Product Design

Fig.1 Flow chart of E. coli carbon utilization system

1. Overview

In this project, we, the NCKU Tainan Team, have proposed an alternative way to reduce the emission of Carbon dioxide (CO₂). Referring to the opinions and feedbacks from many industry experts and professors, we design a new factory flow to capture CO₂ by E. coli Not only our device meets the specs to commercialize, but it also demonstrates high cost performance.

The emission of CO₂ has been a serious problem for a century that causes global warming and severe climate change. Even though many ways have been tried to reduce it, the generation of CO₂ primarily from industry is still overwhelming. Therefore, scientists and governments have been working hard to find solutions to tackle the problem.

2. Control System


Fig. 2 Overview of the control system

There are many aspects we need to consider. First, we calculate the emission velocity of CO₂ from the factory, as well as the medium exchange rate and the growth rate of our E. coli.

Fig. 1 is a process of whole E. coli carbon utilization that we design for industrial application. We simplify it into three parts which shows in Fig. 2 to explain more clearly. Three switches control three parts, named A, B and C. Basically, the factory replaces the medium twice a day. At one hour before replacing the medium, the user needs to turn on switch C to discharge ninety percent of the medium. When it is time to replace the medium, switch C will be turned off and switch B will be turned on to refill medium. When sufficient medium is added, switch B will be turned off and switch A will be turned on to let CO₂ in. Just like the animation showed on Fig. 1.

Considering the cost, the growth time of our E. coli and the floor area, we optimized replace time of the medium, replace it every twelve hours and with 72 parallel bioreactors. Next, we are going to have more detail description on three parts, which are Gas preparation system and flow system, Medium preparation, and Downstream products purification and biosafety.

A. Gas preparation system and flow system



Fig. 3 Diagram of gas preparation system and flow system

According to IGCC (Integrate Gasification Combined Cycle) flow diagram, the fuel is first converted to syngas which is a mixture of H₂ and CO. The syngas is then burned in a combined cycle consisting of a gas turbine and a steam turbine with a heat recovery steam generator (HRSG). After CO₂ / H₂ separation, IGCC can reach the demand of CO₂ purity including low SOx and NOx emission fraction of allowable limits of bacteria. Finally, the produced flue gas could enter the pipeline leading to the bioreactor.

In E. coli utilization system, the air is pumped in to neutralize the concentration of CO₂. A controlled valve is used to control flow rate and split distribution. When the switch a is turned on, the switch b will be turned off, and vice versa. As for the CO₂ inlet and outlet, it will maintain an open system of bioreactor. In other words, CO₂ will enter continuously and cause some non-reacted CO₂ emitted.



Fig. 4 IGCC process flow diagram. Source: Vattenfall. (2010) Syngas has been treated by sulfur and nitrogen removal, as well as heavy metal removal and cooling tank. Through IGCC process, purified CO₂ in flue gas is allowable for E. coli CO₂ utilizing.

B. Medium preparation



Fig. 5 Diagram of medium preparation

At this stage, we have two sections to consider, medium storage and medium preparation before replacing time.

The medium is composed of M9 salt and xylose. For storage, we will convert it into powder with the required proportion. At one hour before replacing time, pour the powder into the medium tank and turn on the water injection switch. Turn on the stirrer of medium tank to have medium powder and water perfect mixing. The outlet of bioreactor (switch c) will be turned on at the same time, letting ninety percent of the medium in the bioreactor flow out . When the medium have prepared well, turn on the switch a and switch b for replacing medium in bioreactor, while the switch c will be turned off.

We also consider the process of raw materials, especially xylose, which is the key source of our pathway. Since xylose is one of the products of agricultural waste degradation, we visited the 2018 Tainan Biotechnology and Green Energy Expo to consulted with researchers from National Energy Program-Phase II, whose projects was biofuel and biodegradable plastic production via agricultural waste. They had developed technique that degrade cellulose and semi-cellulose by ion solution.

Besides, we have opportunity to collaborate with UESTC-Chian team . They work for degrading straw with synthetic biology and convert the product into bio-fuel. One of the product from straw degradation is xylose. These techniques are eco-friendly and low-energy-require. Therefore, the process development of xylose production will be a low-carbon-emission process.

