Team:NU Kazakhstan/Implementation
Background
IDEA
Rhamnolipids are biosurfactants that have a wide range of applications in industry and research, and are most competently produced by bacteria Pseudomonas aeruginosa. Unfortunately, this bacteria is virulent and produces toxins such as pyocyanin during rhamnolipid production. It is possible to engineer other non-virulent bacteria to produce rhamnolipids, for example Pseudomonas putida.
Prior to our project, there were successful attempts to engineer Pseudomonas putida in a way that would allow rhamnolipid expression. However, there were difficulties with constructing metabolically-enhanced mutants with the potential for combined expression of respiratory and rhamnolipid genes. This is due to the lack of cloning systems functional in this organism that allow the efficiently controlled expression of more than one gene since most of available vectors have a single cloning site under the influence of a constitutive or an inducible promoter system.
We fix this problem by employing a novel dual-inducible duet-expression system for gene overexpression in P. putida to construct combined expression of NAD and rhamnolipid synthesizing genes. Moreover, by putting engineered bacteria under electrofermentative conditions using bioelectrochemical set-ups, we will enhance the production of rhamnolipids.
APPLICATIONS
END-USERS
We have thoroughly studied rhamnolipid applications in a variety of fields to identify key areas of focus. Based on a table by Irorere (2017), we could align potential end-users with fields of rhamnolipid usage. However, the majority of these applications were not feasible in Kazakhstan due to the lack of end-users, specifically medical use, MFC, and pest control. Crop protection and food processing industries of Kazakhstan were eliminated as well since there is no demand for rhamnolipids specifically.
Kazakhstan has one of the largest oil & gas reserves in the world (top 15) and this industry’s share in the GDP of the country amounted to 17%. Having more than 250 oil and gas fields with the majority of them being located in highly endemic areas such as the Caspian Sea raises the need for environmentally safe ways of managing resulting oil spills.
Considering that our project is aimed to increase the production of rhamnolipids in a safe way the resulting product can be used directly on the spillage sites, allowing us to emulsify oil safely and quickly to neutral organic compounds. Therefore, we decided to explore Microbial enhanced oil recovery (MEOR) and Bioremediation of petroleum at contaminated sites, and contact our end-users for feedback.
FEEDBACK
Oil spill response and bioremediation are complex processes closely regulated by the government and oil companies. By meeting with government representatives from the Ministry of Ecology and non-governmental ecological organizations we realized that we need to speak with companies that provide bioremediation and oil spill response services to learn about the current tendencies. Meetings with leading companies in the oil & gas industry as well as a couple of local waste management companies confirmed that there is an acute shortage of safe, effective, and affordable bioremediation methods. The application of rhamnolipids for bioremediation of crude oil was in demand and could be successfully commercialized. Therefore, we needed to construct our business model to be proposed for implementation.
BUSINESS MODEL
By participating in iGEM EPIC Summer Bootcamp our team members acquired valuable skills in commercializing our project. Specifically, Business Model Canvas and the Pitch Deck were used extensively in preparation for meeting with companies and participating in business incubation programs. With respect to our Integrated Human Practices activities, we modified the draft of our Business Model Canvas up until its recent draft. Although the project has a long way to go, you can observe the changes that have been made so far.
Moreover, we have troubleshooted our business model on a local incubation program hosted by Nazarbayev University Research and Innovation System (NURIS) - ABC Incubation. There we pitched our project and received valuable feedback from experts in this field, venture investors, and other participating start-ups. Although they generally referred to our project as being socially relevant and potentially applicable, lack of technical explanation of how are we going to scale-up our project to industrial demands was questioned. You may see our presentation for this program below that contains some data on market analysis and competition section(translated from Russian):
BIOPROCESSING
To upscale the bioprocessing of RemiDuEt up to an industrial level we need to go through upstream and downstream processing steps. For upstream bioprocessing, we will determine the growth kinetics of engineered Pseudomonas putida, propagate an optimized cell line, prepare and expand this inoculum. We will use a column reactor with steel yarn inside as a working electrode to provide electrofermentative conditions for bacteria inside. Steel yarn will also be used as a surface for biofilm growth since biofilms can be better for cell immobilization and some studies report that they are better for bioremediation purposes in comparison to free-floating planktonic cells. It is advantageous that vast regulatory apparatus empowers P. putida with high flexibility to quickly adjust to steady changing conditions, which is especially desirable in a large-scale bioreactor with heterologous microenvironments (Kukurugya et al. 2019; Sudarsan et al. 2014). Moreover, it should be noted that a very broad spectrum of cheap and renewable substrates can be used as feedstocks, including petrochemical-derived wastes.
In downstream bioprocessing, we need to separate cellular products and optimize the purification of obtained rhamnolipids. Although acid precipitation is one of the most commonly used ways of downstream processing methods for rhamnolipids, the percent purity of an end product is ~50.9 percent. Therefore, we aim to conduct alcohol precipitation prior to acid precipitation, which will give a product with ~89.9 percent purity, which can be increased up to ~95.4 percent if followed by calcium precipitation (Invally, 2018).
