Implementation
Background
First, from the aspect of sociology, the soaring in gasoline prices causes more and more people to choose to explore new energy types, which coincides with our research project--producing n-butanol. N-butanol is a new bio-fuel with great potential and has been called the second generation biofuel[1]. The new method of synthesizing n-butanol with Lactobacillus which we are currently studying can efficiently solve the problems of terrible pollution and the high cost of traditional production methods, which is conducive to the low-carbon life vigorously advocated by the country. Together with the support of several experts’ opinions we interviewed, we believe that the method of synthesizing n-butanol with Lactobacillus has certain development prospects.
In order to get a preliminary understanding of the public's awareness of n-butanol and serve for further publicity, we conducted surveys through online questionnaires and offline campus interviews and found that most people don’t have any knowledge of n-butanol and Lactobacillus.
Advantages of N-butanol
Compared with ethanol, butanol has a lower hygroscopicity and greater water solubility with a weaker corrosiveness. Butanol has a calorific value of about 130% of that of ethanol, while the volatility is only about 1/6 of that of ethanol. The higher boiling point and flash point also make butanol safer[2]. In addition, butanol as a fuel has been proven to be used on the current petrol cars with just a little modifying renovation[3]. All these factors make butanol an excellent ingredient in both laboratory and industrial manufacturing as well as a potential fuel, which can be used to alleviate the stress caused by energy shortages to a large extent.
Advantages of Lactobacillus
American scientists have used colibacillus to successfully synthesize n-butanol[4]. But due to the toxicity of n-butanol, this method cannot achieve industrialization with great yield. In contrast with colibacillus, Lactobacillus has a higher developmental ceiling, because Lactobacillus as a carrier is more stable and is not easy to break (which also triggers more difficulties in successfully changing its gene). This might enable the biosynthesis-method to industrialize. The successful industrialization would result in huge cost savings and would reduce annual CO2 emissions by 12m tonnes. Besides, the production cost of n-butanol by Lactobacillus is much lower than that of chemical synthesis, so it can promote the economic development of the domestic chemical industry.

According to above analysis, our method of producing n-butanol by Lactobacillus is relatively environmentally friendly and clean, so the benefits to the whole society are great. Comparing to the traditional way of manufacturing n-butanol by the chemical industry which causes significant pollution, this new method of producing n-butanol has incomparable advantages in alleviating pollution.
Target Users
According to the interviews we have conducted with several experts and our literature investigation, n-butanol we produced can be used in many industries..
1. Partners or Competitors
Since our goal is to provide a new production method of n-butanol through biosynthesis and biotechnology, our potential partners will be those traditional chemical plants, like BASF-YZ Company, which are also competitors in some way. According to our investigation, these traditional chemical plants are also looking for a new production method that will be more eco-friendly, more sustainable. So we are working on this project to give them an opportunity to open a brand new era in chemical material production.
2. Application Fields and Business Partners
Per our literature investigation, n-butanol has low volatility but strong adaptability, it is commonly used as a solvent in many industries, like cosmetics, pharmaceuticals, food processing, scientific experiments et al. Therefore, our product, n-butanol could supply to those cosmetic factories[5], pharmaceutical companies, food processing factories, and scientific institutes. On the other hand, n-butanol is also considered a very competitive renewable biofuel to replace gasoline given its many advantages, it could be used in many gasoline-driven engine machines which means it might have a big impact on the automobile industry. Therefore, our n-butanol could also supply to those fuel supply companies.
3. End Users
Based on the above analysis, the end users are also the customers of our business partners. They will include the buyers of cosmetic products or some manufactured food, patients who purchase some medicine from pharmaceutical companies, and researchers who conduct experiments in the laboratory. Of course, the end users of our n-butanol also include those customers who drive cars with n-butanol as the fuel resource.
Our product will be applied to every corner of society and everyone will be a potential user of our product.
Implementation Procedure
1. Bio-synthesis system of n-butanol
Figure 1. The metabolic pathway of N-butanol synthesis in Lactobacillus Brevis ATCC367.
Genes introduced into L. brevis are shown in red (Figure 1). The thl gene encodes mercaptan, hbd encodes β -hydroxybutyrate CoA dehydrogenase, crt encodes 3-hydroxybutyrate CoA dehydrase, and ter encodes trans-enol CoA reductase. Only when the four enzymes are expressed together can the engineering strain achieve the metabolic process from glucose to N-butanol.
Figure 2. General experiment procedureFigure 2. General experiment procedure
First, we constructed plasmids pLY15-opt and transferred them into Streptococcus Brevis. Then, erythromycin was used to select whether the plasmid was inserted into the competent bacteria Streptococcus Brevis ATCC367 and screened. Finally, when our bacteria grew, we measured the growth curve and detected the yield of N-Butanol (Figure2).
2. Performance Teste
As shown in Figure4, we successfully detected a higher yield of N-butanol. With the growth of the Streptococcus Brevis, the yield of N-butanol is also increasing. This result is really encouraging. It can be inferred from the below result that with the engineered strain, we can finally produce enough N-butanol.
Figure 3. After pLY15-opt was transformed into Streptococcus Brevis, N-butanol production of ply15-opt strain was measured at different times (48h, 69h, 95h, and 159h)
Safety and Challenge
Our experiments were conducted in the laboratory of the Nanjing University Of Chinese Medicine and under the supervision of our instructor Dr. Lv. We strictly followed the protocols and regulations. The bacteria (Streptococcus Brevis, Escherichia coli) and plasmids exposed to during the experiment are harmless and cannot infect humans.