C. Downstream products purification and biosafety



Fig. 6 Diagram of downstream process

We will discharge 90% of the used medium in the bioreactor one hour before new medium flows in. Which means that we let 10% of the reacted bacteria remain in the bioreactor to maintain a steady cell density condition of in the bioreactor. The effluent medium will be sterilized and filtered in the downstream clean-up tank. At this step, we harvest the bacteria by centrifuging and extracting the by-product such as amino acids, proteins, medicine or bio-fuel. Different extracting process designed depends on different by-product.

Besides, we try to reuse the waste heat of factories for sterilizing. The waste water can be recycled as well. Through Removing toxins and adjusting pH value the effluent could return to the medium tank. As for energy require for this system, renewable energy helps us to reach near -zero carbon emission process.

Entrepreneurship : China Steel

Fig.7 Picture of CSC interview

Meeting with experts and stakeholders is important in shaping our project to fulfill the needs of our target user. China Steel Corporation is the largest integrated steel Manufacturer in Taiwan. Also, they had been adopting the algal bio-sequestration by cooperating with the research group at our university. Click here to know more in Entrepreneurship：Process, Suggestion and question and Interview record.

Process

We were given the opportunity to meet with the senior executive of China Steel Corporation to gain invaluable insight for our research. The meeting commenced with our presentation. During the presentation, we introduced our project, including the bioreactor design and the industrial model. By listing out all the aspects we had considered, we would like to obtain advice on the practical and social considerations involved in the application of our project in industry.

Suggestion and Question

Will the high concentration of CO₂ retard growth of engineered bacteria?

Microalgae is reported resistant to SOx and NOx. Does E. coli survive under such conditions?

The best condition for engineered E. coli to capture CO₂ is a lower CO₂ concentration without too much SOx and NOx particles. However, we won’t be able to provide an ideal culture condition in Industrial application. After testing the tolerance of E. coli, we conclude that E. coli is possible to survive under that kind of condition in factory and the only effects its expression. It may not capture as much CO₂ as culture in the lab.

It is important to define a specific commercial product that can be truly produced since your user may consider its economic viability. They stated that a product that can be widely used is better. At the same time, we should consider current GMO legislation if we want to commercialize those products. The actual condition is not as ideal as in the laboratory, we should optimize the condition to maximize the carbon fixation ability of the microbes.

Interview record

The record can be separated into two parts. One is about their feedback after interview, another one is our customer investigate questions. We use CSC represent China Steel.

Part1. Interview record

Date：September. 15, 9 am.

Location：China Steel meeting room

CSC： What is the adaptability of E. coli for the corporate? Do you have any doubt about the actual application?

It can be explained from the following points:

1. Concentration:

Bacteria can tolerate the increase of CO₂ concentration. However, there is limit in the input, and our team is targeting this system.

A shunt is designed to slow down the rate of input to enter the bacteria rapidly.

2. Temperature:

In this system, 42 degrees Celsius is our limit, and we need to overcome by technology in the high temperature.

The problem is that our team will lower the temperature through other devices.

3. Waste:

Our team solves the problem of waste by recycling and filtering out.

CSC ：From the perspective of the company, how much additional benefit can it bring to the output value of the products in their downstream of system?

At present, the product of downstream in our system is glutamine, why we choose is because glutamine is accessible and easy to operate for us. Its additional benefit refers to the different application. Take the market value of glutamine as example, the additional benefit can reach 10 times larger of the E. coli culture cost, ignoring the fixed cost of the whole system.

Besides, E. coli was regarded as high potential species to produce all kinds of protein. Including essential amino acid that cannot be synthesized by organism, or forage for stock farmer. Therefore, our system has high potential output value to bring great additional benefit.

CSC：China Steel is the second largest carbon consumer in our country. It needs two-thirds of Taiwan's area to balance one-tenth of the current emissions. In practice, it is still too far away. Is it possible to match the materials with 3D layout?

