Increasing demand for oil & gas production in modern days pushes for higher production and distribution rates. The occurrence of oil spills in such processes is not unusual and the vast majority are left unaccounted for. Existing treatment methods are only efficient for localized spills shortly after the accident, while in the long-term they are less cost-effective and hazardous to Kazakhstani endemic flora & fauna. We propose a solution to this problem by developing a novel agent for crude oil bioremediation - “Remi, du et!”. Specifically, we targeted to modify nonvirulent Pseudomonas putida using a dual-inducible system for overexpression of genes nadE and rhlA/B, coding for NAD synthetase and rhamnolipids, respectively. The hypothesis is that the metabolically-engineered bacteria should demonstrate a high yield of rhamnolipids when grown as biofilms under electrofermentative conditions. Since produced rhamnolipids are biosurfactants that emulsify crude oil, the resulting product can be used directly for the treatment of oil spills in ecologically-sensitive areas.
Our team aimed to develop a new process for crude oil remediation called "Remi, du et!". To achieve our goal, we planned on modifying nonvirulent Pseudomonas putida using a dual-inducible system for overexpression of nadE and rhlA/B genes, coding for NAD synthetase and rhamnolipids, respectively; under electrofermentative conditions. However, currently, we have focused on using a P. aeruginosa model that allows the functioning of nadE and rhl genes, with this we wanted to achieve elevated expressions of rhamolipids by induced overexpresion of nadE.
WHAT IS THE PROBLEM?
The most common ways to remove oil from the environment involve mechanical, manual labor, chemical dispersants, absorbents, and in-situ burning. However, to a certain extent, existing methods are hazardous, inefficient, and threaten biodiversity. This is especially problematic for Kazakhstan which has more than 250 oil fields where oil leaks are frequent. This tendency demonstrates the necessity for a more efficient way to deal with oil spills.
WHY PSEUDOMONAS PUTIDA?
One of the promising methods of oil clean-up is bioremediation. Our project involves using biosurfactants, particularly rhamnolipids, which decrease surface tension between crude oil and water and degrade oil into smaller oil droplets, which endogenous microorganisms can degrade. Pseudomonas aeruginosa is known to produce rhamnolipids; however, the usage of this bacteria is restricted due to its pathogenic nature and production of virulence factors such as exotoxins cytotoxins, elastase, and other pathogenic proteins .
We first want to prove he model where overexpression of nadE in P. aeruginosa results in higher yields of rhamnolipid production. NAD synthetase gene (nadE) and rhamnolipid genes (rhlA, rhlB) will be extracted from Pseudomonas aeruginosa PAO1 and inserted into pRGPDuo2 plasmid provided by Dr. Rahul Gauttam, Postdoctoral Research Fellow at Berkeley Lab. Obtained plasmids are going to be transformed into P. aeruginosa. We expect that overexpressed nadE gene will lead to increase of NAD synthetase which will increase the NAD/NADH pool in the bacteria; consequently, internal electron pool will expand. The given electron pool can be used to form more ATP and expanded ATP pool will induce more gene expression and bacterial growth of P. aeruginosa. Overall, stated processed will result in increased rhamnolipid production. Furthermore, under electro fermentative conditions external electron pool of the bacteria will be also enlarged. However, due to the toxicity of P. aeruginosa, it can not be used as a model for scale application. Therefore, after proving the increased rhamnolipid production model by nadE overexpression in P. aeruginosa, we will aim to modify standard non-pathogenic Pseudomonas putida KT 2440 to increase rhamnolipids synthesis. P. putida is a BSL-1 microorganism commonly found in soil and has a well-established genetic system.
Furthermore, to minimize possible risks such as horizontal gene transfer with other bacteria or mutations, genetically modified P. putida will not be used on the oil spill site. Synthesized rhamnolipids will be separated from bacterial strains and used for oil emulsification.
STEPS OF THE PROJECT
While the theoretical scenario of our project seemed to be well planned, COVID-19 had a plan for our team on its own:
Alonso, B., Fernández-Barat, L., Di Domenico, E. G., Marín, M., Cercenado, E., Merino, I., de Pablos, M., Muñoz, P., & Guembe, M. (2020, December 11). Characterization of the virulence of Pseudomonas aeruginosa strains causing ventilator-associated pneumonia. BMC Infectious Diseases. Retrieved October 18, 2021, from https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-020-05534-1.
Soberón-Chávez , G., Lépine, F., & Déziel, E. (n.d.). Production of rhamnolipids by Pseudomonas aeruginosa. Applied microbiology and biotechnology. Retrieved October 18, 2021, from https://pubmed.ncbi.nlm.nih.gov/16160828/.