Besides the safety during the development inside the experiment we considered, we also concern about the safety when we implement our project in the real world and other challenges.

1. N-butanol is a little toxic, and its vapor has a pungent odor. Therefore, it should be well sealed during preservation and shipment.
2. The biosynthesis method (cultivating Lactobacillus to produce n-butanol) may require a large investment on development.
3. N-butanol is flammable. The mixture of its vapor and air can form an explosive mixture, which can blast when contacting fire or being at a high temperature.
4. Utilizing n-butanol as fuel is impossible to prevail without permission and support from the government.
Future Plan
As the production of n-butanol by Lactobacillus is environment-friendly, clean, and pollution-free, which represents future trends in the future chemical product manufacturing industry, and is beneficial to promoting social and economic development and protecting the environment. This is our initial purpose but also our most competitive selling point.
Our marketing promotion will focus on the aspect of environmental protection and highlight the advantages of the biosynthetic method of producing n-butanol by Lactobacillus. Make it clear to the public that the new method of biosynthesis has a positive impact on the environment and its advantages in alleviating pollution are better than other production methods.
Meanwhile, we will also work on the advertising promotion of the broad application of n-butanol. It is also an indispensable part to remind the entrepreneurs of the superb prospect of the utilization of n-butanol as a fuel.
In addition to the advertising works, we will enhance productivity by continuously upgrading our production technology, like conducting more tests to confirm the optimum culture conditions of our engineered strains, and applying for patents so that more enterprises will see our growth and purchase our patents.
Reference
[1] Chao Jin and Mingfa Yao and Haifeng Liu and Chia-fon F. Lee and Jing Ji. Progress in the production and application of n-butanol as a biofuel[J]. Renewable and Sustainable Energy Reviews, 2011.
[2] 刘娅, 刘宏娟, 张建安,等. 新型生物燃料——丁醇的研究进展[J]. 现代化工, 2008, 28(6):5.
[3] Dürre, P. Biobutanol: An attractive biofuel. Biotechnol. J. 2, 1525–1534 (2007).
[4] "美科学家:利用大肠杆菌高效生产生物燃料正丁醇." 科技与生活 (2011).
[5] Green, E.M. Fermentative production of butanol—the industrial perspective. Curr. Opin. Biotechnol. 22, 337–343 (2011).
[6] Qi Li, Meixian Wu, Zhiqiang Wen, Yuan Jiang, Xin Wang, Yawei Zhao, Jinle Liu, Junjie Yang, Yu Jiang, Sheng Yang, Optimization of n-butanol synthesis in Lactobacillus brevis via the functional expression of thl, hbd, crt and ter, Journal of Industrial Microbiology and Biotechnology, Volume 47, Issue 12, 1 December 2020, Pages 1099–1108, https://doi.org/10.1007/s10295-020-02331-2