Part One: Discovery and Analysis of the Problem

Brain Storm

None Last year, at the beginning of our journey, we put our heads together for a long time, and invited some students for help. Others with a cross-disciplinary background at NPU, Mr. Geng Tian, Mr. Jindou Li, Mr. Qijing Lai, Mr. Wu Gao and Mr. Di Wang gave us some ideas as follows.

In the process of communication with them, we broadened our scope of mind, and after several plenary meetings of our team, we paid more attention on persistent organic pollutants (POPs) when choosing the topic.

Figure 1: Brainstorming with other people at the beginning of our journey

Information Retrieval

It was important for us to learn about the current situation when determining the project topic. We searched public materials and analyzed some significant issues related to POPs.

On one hand, the Stockholm Convention is a global treaty that aims to protect human health and the environment from the effects of persistent organic pollutants (POPs). The Convention entered into force on May 17, 2004. The current scope of the Stockholm Convention is 30 kinds of POPs, which are pesticides, industrial chemicals and/or byproducts. Any Party may submit a proposal for listing a new chemical to be listed in Annexes A, B and C to the Convention. The scientific subsidiary body, the POPs Review Committee, evaluates the proposals and makes recommendations to the Conference of the Parties on such listing. At its ninth meeting in 2019, the COP to the Stockholm Convention decided to eliminate one pesticide and one group of toxic chemicals, which together total about 4000 chemicals, listed in Annex A to the Stockholm Convention, namely dicofol and perfluorooctanoic acid (PFOA) and its salts and PFOA-related compounds.

The U.S. Department of Health and Human Services (HHS) announced the Fifteenth Report on Carcinogens on December 21st, 2021. Some substances such as benzene, benzidine, cyclophosphamide, cyclosporin A, are known to be human carcinogens.

On the other hand, POPs are essential for various applications such as flame retardants, fire suppressants, heat transfer agents, surfactants, cosmetics and pesticides due to their less reactivity and stability. POPs are referred to as forever organic compounds due to their resistance to degradation via physical, chemical, or biological methods. POPs are primarily found in dust and air, and they enter the human body by ingestion or inhalation and leave toxicity there. Polyaromatic hydrocarbons (PAHs), a broad class of POPs, are widely available as they can be released into the environment by nature or by human activities. Bio-combustion, such as the combustion of forest and grassland, is the main source of PAHs. In addition, the emission of PAHs can be also caused by the combustion of gasoline, coal and industrial waste.

PAHs can exert toxicity in humans through the mechanisms of DNA adducts and the formation of reactive oxygen species, and their adverse health effects are usually proportional to their molecular weights. Some PAHs, including naphthalene and phenanthrene metabolites, were observed in the urine samples of children aged between 6 and 11 years which decrease lung function and cause asthma.

Based on these findings and global public policy, at one plenary meeting of our team, we made a decision on PAHs as the topic of our project this year.

Meeting with Dr. Ming Li

To understand the specific PAHs organic pollutants at this stage, we contacted Dr.Ming Li , a professor at the College of Natural Resources and Environment, Northwest A&F University. Dr. Ming Li mainly studies algae and the environment of rivers and lakes. We interviewed him about the harm of organic pollutants to water basins and the treatment plan. He affirmed the significance of our project, and we also summarized the points that need special attention from the discussion with him.

1. We need to have complete data model results.

2. We need to consider the survival of the strain itself.

3. We need to pay attention to whether the strains designed by genetic engineering have good genetic stability.

Figure 2: Online meeting with Dr. Ming Li

Dr. Ming Li, male, doctoral supervisor, conducts research in watershed ecology and aquatic ecology and their intersections. His research interests include: (1) eutrophication and cyanobacterial bloom prevention and control (2) Algal ecology and algal biotechnology (3) Traceability of non-point source pollution and health assessment of rivers and lakes (4) Ecotoxicology (5) Migration and transformation of biomass elements (carbon, nitrogen and phosphorus).

The detailed interview transcript is as follows.

Q: What is the current situation of organic pollutants in water? How does it affect ecological waters?

A: At present, organic pollutants exist in almost all water areas. Although the concentrations of each type of organic pollutant are small, the effects on ecosystems have not yet been felt. However, this potential risk and the toxicity risk of the combination of multiple organic pollutants are still large. For example, some environmental concentrations of triclosan, although only a few micrograms per liter, have been shown to be toxic to algae.

Q: What is the impact of organic pollution in water on human life and the ecosystem?

