As a part of iGEMers, our team wishes to bridge the gap between laboratories and industry. In order to obtain more information about CO₂ biofixation, we visited microalgae cultivation demonstration plant in An-Nan Campus of National Cheng-Kung University. The microalgae cultivation demonstration is a project managed by Professor Jo-Shu Chang, an expert in biofixation of CO₂ from industry using marine algae. We toured their microalgae cultivation systems, and were impressed by their idea of converting microalgae into functional products. This inspired us to synthesis commercial products from CO₂ in modified E. coli. They also provided us with a wealth of knowledge about the considerations needed to design a bioreactor for utilization of CO₂ and evaluated cost effectiveness of our project. CO₂ concentration in flue gas can be as high as 20~30%, thus it is necessary for us to lower the CO₂ concentration level before entering bioreactor to increase carbon fixation efficiency. With the advices given, we modified our bioreactor design for practical application in industry.

China Steel Corporation (CSC), located at Kaohsiung, Taiwan, was founded in December 1971. With annual production (in terms of crude steel) around 10 million tons, CSC produces a range of products including plates, bars, wire rods, hot and cold rolled coils, electrogalvanized coils, electrical steel coils, hot-dip galvanized coils, and Ti/Ni-base alloy. The domestic market takes roughly 69% of CSC’s production and the exports take the remaining 31%. CSC is the largest steel company in Taiwan, enjoying more than 50% of the domestic market. Major export destinations are Mainland China (including HongKong), Japan and Southeast Asia.^[1]

After participating at the Asia-Pacific conference, we organized some questions and made a report to CSC. We consider the implementation of industrial and have a presentation to CSC. We also got some feedback from them. Their concern is that the smoke emitted by the factory is not completely carbon dioxide, but will contain some impurities like SOx and NOx. So they asked more about whether E. coli can survive and react in this environment. In addition, they also praised our idea of removing carbon and solving the disadvantages of microalgae space. They even said that they are very supportive of young people’ s ideas and there are opportunities for cooperation in the future.

After the interview, we thought more about the growth environment that E. coli can tolerate, including excess CO₂ concentration , SOx, NOx and even metal particles. Therefore, we hope to design a system that processes metal particles and cooling gas beforehand in the factory, and recycle the used waste medium.

[1] ChinaSteel Basic Information

Prof. Ng is the mother of NCKU-iGEM team. Since the establishment of NCKU-iGEM in 2016, Prof. Ng has brought synthetic biology as well as iGEM to the spotlight of NCKU campus. Prof. Ng always inspires team members by asking us some critical questions without telling us the answer directly. She gave us advice from different aspects. She initially gave us many critical suggestions during the brainstorming. Except for teaching us knowledges about synthetic biology, she always reminds us to think more about the real application of our project.

Various useful advice was given by her. She emphasized the advantage of protein production by E. coli, she also suggested us to study more on high cell density fermentation. Without her timely advice, our team cannot improve our project.

Our PI, Prof. Huang is an associate professor of the institute of Basic Medical Sciences in National Cheng Kung University. His major field is Bacteriology, Bacteria Genetics, and Molecular Biology.

We invited Prof. Huang to attend our first presentation. During our first presentation, he provided us a lot of useful feedback and we could feel the intention of Prof. Huang. So, our team thought that Prof. Huang could guide us for all our project and we wanted to invite him as our PI. Luckily, we have succeeded!

Since the day Prof. Huang joined us, he provided us all the resources that we needed or could make it more convenient. Secondly, he gave us many ideas when we had met difficulties or junctions in our progress whether it was dry lab or wet lab. We really appreciated what he has told and taught us.

Our secondary PI, Prof. Hashimoto Masayuki is a project assistant professor from Institute of Molecular Medicine.

The host organism in our project is E. coli. Prof. Hashimoto is familiar with the genetic manipulation of E. coli, therefore we introduced our idea to him. One of his research is about the effect of carbonic anhydrase (CA) to the growth of E. coli. Since we wanted to introduce heterologous CA into the E. coli, he suggested us to consider whether it will compete with the native CA of E. coli, which might affect the function of the heterologous CA. Also, Professor Hashimoto introduced the concept of homologous recombination to us as we had the idea to insert our construction into E. coli chromosome. However, we were suggested not to do the homologous recombination due to the limited time we had.

After the discussion, we started to review journals and found out the feasibility of expressing both native and non-native CA in E. coli. We found out that there were research which expressed heterologous CA successfully. Therefore we decided to clone the CA gene into the E .coli.

This year, our project is related to carbon sequestration. Therefore, we grab the chance to discuss our idea with Prof. Bieng-Zih Hsieh. Prof. Hsieh is an associate professor in the Department of Resources Engineering. One of his research topics is about CO₂ sequestration, which is a topic that related to our project, the difference is that he employs a physical method in this aspect, while we use a biological method.