We want to save the space and culture in high density concentration:

1. Reduce the volume of culture material
2. Stacking the bioreactors

CSC： How to deal with the waste of this system? Is there a problem with super Cryptococcus neoformans?

The protein needs to be separated before produced. At the same time,this process will produce the bio-waste. The special process is high temperature and high pressure. It can be used in the factory's original waste system under the high temperature and high pressure environment.

We use the general strains, and there is no possibility of mutations. In addition, with the monitoring of environmental, the probability of mutation is greatly reduced to reach biosafety.

CSC position description：

Algae is one of the implementation of the CCS plan, and they always want to build a multi-system. Each system has its advantages and disadvantages. Therefore, what we proposed was a one more choice for them and they were glad to hear that E. coli and contribute to CCS&U (Carbon Capture Storage and Utilization).

Part2. Customer demand investigation

1. The research and development of new technologies, which level will be considered to mature and worthy investing specifically?

There are three conditions:

1) Feasibility of laboratory technology: It’s ok with technical confirmation.

2) Feasibility of engineering: It’s feasible under engineering equipment construction, application of space and on-site environmental conditions.

3) Feasibility of economic: total cost (input, output) must be positive benefits.

2. There is a problem of limited space in Taiwan, how much space did we need to reduce at least in the enterprise?

This proposition should be how much CO₂ the technology can absorb per unit area. Based on this basis, Industrial will evaluate the existing space of the factory, consider how much CO₂ can be absorbed, investment cost of equipment, the amount of CO₂ that can be reduced, and calculate the input and output to evaluate whether there is positive benefit.

3. We will consider the secondary cost of waste disposal, just like the application of your company unit in basic-oxygen-furnace slag, will you consider the cost of waste recycling be beneficial? Or is there a problem caused by China Steel and secondary pollution?

This part cannot be provided due to operational confidentiality. It is recommended that this proposition should be turned into be directly used as a marketable product. The cost of the resource should be assessed by the Life Cycle Assessment (LCA) as a whole.

4. Since our project is facing the problem about the higher cost of culture medium, we would like to ask you about the benefit of carbon fixation and cost of carbon fixation method.

The cost of carbon fixation depends on the carbon capture and storage methods used. For example, the calcium circuit developed by the Industrial Research Institute is used to capture carbon. The recent cost of carbon capture is intended to be reduced to US$30 per ton, and US$10 per ton of geological storage is required. Competition between carbon capture methods can be assessed by cost and overall utilization of reuse.

5. Regarding the part of industry-university cooperation, I would like to ask why China Steel chose to cooperate with Annan Campus in NCKU for microalgae carbon fixation.

When the former academic research unit strives for the NEP project (National Energy Program), the technology that the audited authority usually requires that project must be adopted by the industry. Therefore, both the academic research center and the industry usually sign the cooperation letter of intent for review. For China Steel, it is willing to support the academic research community to conduct forward-looking technical research with national resources to provide the technical information needed to evaluate feasibility.

6. The medium we need will still consume energy in the process of preparation, and it may cause carbon emissions simultaneously. We wonder how to regard upon overall carbon footprint may be increased from the perspective of enterprise.

If the overall footprint of the carbon fixation process developed may be positive (increased), in general, from the perspective of carbon reduction within the enterprise, there is no possibility of application. If the derived external carbon reduction benefit is greater than the internal carbon loss, it proves to have a positive net benefit to the environment. As long as it meets the feasibility of engineering and economic, the enterprise is willing to adopt it.

7. Research on carbon fixation, what is the driving force for China Steel in addition to economic benefits?

Regulatory requirements, corporate identity and social responsibility.

Part3. Picture Record

Business Model

Cost Evaluation

The cost evaluation is always crucial for product being on the market. To compare our engineered E. coli to microalgae, we calculate how much the cost it would be when capturing 1000 kilograms CO₂.