A: On the one hand, organic pollutants in water will be enriched through the food chain, especially into a variety of aquatic products, and there will be health risks after being eaten by human beings. On the other hand, organic pollutants such as antibiotics in water will change the production and distribution of resistance genes in the environment, especially increase the presence of resistance genes in the environment, which will reduce the effectiveness of antibiotics in the future and indirectly harm human health. In addition, organic pollutants in waters may have an impact on aquatic biomes and harm the ecological health of waters.

Q: What are widely used treatment methods for pollution problems?

A: At present, we do not have very effective and extensive treatment methods for organic pollutants. The treatment rate of some organic pollutants in sewage treatment systems is not high. To address the problem of organic pollutants pollution, we must first discharge them from the source and strictly control and reduce the discharge of such pollutants. At the same time, we should increase the supervision of organic pollutants in the sewage treatment system, and carry out technological transformation to improve the removal efficiency of organic pollutants in the sewage treatment system.

Q: What are the conditions or final results of the current ideal solution to the pollution problem?

A: The ideal solution is that the organic pollutants produced by human beings, where they are used, how much they are treated, and how much they are discharged into the environment can have a complete set of data, strict supervision, and continuous improvement of the end processing capacity.

Q: If we want to design an artificial bacteria that can degrade polycyclic aromatic hydrocarbons through genetic recombination, what should we pay attention to in addition to?

A: The performance of bacteria in degrading organic pollutants in indoor environment is very good, but whether these bacteria can survive in natural environment and whether the ability of bacteria to remove organic pollutants has genetic stability is the most important research content of this topic.

Social survey

We wanted to know the level of public knowledge and opinion about organic pollutants, so we conducted a public questionnaire. We found that most of the respondents in the feedback questionnaire for undergraduate students but for PAHs have as much as 19% of people don't understand and about 44% of people only know is a kind of chemicals. Only about 15% of them know about PAHs.

Although the public's understanding of PAHs is not very sufficient, the vast majority of people believe that PAHs are harmful to human body to varying degrees, which confirms the design significance and value of our project. PAHs, which exists in every aspect in people's lives, so it brought great push for our project. We can through the project design and popular science propaganda to increase the public for PAHs and the awareness of environmental protection. At the same time, by strengthening the communication among college students, the possibilities of youth can be fully brought into play, thus providing more solutions to the pollution problem of PAHs in the future.

Part Two: Solution of the Problem

Document Retrieval

The degradation of PAHs can be divided into three categories: physical, chemical and biological degradation.

Physical remediation refers to the use of various physical techniques and means to separate or remove contaminants. It mainly includes heat treatment technology and extraction remediation.

Thermal desorption technology is a way that through direct or indirect heating, by controlling the system temperature and material residence time to selectively promote the evaporation of pollutants, to achieve the goal of the separation and removal of target pollutants. However, the thermal desorption process has certain requirements for temperature control, and the traditional thermal desorption process has some problems, such as long organic matter desorption time, high energy consumption, high treatment cost, and the exhaust gas is difficult to reach the standard and easy to cause secondary pollution[1].

Extraction remediation is a method to elute PAHs into organic solvents and then treat them further. However, in the process of solvent extraction, the extraction agent is often lost and the treatment cost is high. The type of pollutants, the content of inorganic salts, the type of solvent and the operation mode will all affect the extraction efficiency[2].

Chemical repair is a way to transform or degrade PAHs into low-toxicity or non-toxic substances by using the properties of oxidation, reduction and catalysis of chemical substances. It mainly includes Fenton oxidation, ozone oxidation and photocatalytic oxidation.

Fenton oxidation is a process in which H2O2 and Fe2+ produce hydroxyl radicals under the action of catalysts to degrade organic pollutants. However, Fenton oxidation requires extremely high anti-corrosion requirements for equipment, which can easily cause oxidative damage to equipment[3].

Ozone oxidation is the direct oxidation of pollutants by ozone itself or hydroxyl radicals generated by its decomposition to achieve the effect of indirect oxidation of pollutants, but the operation cost is high, it is not easy to large-scale application, and it depends on the concentration of the carrier medium and polycyclic aromatic hydrocarbons[4].

In this project, a bioremediation approach was used to convert PAHs in soil into simple compounds using microbial metabolism. Compared with physical and chemical remediation, bioremediation has low cost, is easy to be processed on a large scale. It has good effect and does not produce secondary pollution. Therefore, bioremediation is a low-consumption, efficient and environmentally friendly remediation technology[5].

[1] W. Chen, M. Chen, C. Sun, T. Chen, Z. Chen, L. Ji, X. Li, J. Yan, Eggshell and plant ash addition during the thermal desorption of polycyclic aromatic hydrocarbon-contaminated coke soil for improved removal efficiency and soil quality, Environ Sci Pollut Res Int, 27 (2020) 11050-11065.

