As the old saying goes, “Through active listening, you can better understand what the other person is trying to say, and can respond appropriately.”

In order to take responsibility for various stakeholders, we did a wide range of communication to consult the advice and feedback for our project, contributing to the gradual amelioration of our project. The group we communicated with includes experts in water and environment, iGEM teams especially those who use the CRISPR-Cas9 system, the provincial Energy Bureau , the various practitioners in the biofuel industry . Significant also, to invite more people to the synthetic biology world and let wider scope of the audience know about the magic power of micro-algae, we did scientific popularization by designing a series of algae and synthetic biology-related classes, attracting more than 90,000 people to attend our educational activities and securing a provincial level award for scientific popularization.

We initially tried to figure out whether the strategy of combining sewage treatment and algae cultivation is feasible or not. After consulting two leading authorities in the water and environment realm, we decided to center our project on culturing algae for biofuel production. Let us view our interviews!

Generally, Professor Zhang Zhaohan found our project technically feasible .

While tricky problems we should take into consideration are that, the components of sewage are so complicated that the algae may find it hard to survive . Based on this, he made suggestions that we should just concentrate on biofuel production instead of cultivating algae in the sewage. If we still want to maintain sewage disposal, we can consider enabling the algae to absorb oil by using genetic engineering.

After referring to related papers and materials, Engineer Ma said that the thought of combining sewage disposal and biofuel production is feasible and intelligent .

Nonetheless, the difficulty and workload are still quite hefty because the subject involves several specialties, thus necessitating several giant technical breakthroughs to achieve satisfactory results. To be specific, from the cultivation of special microalgae to the production of biofuels, from algae properties to its efficiency of absorbing impurity in the sewage, the above-mentioned issues need to cross huge technical barriers, and research expenses would be very costly. Moreover, each direction may not be fully aligned and corresponding efficiencies may be divergent. Therefore, it is recommended to focus on one key aspect. To illustrate, the biological direction should focus on culturing special algae for biofuel production. The environmental direction should center on decontamination ability. The work should be simplified to conduct more in-depth research so as to achieve certain breakthroughs.

As an engineer, he also put forward suggestions from an engineering perspective. To make our subject have engineering value, it is recommended that the research should be as quantitative as possible, so that the "possibility" may rise to impuritieslity". As biomass is related to algae abundance and lipid production rate, etc.; sewage treatment is related to the removal rate of impure components. These all seem to be closely concerned with biomass, so "biomass" may be a good metric to gauge the effectiveness of our project. Accordingly, inside the lab, we did a quantitative analysis in the result of the experiment part called Effects on growth and lipid production of Chlamydomonas reinhardtii under different stress treatments.

Additionally, if we want to step onto the road of comercialization and industrialization, we can refer to the "algae-eating insect" industry and learn from its experience.
He added that, if we want to step onto the road of commercialization and industrialization, we can refer to the "algae-eating insect" industry and learn from its experience.

After consulting two experts and taking technical factors into consideration, we find it virtually impossible to cultivate algae with sewage treatment ability and bio-fuel production capacity at the same time within a limited time span. Consequently, we temporarily decide to quit sewage treatment and concentrate on cultivating special algae for bio-fuel production at the current stage.

As our team used the CRISPR-Cas9 system to cultivate highly efficient algae, we wanted to address related technical problems and expand our way of thinking, a CRISPR-themed meetup was hosted by us and HainanU_China. On the meetup, 7 other teams , including CPU_China, SCU-China, BIT DUT_China, HiZJU-China, NWU-CHINA-A, SCAU_China, and SJTU-software joined our extensive discussion. We also invited three special guests to give comment on our presentation and discussion. They are Yang Jianzhe who acted as iGEM Ambassador in 2021, Liu Jingrong who has been tutoring more than 20 iGEM teams and ShiSonglin who is a member of the Biodiversity Leadership Program and familiar with the CRISPR system. With 8 other iGEM teams and three special guests, we did extensive discussions on experiment, modeling, wiki, and human practice respectively. For our part, after presenting our project in great detail, other teams gave us precious advice on algae cultivation, CRISPR application, eletro-transformation, and so on.

Besides, we also attended CCiC(Conference for China iGEMer Community) to do the presentation of our project, as well as discussing widely with other 84 teams. At this conference, the judge said that our project has vital importance for current energy problems. But we should enhance biosafety levels on the original basis due to the application of gene-editing technology. Significant also, our team captain Wang Xingjie also acted as the judge to give comments and advice on other teams' project.

After attending CCiC conference, we got to know that DUT_China also uses the CRISPR system. We exchanged the title and abstract of our projects through social media and surprisingly found that we were both doing similar modeling for off-target prediction. We kept communicating during the process of constructing our model.

As our team uses CRISPR-Cas9 system to modify the metabolic pathway of microalgae, they gladly invited us to test our model for the prediction of off-target activity. Based on CNN, DenseNet, an outstanding neural networks model was used by DUT_China to screen out sgRNAs with high efficiency. UESTC_BioTech also developed a thermodynamics-based model to predict the potential off-target activity of the selected guide RNA.