Volume

Table 1 Volume require in capturing 1000 kg CO₂

Cost

The most expensive source in the medium of our engineered E. coli is xylose. 1 mole xylose will capture 0.17 mole CO₂, so it would need 20.0535 kilograms xylose and 1 kilogram xylose is cost 2 USD. The total cost for our engineered E. coli is require 40.107 USD for capture 1 kilogram CO₂. In contrast, microalgae need 1000 liter to capture 250 gram CO₂, so it need 4000 liter (about 4 Tons) water and 1 tons is cost 9.78 USD (300NT). The total cost for microalgae is require 39.13 USD.

Table 2 Cost require in capturing 1000 kg CO₂

Future Work

For industrial application design, we focus on manufacturing valuable products using pyruvate and the linkage between our engineered E. coli between factory. We have designed a device containing our recombinant E. coli, constructed a system which links with factory. However, we still look forward to more modifications of our biological pathway and system.

The most important intermediate product, pyruvate, is also possible to be converted to other compounds by E. coli native enzymes or constructed enzymes which is clone into E. coli from other organism. For future work of pyruvate, we expect that it is predicable to produce amino acid, fatty acid, biofuel and even biodegradable plastic. Pyruvate is crucial for central metabolism pathway, the TCA cycle, of most organism and has the potential to become vary biochemistry compounds.

We set our first future goal at producing glutamine, an essential amino acid for human and some animals. We can simply purify it as a nutrient supply. Not only for medical and daily usage for people, but also for animal husbandry. Furthermore, glutamine can easily convert to other amino acid, and potentially produce other proteins.

Fig. 8 Diagram of pyruvate in central carbon metabolism

Furthermore, researchers have successfully constructed pathways produced cellulose and Poly 3-Hydroxybutyrate-co-3-Hydroxyvalerate through the TCA cycle. We are confident of manufacturing more valuable and diverse products from pyruvate.

WAs for the device we designed, we expect that it is possible to modify our device for power generator and other industry. Our device can utilize CO₂ and convert it into various valuable products. With our system, companies can not only reduce CO₂ emission but also make profits.

Reference

1. Fuyu G, Guoxia L, Xiaoyun Z, Jie Z, Zhen C and Yin L. Quantitative analysis of an engineered CO2-fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation. Gong et al. Biotechnology for Biofuels, 2015, 8:86.
2. 張嘉修、陳俊延、林志生、楊勝仲、周德珍、郭子禎、顏宏偉、李澤民 (2015), 二氧化碳再利用─微藻養殖, 科學發展　2015 年 6 月│ 510 期
3. Lawrence Irlam (2017), GLOBAL COSTS OF CARBON CAPTURE AND STORAGE, Global CCS Institute, Senior Adviser Policy & Economics, Asia-Pacific Region
4. Jin Hwan Park, Jae Eun Oh, Kwang Ho Lee, Ji Young Kim, and Sang Yup Lee. Rational Design of Escherichia coli for L‑Isoleucine Production. [ACS Synth Biol.](https://www.ncbi.nlm.nih.gov/pubmed/23656230#) 2012
5. M. KUNDAK, L. LAZI], J. RNKO. CO2 EMISSIONS IN THE STEEL INDUSTRY. METALURGIJA 48, 2009
6. V. N. Kalpana, D. Sathya Prabhu, S. Vinodhini and Devirajeswari V. Biomedical waste and its management. Journal of Chemical and Pharmaceutical Research, 2016
7. Qian Ma, Quanwei Zhang, Qingyang Xu, Chenglin Zhang, Yanjun Li, Xiaoguang Fan, Xixian Xie, Ning Chen. Systems metabolic engineering strategies for the production of amino acids. Synthetic and Systems Biotechnology 2 (2017)
8. Jørgen Barsett Magnus, Daniel Hollwedel, Marco Oldiges, and Ralf Takors. Monitoring and Modeling of the Reaction Dynamics in the Valine/Leucine Synthesis Pathway in Corynebacterium glutamicum. Biotechnol. Prog. 2006
9. Isao Kusumoto. Industrial Production of L-Glutamine. American Society for Nutritional Sciences, 2001
10. Quan Chen, Qian Wang, Guoqing Wei, Quanfeng Liang, and Qingsheng Qi. Production inEscherichia coli of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) with Differing Monomer Compositions from Unrelated Carbon Sources. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2011