[2] T. Kariyawasam, G.S. Doran, J.A. Howitt, P.D. Prenzler, Polycyclic aromatic hydrocarbon contamination in soils and sediments: Sustainable approaches for extraction and remediation, Chemosphere, 291 (2022) 132981.

[3]X.H. Ma, L. Zhao, Y.H. Dong, H. Chen, M. Zhong, Enhanced Fenton degradation of polychlorinated biphenyls in capacitor-oil-contaminated soil by chelating agents, Chemical Engineering Journal, 333 (2018) 370-379.

[4] C. Lin, J.B. Liao, H.Z. Wu, C.H. Wei, Mechanism of Ozone Oxidation of Polycyclic Aromatic Hydrocarbons During the Reduction of Coking Wastewater Sludge, Clean-Soil Air Water, 44 (2016) 1499-1507.

[5] S. Maletic, J. Beljin, D. Tamindzija, M. Grgic, J.M. Jazic, M.K. Isakovski, S. Roncevic, Bioremediation perspective of historically contaminated sediment with polycyclic aromatic hydrocarbons, International Journal of Sediment Research, 36 (2021) 479-488.

Visiting at Shaanxi Huabo Testing Technology Co., LTD.

Although the written or data obtained from various documents are reliable, they are still limited by their timeliness in the context of ever-changing ecological conditions. Human practice is one of the most effective ways to get the most real feedback about the research object. In order to better master the environmental testing technology, we visited professional testing institutions and learned a series of key operations.

Study and Interview

Date: June 24, 2022

Shaanxi Huabo Testing Technology Co., LTD. was established on August 8, 2017. It is a standardized third-party testing organization with independent legal person qualification, which has passed the certification of Inspection and Testing Organization (CMA) and agricultural products Testing Organization Certification (CATL). It is also registered in Shaanxi Province environmental protection industry and Xi 'an environmental protection industry record registration.

Figure 3: Visiting at Shaanxi Huabo Testing Technology Co., LTD.

We visited the professional testing institutions of the testing process and testing equipment. And we compared with the company's technical staff related to interview communication. We inquired about the testing data, the causes for the problems existing in the experiment and the sampling technique and related problems. We said that we wanted to take water quality samples from natural river basins for testing. They told us that it was difficult for individual organizations to obtain more accurate test data results without professional technical training. But they still encouraged us to try and explained to us the randomicity and diversity that we should pay attention to when taking samples.

Figure 4: Communicating with company technician

The interview is recorded below：

Q: How do we detect a thick cyclic compound, such as the PAHs?

A: Polycyclic aromatic hydrocarbons macromolecules are generally used in liquid phase. Instrument detection is divided into categories, such as gas detection generally detects small molecules, temperament detection generally detects small molecules, liquid detection of hydrocarbon macromolecules and some polypeptides. Therefore, it is necessary to detect according to the different classification of instruments. Using the right equipment will make it easier to get the desired results. If you use the wrong method, you may not get the results.

Q: How many tests will be done to verify the test?

A: The average routine food test requires more than two tests, while the average number of environmental tests varies and requires no less than 10%.

Q: If we need to take water samples from the lake, are there any requirements for the sampling operation or tools?

A: The sampling should be based on the sample type, such as metal, microbial, organic or other. If something is unstable in the field, it needs to be tested in the field, such as PH value, acidity of the solution and such detection properties are tested in the field. Some samples are special in nature and need to be sampled separately on site. For example, microorganisms need to be sampled with sterile devices and stored in cold storage. Different samples add different fixatives, for example, metal elements in water will use nitric acid, hydrochloric acid as fixatives. By adding different fixatives, more accurate results can be obtained when the sample is further tested in the laboratory.

Q: What is the reason why the data that we measured in the liquid phase sometimes appears negative value?

A: The first reason may be that the peak map contains some water peaks or some solvent peaks leading to negative peaks. It is also possible that some potentials need to be adjusted, because some peaks are assigned according to the probing potential, and the negative peaks will become positive after the probing potential is corrected. It is also possible that when you make the curve, you set the vertex too high. And the sample itself has some impurities or the sample size is a little bit lower. Accordingly, you have a negative peak.

Results and Discussion

The determination of natural water samples by high performance liquid chromatography did not yield any useful peak data. In this practice, we understood that the technical requirements for natural environment detection are very strict, and it was really difficult for non professionals to succeed. Although we did not get effective results, we also accumulated some experience in environmental detection. There are many studies that have only been fully verified in the laboratory, but further research and exploration are still needed to go from the laboratory to the nature. We should not only consider the security performance, but also consider how to analyze it in the complex background environment in the natural state. Currently, our team does not have the ability to detect the distribution of PAHs in natural environments, which is something we need to consider and learn from in the future.

