Team:NU Kazakhstan/Proof Of Concept

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It was found previously that overexpression of nadE gene leads to increased production of NAD+/NADH cofactors as well as to decrease of electron transfer resistance [1].


This encouraged us to investigate whether overexpression of nadE gene under electro fermentative conditions increases production of rhamnolipids in bacteria.


To prove this hypothesis, we decided to insert a plasmid with additional nadE gene into P. aeruginosa to induce overexpression and production of NAD. We also introduced plasmids bearing rhlA and rhlB genes into P. aeruginosa. This was followed by evaluating rhamnolipid production under electro fermentative conditions and their efficiency in oil degradation.
Further, results obtained from electro fermentation experiments in P. aeruginosa can be used as a model for P. putida.

To prove our concept the following tests were performed:


Genetically modified P. aeruginosa was introduced to the minimal salt media with crude oil and incubated it for 24 hours. Bioelectrochemical experiments were conducted using chronoamperometric (CA) method and cyclic voltammetry (CV).

Cyclic voltammetry (CV) method

Figure 1. Cyclic voltammograms (CV) at 10mV/s scan rate between -400mV and 400mV for the different electro fermentative setups for production of rhamnolipids by the different test P. aeruginosa strains (engineered and wild type). Media used: MSM (minimum salt media) + oil + casein

Cyclic voltammetry (CV) plot shows distinct oxidation (upwards) and reduction (downward) curves which imply that the set-ups are redox active (electrochemically active). Figure 1 depicts that P. aeruginosa with overexpressed nadE (red line) had the highest peak among the test strains after 24 h incubation. This can be explained by the increased aerobic respiratory activity of the strain caused by increased NAD synthetase production. This in turn increases metabolic reactions that involve NAD as an electron carrier. It can be hypothesized that such a scenario will lead to faster cell growth of bacteria thereby inducing an increased expression of rhlB and rhlA genes and resulting in higher yield of desired biosurfactants.

Although other strains of P. aeruginosa with overexpressed rhlA and rhlB genes and wild type strain showed some peaks at similar oxidation- reduction spots, they had less respiratory activity because they did not possess overexpressed NAD synthetase. Therefore, we hypothesised that this increased respiratory activity could be significant in enhanced biosurfactant yields as well.

Chronoamperometric (CA) method

Figure 2. Chronoamperometry data (CA) at 400mV poised potential for the different electro fermentative setups for production of rhamnolipids by the different test P. aeruginosa strains (engineered and wild type). Media used: MSM + oil + casein

It can be observed that all the engineered strains yielded higher current outputs than the wild type strain with highest current generated by the P. aeruginosa nadE set up.

Measurement of total electrical charge production

Figure 3. Total electrical charge production at 24h incubation time for the different electro fermentative setups for production of rhamnolipids by the different test P. aeruginosa. strains (engineered and wild type). Media used: MSM + oil + casein

Total electrical charge in Coulombs produced after 24 h incubation by the different test setups carrying the different strains is shown above. The graph depicts that P. aeruginosa nadE setup produced the highest charge of 10.8 mC at 24 h. The trend in total charge production was the following: P. aeruginosa nadE > P. aeruginosa rhlA > P. aeruginosa wild type > P. aeruginosa rhlB.


Emulsification index test (E24)

Figure 4. Treatment of crude oil contaminated soils with biosurfactants from the various electro fermentation set-ups using the different P. aeruginosa strains

A: Treatment with P. aeruginosa Wild type crude biosurfactant (from conventional fermentation)
B: Untreated control
C: Treatment with P. aeruginosa nadE crude biosurfactant (electro fermentation)
D: Treatment with P. aeruginosa rhlA crude biosurfactant (electro fermentation)
E: Treatment with P. aeruginosa rhlB crude biosurfactant (electro fermentation)

The specific medium for the production of biosurfactants was filtered and assessed as the water in oil type. An equal volume of cell- free broth and hydrocarbon (diesel) was added to test tubes. This was vortexed for 2 min at high speed and kept at rest for 24 h. Following this period, the emulsified oil height (cm) was compared with the total. The emulsification was calculated according to


Where E24 = emulsification index following 24 h (in %);
He= emulsion height
Ht = total height.

Table 1. Analysis of emulsification assay results after 24 hours of incubation

P. aeruginosa Untreated P. aeruginosa P. aeruginosa P. aeruginosa
WT control with pRGPDuo2 + nadE with pRGPDuo2 + rhlA with pRGPDuo2 + rhlB
39.53 - 74.23 33.52 36.71

Emulsification index was identified using the ImageJ software. From the given table it is obvious that P. aeruginosa with overexpressed nadE gene is the most effective in oil emulsification comprising 74.23%.

Fourier transform infrared spectroscopy (FTIR)

Figure 5. FTIR spectra of extracted rhamnolipids from P. aeruginosa strains

The spectra peaks between 1600 and 1740 cm-1 indicate a C=O stretching caused by ester and acidic groups. Spectra bands around 2930 cm-1, 2857 cm-1 and 1460 cm-1 were due to the symmetrical and asymmetrical stretching vibrations of C–H in aliphatic bonds and aliphatic bending corresponding to lipids. A peak at 3430 cm-1 represents the hydroxyl group free O-H stretch as a result of hydrogen bonding. A peak observed around 1380 cm-1 was due to the COO– antisymmetric stretching.

A signature stretching of C-O-C bond was observed at 1042 cm-1 and it was indicative of the rhamnose sugar. This is explained by the fact that C-O-C vibrations in the cyclic structures of carbohydrates were as a result of the presence of bonds formed between Carbon atom and hydroxyl groups present in chemical structures of rhamnose rings. A band was observed around 703 cm-1 which is a signature region identical with rhamnolipids.
These absorption bands were all characteristic of glycolipids, especially rhamnolipids and FTIR spectra are similar to the spectra of previously reported rhamnolipids.

Reference list:

Yong, X., Feng, J., Chen, Y., Shi, D., Xu, Y., Zhou, J., Wang, S., Xu, L., Yong, Y., Sun, Y., Shi, C., OuYang, P., & Zheng, T. (2014). Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell. Biosensors & Bioelectronics, 56, 19-25. 10.1016/j.bios.2013.12.058

Eraqi, W.A., Yassin, A. S., Ali, A. E. and Amin, M. A., (2016). Utilization of Crude Glycerol as a Substrate for the Production of Rhamnolipid by Pseudomonas aeruginosa. Biotechnology Research International.

Deepika, K.V., Sridhar, P. R. and Bramhacjari, P.V. (2015). Characterization and antifungal properties of rhamnolipids produced by mangrove sediment bacterium Pseudomonas aeruginosa strain KVD-HM52. Biocatalysis and Agricultural Biotechnology, 4(4), 608 – 615.



Kabanbay batyr av., 53, Nur-Sultan, Kazakhstan