Human Practices | UESTC-BioTech

Abstract of Integrated Human Practice

The whole process of our integrated human practice was segmented into five parts, namely practical significance, technical consultancy, bio-safety issues, policy and law, and industry research, during which procedures the human practice acts as indispensable guidance for finding the unique value, centering our project, enhancing biosafety, securing the lawful environment and learning realistic problems of our project.

Practical Significance

Our solution to the Global Crisis

Energy sustains every movement and advancement of our earth. However, traditional energy in the form of coal, petroleum, and natural gas would not only be running out but also emit carbon dioxide while burning to make our planet even warmer. Traditional energy once brought prosperity and advancement to our planet, but now we are confronted with dual dilemmas consisting of searing heat and energy running out. Never have we witnessed the following circumstances: Temperatures in Kuwait once reached 74 degrees Celsius, causing no people dare to walk on the street; Mumbai, due to coals' running out, was plunged into a widespread power outage that left the city in darkness at night, with traffic lights out, causing major congestion throughout the city and almost paralyzing even rail operations.

Figure 1. The burning car in Kuwait.

Figure 2. The traffic confusion in India.

The urgent alarm is striking for us to find more sustainable and greener energy. Luckily, here comes our protagonist, microalgae . Through photosynthesis, it has the magic power of absorbing carbon. Although it would still emit carbon dioxide when it is burned, the carbon that it vomits neutralizes with the carbon that it absorbs, which is consistent with peak carbon dioxide emission and carbon neutralization objectives around the globe. In view of this, a project of utilizing microalgae for biofuel production was finalized.

Local dilemma research

Climate crisis and therefore energy crisis is never an abstract term for current global citizen. Triggered by everlasting high temperatures, our hometown, namely Sichuan province in China had also been through an energy crisis in 2022. Never have we been through a cruel summer with inky nights and no air-conditioner in such a land of abundance.

Figure 3. Virtually inky light in the land of abundance, Chengdu City.

To figure out the reasons for our local energy crisis, we send official letters to the Sichuan Energy Bureau for consulting the local energy mix and comment on our project.

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, accounting 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.

Unique value authentication for our project

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 the hydraulic depletion, thus bringing an energy crisis to 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.

After hearing the introduction of our project, a worker from the Sichuan energy bureau remarked highly on our project:

“The strategy of bio-fuel extracted 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.”

Technical consultancy

In the initial stage of our project, we try to cultivate algae in the sewage, which could achieve two objectives, namely bio-fuel production and sewage treatment. In order to figure out whether our technical route is feasible or not, we introduced our project to a professor and an engineer in the water and environment realm for consultancy.

Views from Professor Zhang Zhaohan

General introduction of Pro.Zhang Zhaohan
Deputy Director, Department of Ecology and Environment,Harbin Institute of Technology
A member of International Water Association
Leading figure in water and environment realm

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.

Views from Advanced Engineer Ma Nian

General introduction of Advanced Engineer Ma Nian
Professorial Senior Engineer, T. Y. Lin International
Being marvelous at the protective planning and technology of river ecology
Putting forward advice from an engineering perspective

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 impurities in 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.

Conclusion for technical consultancy

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. We also got valuable suggestions on doing quantitative analysis inside our lab. So we applied Fluorescence Microscope to settle the exact amount of lipid produced by the single algae.

Bio-safety issues

Intrinsic safety of C.reinhardtti

In the experimental part, we choose C.reinhardtti as the chassis, which won't produce poisonous components and has a relatively high level of bio-safety. It is no wonder that C.reinhardtti has been listed as a “generally safe creature” in America, acting as a source of dietary protein. Even if it is intrinsically safe, we still stay cautious about the utilization of genetic engineering technology. The CRISPR-Cas9 system that we use has undergone the optimization of codon originating from C.reinhardtti. Consequently, the system would only take effect in C.reinhardtti under normal circumstances. What is also worth mentioning is that the gene components we select are exclusive to eucaryon, and thus won't pose obvious impacts on other creatures.

Enhancement of biosafety level through extensive communication

Additionally, in the following CCiC (Conference of China iGEM Community) conference and the CRISPR meetup hosted by us, we also discussed the bio-safety issue more thoroughly. In order to enhance our bio-safe level on the original base, we designed a killswitch for our algae, which is under the guidance of our partner Sorbonne_U_Paris. Please click herefor the detailed design the kill switch.