After testing their model, we found that the model built by DUT_China is very different from the model we constructed in terms of the overall objectives and the methods used, which leads to the fact that the results obtained by both parties cannot make direct comparison. Therefore, no quantitative analysis can be given. However, to a certain extent, what could be obtained is that both teams have similar results for off-target evaluation. Therefore, the correctness of our team's model was further verified by the model of DUT_China.

At the same time, during the communication between us, we found that our friends from DUT_China had some deviations from us in the concept of off-target. So, after further in-depth communication, we made our ideas and thoughts understood by our friends from DUT_China, which laid a good foundation for further collaboration. Then, we provided them with enlightenment on the algorithm design for finding potential off-target sequences in the whole genome sequence, and provided them with an optimized solution for the acceleration of their algorithm.

Sichuan province in China had been through an energy crisis in 2022. To figure out the reason for our local energy crisis, we send official letters to the Sichuan Energy Bureau for consulting related information.

The Sichuan Provincial Energy Bureau provided a series of valuable data in a timely and effective manner. According to its data, the energy mix in Sichuan Province has the characteristics of "being abundant in water and gas, being lack of coal and oil".

The hydro energy is mainly distributed in the Jinsha, Yalong, and Dadu River basins, with a technically exploitable capacity of 148 million kilowatts, accounting for 22.4% of China's total output and ranking second in China. Shale gas accounts for 1.19 trillion cubic meters, ranking 1st in China. By a way of contrast, the amount of oil is limited, with proven reserves being 74.61 million tons, accounts for only 0.2% of the overall amount in China. Coal's proven reserves are 15.7 billion tons, constituting only 1.4% throughout the nation.

From the above-mentioned data and interview, what we could conclude was that Sichuan Province has a clean and diversified energy mix in general, showing high dependence on hydroelectricity and natural gas . Sichuan is an exemplary clean-energy province, being affluent in clean energy resources represented by hydro, wind, solar, and natural gas. However, at the same time, the extremely high temperature in 2022 triggered hydraulic depletion thus bringing an energy crisis for Sichuan Province reminds us to keep exploring new and sustainable energy resources to complement the current energy structure, so as to guarantee energy security and sustainability.

According to the director of the Sichuan Energy Bureau, the guidance document of our National Energy Bureau writes, “the technology of micro-algae cultivation and lipid extraction should be continuously optimized, and technological breakthroughs in using micro-algae for biofuel production should be attained as early as possible.” , which means our project is in line with the direction of National Energy Bureau. Therefore, he was exhilarated that our team dedicated themselves to this field.

The director also commented highly on our project: “The strategy of bio-fuel production from micro-algae can serve as a complementary solution to traditional hydro and natural gas power generation in our province, enhancing the cleanliness and diversity of energy usage and contributing to the energy security of our province.”

To figure out our project's stance in the bio-fuel industry and the realistic problems confronted by our project, we did a wide range of surveys covering a professor, an enterprise, and a practitioner.

After interview this practitioner, we found out the common form, application scenarios and preferential policy of biofuels.

The minutes of our interview
Q1: What are the forms of biofuels?
A1: Biofuels could be segmented into solid, liquid, and gaseous fuels.
(1) Solid biofuels
a. Raw material: crop residue, which can be mixed.
b. Way of production: physical mode, extrusion storage.
c. Features.
Pros: being easy to burn, being popular in the market.
Cons:
·Biofuels of this kind require little technology, and the main cost comes from raw materials. As awareness of the surplus value of crops increases, the cost of purchasing crop residues rises accordingly.
·Not that environmentally friendly, for its higher particulate matter output when it is burned compared with gaseous and liquid biofuels.
(2) Gaseous biofuels
a. Way of production: catalytic reaction → fermentation → carbonization → biomass gas
b. Characteristics:
·higher investment in technology,
·less emission
·less entry to the market, mostly used within enterprises
(3) Liquid biofuels
a. Typical product: ethanol
b. Features:
·Investment in processing technology is high
·High added value spared for more profit
·Due to technological limitations, it has limited application scenarios( Lipid biofuels could only be used in automobiles and several other industries).

Q2:What are the specific application scenariosof biofuels ?
A2: I can come up with three major scenarios currently.
·Firstly, the boiler of factories (including textile, food, beverage, and shoe factories).
·Secondly, providing heat for grain drying.(There exists a storage center for agricultural grain drying, where biofuels could be applied.)
·Thirdly, sand and stone extraction.

Q3: Where do biofuels stand in the whole energy mix and energy market?
A3: To answer this question, I should initially introduce our local energy mix. The energy mix is dominated by natural gas and supplemented by biofuels. The affluence of large enterprises enables them to use more natural gas regardless of its cost. While Small and Medium-sized enterprises may need bio-fuels more than natural gas, because of the lower cost of biofuels compared with natural gas.

