacteria can migrate in the soil and synthesize γ-PGA continuously, while using γ-PGA directly is more like a temporary solution and will cost a lot. Moreover, he informed us that measuring the salinity and alkalinity in soil in situ is indeed hard, and we have to do some preprocessing and allow small errors, which cleared up our confusion in the test paper we planned to design.
After so many interviews, we finally decided that our end product will be a kind of bacterial agent which contains our engineering bacteria and essential nutrients for growth. Also, reminded by professors about the production of bacterial agents, we decided to interact with related companies to know about the actual commercialization process. With advice from Professor Zhou, we planned to use Thellungiella salsuginea as our materials in experiments of the interaction between bacteria and plants in the future. Also, having included plant research in the future of our project, we will consult more experts in saline-alkaline tolerant plants and further develop our project into a comprehensive one.
Though we had consulted many experts from colleges, we found our implementation still seemed impractical without detailed advice from the market and enterprises. Therefore, in order to further understand the selection of bacterial agents and actual production process, we interacted with companies in this area to get across the possible future and limitations of our project at this stage.
Fig. 7 Visit the company Sino Green Agri-Biotech
We got in touch with Sino Green Agri-Biotech, a company focuses on research and production of several bacterial agents in the agriculture market. Led by the manager Zhang Lixia, we visited the company's factories and laboratories to learn more about the operation of the bacterial agent company and the production process of related agents. To know more about bacterial agent as product, we consulted her about the feasibility of our engineered bacteria to be turned into real products, possible problems and restrictions in laws. She provided ideas for our bacterial agent design.
Fig. 8 Visiting the factories
After understanding the production process, we wanted to know more about the domestic market of microbial agents, in order to analyze the potential market value of our product and fix it an appropriate price. So we found AGB( an agricultural microbial agent company) in Shenzhen and invited their manager Kong Zongyao to communicate with us online in WeChat. During the communication, we asked about the situation of agricultural microbial agents and possible competitors to our product.
Fig. 9 Interacting with AGB
Through the conversation with Manager Zhang Lixia, we learned that for different environments, there are mainly 4 forms of bacterial agents for us to choose from.
Liquid form is suitable for sprinkler irrigation and drip irrigation. The powder form is insoluble in water for the peat and vermiculite in it. It is feasible to apply the bacterial in powder form in a place where wind and sand is not large, such as a greenhouse, and apply it in a hole or ditch. In places with heavy sandstorm, dusting powder can easily be blown away by the wind, so a granular dosage form is developed, which makes it easier to enter the soil and mix it with chemical fertilizers. There is also a new form of bacterial agent called freeze-dried powder that is easy to store and use, at the cost of high price of course.
During the interview with Manager Kong, we learned that daily field management is another aspect that affects the effect of bacterial agent product. Saline-alkaline soil treatment should be integrated with the local situation, through a combination of fertilizer application, planting crops and other means of treatment. For the recipe of bacterial agents, their company's products are currently pure agents without synergists.
When it come to the market, manager Kong said that growers and farmers have got to know the advantage of bacterial agents and are willing to buy their products to solve the problem of low quality or low yield. However, the present market is in disorder and mistrust, since many manufacturers add synergists into their products and the customers can’t do the quality judgement themselves.
We responded to our interactions with companies by making changes and supplements to our future implementation plans. Originally we had been considering to develop our product in the liquid form, the most common form of bacterial agents. After the discussion with manager Zhang, we now think that the granular form may be another choice for us, since the most serious regions affected by soil salinization in the northwest of China are generally windy and sandy, where granular form performs the best. We plan to arrange some wet lab experiments to test the performance of granular bacterial agents in the future.
Besides, learning the situation of the bacterial agent market, we plan to focus more on engaging with possible end users and public education towards them specifically, in order to earn their trusts, show respects, and lay a foundation for our implementation.
Special thanks to Sino Green Agri-Biotech and AGB for their help and guidance!
Fig. 10 Companies’ Logo
To close the loop between what was designed and what was desired, above all we did surveys and questionnaire towards the public to fully understand what the society expected from our project. More importantly, we get engaged with those involved and prioritized the values accordingly.
Next, we consulted many experts and collaborated with other iGEM teams in troubleshooting to ensure a better project design to satisfy people’s needs, and make adjustment in our design and experiments accordingly.
Following that, we continued to consider the execution and implementation of our project. We did public education and communication work, in which we presented our project, to collect public attitudes towards it. Knowing that the society approves of our project, we went on interviewing related experts for a practical product design. Besides, hearing that the society held worries towards the safety issues of bio-products, we emphasized our safety and security work.
Then, we interacted with related companies to further understand the selection of bacterial agents, actual production process, and the market conditions. With new information learned, we decided that our bacterial agent product would be in various forms to satisfy users’ needs, and we simulated different implementation schemes with the help of Cellular Automaton Model, to present to the public that our future product is able to live up to their expectations.
In short, we responded to what the society desired in our project by upgrading project design with experts’ advice, carrying out public communication frequently to collect public opinions, and polishing our implementation plans with help from companies and mathematic models.
Fig. 11 How we close the loop