The laboratory is like our greenhouse, but we must learn to get out of it. Through a series of challenges to bring synthetic biology to our side and give our project a real soul.

Part Three: Assessment of the Design and Plan

Meeting with Dr. Jun Wang

In the process of the project, we found by SDS-PAGE that most proteins produced by artificial bacteria appeared in inclusion bodies, which had a great impact on the structure, function and activity of proteins. According to this result, we immediately discussed with Dr. Jun Wang and modified the project.

Dr. Jun Wang: The best solution should be to replace the plasmid, reconnect the target gene to the new plasmid and then induce expression. Considering the time factor, you may have no time to manipulate the gene level. Therefore, you can choose to reduce the concentration of the inducer IPTG and heat up before inducing expression, so that the bacteria can produce heat shock protein to help the target protein fold correctly and eliminate inclusion bodies.

Meeting with Dr. Qiang Wang

Regarding the issues related to inclusion bodies, we not only discussed with Dr. Jun Wang, but also interviewed Dr. Qiang Wang, a professor of synthetic biology. We also discussed with him our thinking on the design of the foundation modules and bio-safety considerations.

Figure 5: Online meeting with Dr. Qiang Wang

Dr. Qiang Wang, professor and doctoral supervisor, received his PhD in 2002 from Beijing Institute of Botany, the Chinese Academy of Sciences. From October 2002 to July 2010, he conducted independent research at Hebrew University of Jerusalem, University of Tennessee, Knoxville, University of Arkansas and Michigan State University.

Research interests: Algae biology, photosynthetic environmental stress response and synthetic biology.

The interview transcript is as follows

Q: In our module design, laccase gene from Bacillus subtilis and lignin peroxidase gene from white rot fungus were introduced into Escherichia coli to achieve heterologous expression. So far, the results of phenanthrene degradation by bacteria are not very satisfactory. Are there any infrastructure issues with this design?

A: Firstly, whether codon optimization is carried out during heterologous expression, codon preference would affect the expression of enzyme; Secondly, the lignin peroxidase of white rot fungi is derived from fungi, while Escherichia coli is a prokaryotic microorganism, which lacks a post-translational modification system. If the lignin peroxidase needs post-translational modification such as glycosylation to function, the treatment effect may be affected. Thirdly, whether to use E. coli whole cells or crude enzyme solution after cytoclast to degrade phenanthellae compounds. If it is a whole cell, whether phenanthrene compounds can enter the cell and interact with related enzymes.

Q: After experimental verification, we found that most of the proteins produced by thallus were inclusion bodies. How can we improve this phenomenon?

A: First, adjust the induction conditions, such as adjusting the temperature to 16oC overnight induction or reducing the amount of inducer added; Second, co-expression with molecular chaperone to improve the probability of correct protein folding.

Q: If we eventually hope to use the strain in future product development, what aspects should we pay special attention to?

A: First, the molecular manipulation of microbial strains carrying genetic manipulation tools, released into the environment there is a risk of leakage into the environment, need a safe handling mechanism; Second, The competition between genetically engineered strains and background microorganisms in the environment needs to be evaluated. The degree of impact on environmental microorganisms shall also be assessed.

Social survey

After discussing with Dr. Qiang Wang about the possible leakage of strain genes, in order to further understand the public's views on biological safety and improve the project design, we simultaneously released a questionnaire during the science popularization activity. We explained the basic route of the project design and related information to the public, and counted their concerns on our project.

We found that about 75% of people pay special attention to the biosafety of microbial products and about 50% of people are willing to try to learn and innovate on microbial degradation of PAHs. Therefore, we began to shift part of our research focus to biosafety, and continue to try to study suicide module systems to ensure that genetically engineered organisms do not cause genetic pollution.

Communications with Members of Freshwater Bioremediation Alliance

In the process of the project, we found by SDS-PAGE that most proteins produced by artificial bacteria appeared in inclusion bodies, which had a great impact on the structure, function and activity of proteins. According to this result, we immediately discussed with Dr. Jun Wang and modified the project.

During the design process, we were constantly communicating and learning from other teams. We held a meeting on biosafety with members of the Freshwater Bioremediation Alliance, please visit collaboration for details.

Part Four: Outlook and Impact on the Future

Proposed Implementation

We have identified application scenario and purposed users based on our preceding work on human-practices. We envision a GMOs release in the wastewater and our intended end users are water company and environmental company.

Our major concern is that the impact on the environment is risk assessed and risk managed with a view to limit the dissemination. We need to develop our existing genetic circuit in order to meet the requirements of US NIH guideline for GMOs release. Further guidance and regulations from EU, Singapore and our motherland China are also referred to by us. Please visit proposed implementation for details.