Q4: We roughly know that our country supports the production of biofuels. What do you know about our national preferential policy towards producing biofuels?
A4: Our nation supports the production of biofuel largely. When the production of biofuels reaches a certain amount, the VAT(Value-added-tax) will be refunded by 80%.

Q5: What is your suggestions for improving our project?
A5: It is recommended to determine the oil composition and select specific application scenarios. If it can be used in the food and drug industry, it will have greater added value and more profit margin than the pure biofuel.

General introduction to Pro. Liu Tianzhong

From the key laboratory of bio-fuels in the Chinese Academy of Sciences

His research focused on the cultivation, processing, and utilization of microalgae specialized for energy.

He knows well about the current situation of using microalgae for biofuel production.

The minutes of the interview

Q1: Are there any commercial attempts to use microalgae to produce biofuels and dispose of sewage?
A1: At present, there is virtually no mature business case of using microalgae for biofuel production. But there are certain business cases for applying algae to wastewater treatment, and most of them are treated by algal and bacterial symbiosis.

Q2: What are the reasons that hinder its large-scale commercialization?
A2: The core issue that hinders its large-scale of commercialization is cost. The cultivation efficiency is not high enough and the production cost is too costly.

Q3: Is there any solution to existing problem that the strategy of using microalgae for biofuel production cannot be widely popularized?
A3: From my experience in studying microalgae and biofuel industry

The cultivation of excellent, oil-producing, and fast-growing algae species is the basis.

Efficient cultivation technology is the key.

Comprehensive utilization to realize multi-component hierarchical utilization is the strategy.

The company previously had an attempt at oil production by microalgae but later quitted it, and now it mainly recycles wasted oil. This company dedicates itself to producing bio-diesel. The company makes use of peanut waste oil, waste oil from the gutter in the catering industry, and animal fat to produce bio-diesel. The two methods of enzyme and chemical method and various technological conditions were mainly adopted, the quality of biodiesel produced was superior to that of 0 petrochemical diesel in China. At present, the production technology of our company has reached the domestic level, which has filled a blank of biofuel production in Qingdao, Shandong province.

Q1: Why is the strategy of waste oil extraction adopted?
A1:Going to food factories, restaurants for on-site recycling waste requires no site fees and low recycling costs. At the same time, the amount of waste oil is large.

Q2: Why does your company give up the idea of oil production by microalgae?
A2: The prospect of this technology is not clear an it requires large scale of investment in the early stage because of its technology immaturity. Furthermore, the cultivation of algae needs the site, which even drives price higher. The fragility of algae also adds the fee for researching in this field.

Q3: Are there any suggestions for our project?
A3: Reducing costs and increasing production will guarantee a relatively rosy prospect for your project.

From the extensive communication centering on the biofuel industry, we could roughly draw a picture of the biofuel industry to prepare for our project in real circumstances of utilization. And we also attained realistic problems which impede the large-scale commercialization of microalgae, namely low lipid output and high investment in algae cultivation. So exploring experiment routes for efficient algae cultivation and high amount of biofuel production becomes a requisite. After viewing numerous related papers and discussing with our PI, we established a relatively mature technology route to culture algae. To increase the cultivation efficiency, we added another antibiotic called Tim to protect algae from bacteria contamination, facilitating the better growth of microalgae. We also found that little or no Fe condition could contribute to the faster growth of algae in the first 24 hours. With regard to increasing lipid output, we utilized various stress conditions in combination with transcriptome analysis to select the appropriate genes to be knocked down by the CRISPR-Cas9 system. At the same time, to predict the off-target of sgRNAs, we constructed off-target modeling for better performance of the CRISPR-Cas9 system. We really responded to the real world's needs by designing such experiment routes.

We especially designed an algal class to cover microalgae related scientific knowledge, during which process we unveil how synthetic biology adds power to microalgae. Additionally, we created a song for this algal class and a set of card games to imitate the algae cultivation lab work and integrate synthetic biology knowledge. [For more information, you can turn to the card page] Our educational activities cover more than 90,000 audiences through cooperating with provincial authorities and got the provincial award for scientific popularization.

The municipal authority, Chengdu ecology and environment Bureau held a forum called Rongchengzhitan, which means wisely making a contribution to reducing carbon emissions. On this forum, we shared the inspiration, the practical significance, and technical cores of our project, trying to provoke the wisdom among citizens towards reducing carbon emissions by utilizing small stuff around us, just like using microalgae for bioduel production. Significant also, our PI, professor Tang Lixia, also did an online sharing whose topic is “Bio-based plastic degradation technology”. This sharing given by our team received 26,000 likes in total. Many citizens who participated in this online forum expressed their appreciation to our environmental protection technology. An official from Chengdu ecology and environment bureau commented that sharing of this kind could inspired some youth to study harder and learn cutting-edge technology to make beneficial contributions to our society.

Due to many iGEM products being GMOs, it is of vital importance to educate and benefit the wider community by introducing GMOs regulations in different countries, so as to promote international communication and education in this realm. This is our video for introducing GMOs regulation by cooperating with iGEM teams in France and America.

This is our video.