At first, we defined our project as carbon capture. However, he introduced the idea of carbon utilization to us as he thought it is more suitable for our project. Prof. Hsieh introduced some carbon capture or utilization strategies that implemented in the industry currently and suggested us to do more research on those methods. He also reminded us to compare our system with other biological approach to figure out our strength and weakness.

Prof. Chao-Chung Peng is an assistant professor in Department of Aeronautics and Astronautics and International Bachelor Degree Program on Energy. He used to work in China steel corporation (CSC) so he gave us a chance to contact CSC.

We met up with prof. Chung to get some suggestions which more helpful for our direction of our projects. Prof. Chung is proficient in energy field, different from giving us experimental technical advice, and he gave us more about suggestion for action on device and ideas.

He thought that when we look for our advantages, we can take apart each of our research processes and experimental steps, and find every possible point that is less efficient. The benefit of the whole project is very great. For example, what kind of way we can use to let every E. coli to eat most CO₂, the way we replace our cultured E. coli. It is true that there are existing ways we can think more about how we can improve. He also emphized that we must be very understandable about our project, it included our advantages, disadvantages, potential threat, our whole project process, and the most optimum environment we should provide for our E. coli. The more we realized our project, we could find more possibility of development. After talking with professor, we inspired more ideas and found direction about our project more clearly.

He gave us some advice in engineering viewpoint. Therefore, we tried to rewirte a big scope of our project and defined the points that we have to fulfill.

We had an opportunity to speak with Prof. Chieh-Chen Huang from National Chung Hsing University (NCHU) in the Asia Pacific Conference, whose work includes the reverse TCA cycle of engineered carbon capturing E. coli. Upon our discussion, Prof. Huang raised the issue of the low efficiency of one of our enzymes, Rubisco and suggested that we cultured our engineered E. coli in an anaerobic condition. Since Rubisco, the ribulose-1,5-biphosphate carboxylase/oxygenase is an enzyme catalyzing the reaction of either carbon dioxide or oxygen with ribulose-1,5-biphosphate (RuBP) as the name itself suggests, putting our system in an aerobic condition would significantly lower the efficiency of capturing carbon dioxide due to the similarities between gaseous carbon dioxide and oxygen.

We discuss with the senior, Hai-Xuan Chen. He is the PhD student from the lab of Prof. Liang-Ming Huang in the department of Environmental Engineering. His research is about biological modeling.

We chose Multi-fuel pure oxygen combustion reduction and Advanced carbon capture combustion technology which have closely related to our project. Interestingly, we found that in such business matchmaking conference involved in many vendors, most of them focused more on what benefit they create rather than the detailed technology process.

Our discussion is about biological modeling. At first, we have to collect the experiment data, so that we can build the model from our experiment. After that, we improve our parameter of model to make our modeling more accurate. In this way, we can operate our model before we start doing our experiment.

After discussion, we have to consider more possible pathways from xylose to pyruvate. Therefore, there is another pathway we have to figure out and build the model to check which pathway our e.coli will go. On the other hand, we can know pyruvate where it comes from by node analysis, and we can realize pyruvate from xylose or carbon dioxide uptake afterwards.

Most important of all, it will produce carbon dioxide in glycolysis. Hence, we have to consider the carbon dioxide we produce, and then compare the carbon dioxide we fix. In this discussion, we find out several ways to improve our project including more pathway we consider and node analysis we can calculate.

Prof. Jo-Shu Chang has a lot of specialties of Biochemical Engineering, Microalgae Biotechnology, CO₂ reutilization, Cecycling economy, Biomass Energy, Environmental Biotechnology, Applied Microbiology. He completes a microalge carbon utilization system in An-nan Campus of NCKU which helped the largest energy company in Taiwan (CPC, Taiwan) and the largest steel maker in Taiwan (CSC) to develop the research platform of microalgae-based flue gas CO₂ biofixation technology. Besides, Since 2009, Prof. Chang served as the PI of National Energy Program (NEP) projects and focused on microalgae-based CO₂ capture and utilization for biofuels production and biorefinery.

Our project is to reuse the CO₂ emitted in the industry with a biological way. Although the pathway of carbon flow in E. coli and in microalge are quite different, it is really important for us to understand the current method of biological carbon utilization before we begin our research. We want to deeply understand the whole microalgae culture pool system including the position of CO₂ pipeline, downstream process, pool cleaning and how is the progress of a research in lab turning into real application.What's more, we He subsequently proposed a 'zero emission' biohydrogen system integrating unit operation of cellulose hydrolysis, darkfermentation, photofermentation, microalgae CO₂ fixation, microbial fuel cell and hydrogen fuel cell.