Policy and Laws

In order to learn about the policy environment of biofuels, so as to prepare our project in the real circumstance of utilization, we consulted a worker who had been in the biofuel realm for a decade. Also, we inquired staff in the Sichuan Energy Bureau about our local regulations for biofuels.

80% of the value-added tax return

The worker who had been in the bio-fuel realm for a decade told us that our nation largely endorses the production of biofuels. When the production amount of bio-fuels reaches a certain level, 80% of the value-added tax would be returned to the producer.

In consistency with the guidance document of National Energy Bureau

In our local community, namely Sichuan province in China, a director in the Sichuan Energy Bureau provided us with related laws and regulations. According to the director, 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.” Therefore, he was exhilarated that our team dedicate ourselves to this field.

Figure 4. The guidance document of our National Energy Bureau provided by the director.

Lack of supervision and corresponding suggestions

The director of the Sichuan Energy Bureau also admitted that due to the less developed level of the biofuel industry in Sichuan province, the related administration and laws of bio-fuels are rather than complete, which means the responsibility of supervising the biodisel remains unclear in the Sichuan Energy Bureau. In view of the rosy pictures demonstrated by bio-fuels, we decided put forward certain suggestions online for our government to fully delineate the regulative responsibilities in the biofuel industry. The worker from Sichuan Energy Bureau extended his appreciation for our suggestion.

Picture 5. Making suggestion on the government's website.

Conclusion of biofuel regulations

Our nation largely endorses the advancement of utilizing micro-algae for biofuel production, for the reason that it is in conformity with sustainable development. However, the regulations in Sichuan province to supervise bio-fuels remains unclear, so we put forward suggestions online to promote the management in this field accordingly.

Realistic problems and countermeasures inside our lab

To figure out the realistic problems confronted by our project, we did a wide range of surveys in the biofuel industry. After researching, two tricky problems impeded the real-life application of our project, namely the high cost of algae cultivation and low lipid output . In order to solve the problems, inside the lab, we used various stress conditions in combination with transcriptome analysis and CRISPR-cas9 system to modify the metabolic pathways of C.reinhardtii, trying to cultivate micro-algae with high capacity for efficient lipid production.

Interviewing a professor from the key lab of biofuels

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?
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 high-quality, oil-producing, and fast-growing algae is the basis

Efficient cultivation technology is the key

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

An interview with a bio-fuel company based in Qingdao, ShanDong province in China

Introduction to Qing Dao FuRuiSi biodiesel enterprise

This company dedicates itself to bio-diesel field, which is an extraordinary and well-developed bio-fuel company in China. The company previously had an attempt at oil production by microalgae but later quitted it. Now they mainly make use of peanut waste oil, waste oil from the gutter in the catering industry, and animal fat to produce bio-diesel. At present, its production technology has filled a blank of bio-fuel production in Qingdao, Shandong province.

The minutes of the interview:
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 cost . 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. Is there any suggestions for our project?
A3: Reducing costs and increasing production will guarantee a relatively rosy business prospects.

Our countermeasure for the realistic problems we learned from the outside

From the previous interviews, we can conclude that there is virtually no mature business case to use microalgae for biofuel production. Two core factors impeding its industrialization is the cost on cultivation and low lipid output. So exploring experiment routes for efficient algae cultivation and high 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.

Conclusion of the integrated human practice

The project we selected is closely related to the global context of energy and climate crisis, and its unique value was further testified by our local dilemma research. To find whether our initial temptation of combining sewage treatment and biofuel production is feasible, we consulted leading authorities in the water and environment realm for consultancy, thus centering our project on biofuel production. Significant also, policies and laws for biofuel were also researched to prepare our project in real circumstances of usage . To figure out the current situation of using micro-algae for biofuel, we consulted stakeholders consisting of a biofuel company and a professor in this field, after which process the realistic problems of our project were secured. Two tricky problems impeded the real-life application of our project, namely the high cost of algae cultivation and low lipid output . After getting feedback from the outside, inside the lab, we use various stress conditions in line with transcriptome analysis and CRISPR-cas9 system which has been combined with modeling for its better performance to modify the metabolic pathways of C.reinhardtti, trying to cultivate excellent micro-algae with the capacity for lipid output of high efficiency and large amounts. In the future, we will also take using sewage as the culture medium into consideration, so as to cut down the cultivation cost.

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Overview