CHALLENGES
There are still many challenges that need to be overcome before RemiDuEt can be used as an oil recovery agent in the Caspian Sea.
Firstly, a lack of transparency in data or even absence thereof regarding oil spill incidents, their scale, and reports on their management from Oil & Gas companies in Kazakhstan significantly limit the scope of our work since we cannot even nearly quantify the scale of caused environmental damage. The situation is worsened by the ambiguous legislative policies by the government that do not oblige these companies to report caused environmental harm nor to contribute resources for recovery of damaged areas. These factors are important in identifying why companies are not eager to employ more environmentally safe clean-up methods since it does not benefit them in any way and they will not face consequences regardless. As a result, it is extremely hard to reach local companies: out of 27 contacted companies, only 4 responded with 2 of them being rejection letters. To advance in the promotion of the utilization of safe and sustainable oil spill management methods, we rely heavily on governmental support. In technical aspects, we face challenges with having access to necessary facilities in the long-term since we cannot operate commercially on Institution owned laboratories. In the process of upscaled production of rhamnolipids, excessive foaming, the small surface area of the electrode (walls of steel yarn), and production costs can be challenging as well.
We believe that by tight cooperation and support of one of the largest oil & gas companies of Kazakhstan (KMG) that is owned by the government, we can make our project applicable in real life since the majority of outlined challenges are dependent upon the presence of support from major players in the industry and governmental support. Technical challenges could be resolved in continuation of our research work and collaboration with KazEcoSolutions.
SAFETY
The main idea of implementation lies in the production of rhamnolipids and separating them from bacterial strain to avoid interaction of genetically engineered P.Putida with other bacteria in the site of the spill, which can lead to mutations or horizontal gene transfer with other bacteria. Therefore, the implementation of modified P. putida in industry to make pure rhamnolipids requires the construction of specific bioreactors suitable for isolated growth of this bacteria. Notably, before starting our work with this strain we conducted an independent review of available literature on the toxicity of P.putida to the surrounding environment to ensure that our project will not cause significant environmental damage even in case of emergency, where bacteria get released into the environment.
SUMMARY
Overall, the end product of our project can be used in a multitude of ways in different fields, ranging from medical devices to pest control. Our team has focused on a more locally relevant application of rhamnolipids - bioremediation of crude oil. Through our interactions with local stakeholders, companies, and government representatives, it was concluded that our project is feasible and can be successfully implemented in real life. Besides, we confirmed that our project can be upscaled for industrial manufacturing and have signed a memorandum of cooperation with one of the largest companies in Kazakhstan. Though many applications are left unexplored due to local and financial constraints, rhamnolipids produced by our engineered bacteria can be distributed worldwide among companies from different industries.
Reference List:
Askitosari, T. D., Berger, C., Tiso, T., Harnisch, F., Blank, L. M. and Rosenbaum, M. A. (2020). Coupling an Electroactive Pseudomonas putida KT2440 with Bioelectrochemical Rhamnolipid production. Microorganisms 8: 1959
Bodey, G. P., Bolivar, R., Fainstein, V., & Jadeja, L. (1983). Infections caused by Pseudomonas aeruginosa. Reviews of infectious diseases, 5(2), 279-313.
Gauttam, R., Mukhopadhyay, A. and Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid 110: 102514. https://doi.org/10.1016/j.plasmid.2020.102514
Gauttam, R., Desiderato, C., Jung, L., Shah, A., Eikmanns, B.J. (2019). A step forward: compatible and dual-inducible expression vectors for gene co-expression in Corynebacterium glutamicum. Plasmid 101, 20–27.
Kukurugya MA, Mendonca CM, Solhtalab M, Wilkes RA, Thannhauser TW, Aristilde L (2019) Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida. J Biol Chem 294(21):8464–8479. https://doi.org/10.1074/jbc.ra119.007885
Invally, K., Sancheti, A., & Ju, L.-K. (2019). A new approach for downstream purification of rhamnolipid biosurfactants. Food and Bioproducts Processing, 114, 122–131. https://doi.org/10.1016/j.fbp.2018.12.003
Sudarsan S, Dethlefsen S, Blank LM, Siemann-Herzberg M, Schmid A (2014) The functional structure of central carbon metabolism in Pseudomonas putida KT2440. Appl Environ Microbiol 80(17):5292–5303. https://doi.org/10.1128/aem.01643-14
Schmitz S, Nies S, Wierckx N, Blank LM and Rosenbaum MA (2015) Engineering mediator-based electroactivity in the obligate aerobic bacterium Pseudomonas putida KT2440. Front. Microbiol. 6:284. https://doi.org/10.3389/fmicb.2015.00284
CONTACT
igem@nu.edu.kz
Kabanbay batyr av., 53, Nur-Sultan, Kazakhstan
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