Prof. Chang lets us visit how his project work in An-nan campus. Prof. Chang's team has been working with CSC and CPC, Taiwan to develop innovative microalgae-based flue gas CO₂ reduction and biofuels production technologies. Therefore, they shared the progress of the whole technologies coming out. From the beginning, Prof. Chang was focusing on the conversion of biomass feedstock and waste materials to energy. Large scale microalgae cultivation plants were constructed in CSC and on An-Nan campus of NCKU.

We visited Prof. Chang to discuss about our carbon capture system. We had a great time discussing and he gave us three pieces of advice. One is to define the velocity of CO₂ input in which scale of CO₂ we want to solve with. Another one is to confirm a commercial product like proteins which can bring us some income to balance the cost. The last one is to calculate the whole carbon balance from the source of xylose to the process of waste. We were really appreciated that we can have a short meeting with prof. Chang.

Profr. Veerabhadran Ramanathan is a Distinguished Professor in Scripps Institution of Oceanography at UC San Diego, and UNESCO Professor of Climate and Policy at TERI University, New Delhi, India. Prof. Ramanathan discovered the greenhouse effect of halocarbons, particularly, CFCs in 1975.

He has won many prestigious awards, such as the Volvo Prize, the Rossby Prize and the Tyler Prize, the highest rated environmental award given in the United States. In 2013 he was awarded the United Nations Top Environmental Prize.

He was invited to share his thought about climate change in NCKU as the 2018 Tang Laureate for sustainability science. Using the topic ‘Bending the Curve’, he would like to raise the public awareness on climate change issue. His speech inspired us a lot. He not only introduced the ideas of super pollutants such as HFCs, CH4 and black carbon, but also proposed the solutions for tackling climate change.

After his speech, we briefly introduced our project to him. Although his work focuses on the impact of non-CO₂ gases on climate change, he thinks that human beings need more alternatives to tackle carbon dioxide emission problem, and our project may be one of the solutions, which makes us have faith in our project. He was glad to know that undergraduates have worked hard to deal with this issue.

Through this meetup, we realized that ‘everyone is responsible to make the world better, no one should be excluded.’ Unity of the entire society is the most effective way to mitigate climate change. Therefore, we put more effort in our human practice activities by engaging with government entities, local enterprise, institution and the public.

We were lucky enough to arrange a meeting with Dr. Cheng-Hsien Shen, manager in the ‘New Energy Technology Division’, who is currently working on Carbon Capture & Storage Application Project at Industrial Technology Research Institute (ITRI). ITRI is a national-level, non-profit organization for applied research which accelerates industrial technology development in Taiwan. He introduced us to current carbon capture methods and technologies in Taiwan. Storing carbon dioxide underground into deep geological formations is believed to the best way to return carbon dioxide to the Earth. However, considering the active fault lines across Taiwan and the frequent occurrence of earthquakes, it may be risky to have largescale CCS project here. He was amazed by our project, as we developed a biological solution of capturing CO₂ emission in industry. The highlight of this method is engineered E.coli could efficiently capture and utilize CO₂ into products with great market potential. Shen pointed out that gaseous emitted from heavy industry contains a lot of toxic elements, which is not suitable to be recycled into esculent products. We take this consideration into our project.

During the meetup, we asked her some problems about our robot that we have met in this morning and she answered us that there were still some components being shipped so hopefully we could wait until those components to arrive. For the software problems, she built a forum for all the iGEM teams which got the robot so that we could discuss online or sharing all kinds of protocol with each other.

We also briefly introduced our project but since we didn't have too much time so she could only suggest us to study more related topic of iGEM. She is willing to have next video meeting with us and expertly we could show her our device next time. We were so glad to have this talk that day.

We had a video meeting with Tec-Monterrey. After introducing our project to them, they had pointed out some aspects that we need to think more about. They reminded us to think about our end product which can make our project more complete.

During the meeting, we came up out with the ideas to collaborate with each other. Their project has two parts. The main part is E-coding, which uses bacteria to record the data. Another part is to sense the pollutant in river of Mexico, which is the part that we could link our work. We had constructed a pH alert system for low pH sensing, they suggested us to incorporate our pH sensing construct in their pollutant sensing system, making it become more complete.

We had a nice talk with members from Tec Chihuahua. First, we introduced our project to each other.

During our talk, we received many useful recommendations and advice especially for our constructions and cost of our main device. The considering these aspects which also led us to more focus on the value of our project to be applied on the industries sides. Eventually, as we were communicating for our main ideas and project, we found that Tec Chihuahua seems to have a great idea on solving the problem of larvae-killing bacteria. According to researchers, the extinction of bee will also lead the human society to face the acute problem which is the shortage of food sources. By considering this knotty dilemma, team Tec Chihuahua has outcome this with the technologies of synthetic biology. In addition, we also give our suggestion such as constructing a control system on their project so that there will be an awareness on the shortage of material or overusing of their materials.