Difference between revisions of "Team:BGU ISRAEL/Results"

The bacterium R. ruber, known to utilize PE (polyethylene) as a carbon source, was grown in various liquid growth media with and without amino-acids and containing PE or PET pellets for a period of 30 days.

For control we used a SM liquid growth medium with glucose, glucose and amino-acids and a SM medium with no carbon source (for detailed instructions on preparation of the media see Protocols ).

As expected the results show that Rhodococcus ruber`s growth on glucose media was substantially faster and yielded high O.D. measurements throughout the experiment. We have observed similar results when the bacterium was grown on media containing amino acids (AA), suggesting the bacteria can utilize the AA as a carbon source. The media containing plastic as the sole carbon source (PET, PE), reached higher O.D values from that of a medium with no carbon source (SM medium) but lower O.D measurements than on glucose media or plastic enriched with AA media. All samples have shown a steep rise in the measurement in the first few hours of the experiment. We can assume that these results are due to the trace of rich medium that was left from the starter that used prior to the start of the experiment. This might also explain the increase in O.D values in the SM medium. Lack of values of certain samples is due to contaminations or broken equipment.

After 30 days we ceased measuring O.D values, but continued monitoring our samples for bacterial growth. Samples were left to incubate unopened for 3 months to test for the formation of biofilms.

As seen in the photo, after 3 months R. ruber developed a biofilm surrounding the pellet (in this case – PE). The orange color is a unique property of R. ruber.
We have witnessed similar results with PET pellets, suggesting R. ruber can utilize PET as a sole carbon source.

We took samples from both PET and PE media (with pellets) and examined them with a Phase-contrast microscope and scanning electron microscope (SEM):

First, we could see that Rhodococcus ruber was present in its planktonic form in the media.

After further examining the images we noticed the formation of vacuoles close to aggregates of bacteria. We assumed the vacuoles are made up of Extracellular polymeric substance (EPS) that was secreted by the bacteria into the media. We assume that in this case the EPS is used for the absorption of the PET micro partials and allows the accumulation of secreted enzymes. The enzymes are used to catalyze the initial cleavage of the polymer and allow absorption of nutrients (Laspidou et al 2002). These photos may suggest a mechanism to improve degradation by increasing the extracellular enzyme concentration.

More samples from the same experiment were analyzed using a scanning electron-microscope:

As seen from the images above, there is a substantial difference between the surfaces textures. The control's surface is rough while the treated one is smoother. It can be explained by the ability of the bacteria, mainly its enzymes, to degrade exposed and less dense polymers with more ease, smoothening the outer layer.

We made the same imaging to the PET samples in order to observe if the same effect on the polymer surface is induced by the bacteria:

Contrary to our expectation, the untreated PET surface is smooth while the treated sample’s surface seemed to have gone through erosion.

Furthermore, we have come to notice the appearance of crystalline rods near bacteria aggregation sites. That can be explained by the preferential degradation of the amorphous material exposing the crystallized material that goes through a slower degradation (Shah et al 2008).

Our results may suggest that Rhodococcus ruber is able to degrade PET, having presented substantial changes before and after incubation with PET. It should be mentioned that it is not yet proven that Rhodococcus ruber is able to utilize PET as a carbon source. In order to verify this assumption, more tests are needed.
After identifying R. ruber's mechanisms of plastic degradation such as secretion of polymer degrading enzymes and forming vacuoles from EPS to increase the extracellular enzyme concentration, we decided to concentrate our efforts on improving a PET degrading enzyme.

References

1. Laspidou, C. S., & Rittmann, B. E. (2002). A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass. Water Research, 36(11), 2711-2720.
2. Shah, A. A., Hasan, F., Hameed, A., & Ahmed, S. (2008). Biological degradation of plastics: a comprehensive review. Biotechnology advances, 26(3), 246-265.

One method to assess the activity of our designed mutants was using the pNP-Butyrate degradation assay (see Protocols).
We ran the tests on all 5 variants of the LC-Cutinase protein with different concentrations of the substrate:

As seen from these results, all of our variants, including the codon-optimized Cutinase, have shown increased pNP-B degradation activity compared to the W.T. LC-Cutinase in all concentrations tested.
We can also see that the highest activity is displayed by the codon-optimized(CO) Cutinase, followed by the F4 mutant.

These test were preformed by incubating the substrate with a supernatant from LC-Cutinase expressing bacteria and not taking into consideration the different expression levels of each variant. Even though later tests with R. ruber show that the concentration of enzyme is an important part in efficiency of degradation, we still decided to run these tests again, with equal enzyme concentrations on our 2 fastest variants - CO and F4:

Again, we can see that the CO and F4 variants have higher activity in regards to pNP-B degradation than the W.T. Cutinase. The CO Cutinase is once again with the highest activity, suggesting that expression levels are not a major determinant in its pNP-B degradation activity.

We can conclude from all these results that we managed to dramatically improve LC-Cutinase's activity, with regards to pNP-Butyrate degradation.

To further characterize LC-Cutinase's PET degradation activity we used Scanning-electron microscopy of PET pellets incubated with E. coli expressing LC-Cutinase.

Control:

One control was PET pellets incubated for 2 days in LB liquid media with no bacteria.
As seen from the images, the surface of the PET pellets is relatively smooth, there are some high crystallinity rod shaped structures but they are fairly covered.

As a second control we used E. coli transformed with the pACYC vector with no insert (the backbone alone), to assess the effects of bacteria themselves on the PET.

PET incubated with LC-Cutinase expressing bacteria:

As seen above, after incubation with the LC-Cutinase expressing bacteria, the surface of the PET is rougher than the control. We also notice that the surface of the PET looks more porous and the rod shaped, high crystallinity PET, is more exposed than in the control.
Moreover, when comparing the bacteria expressing the LC-Cutinase gene and the bacteria transformed with an empty vector backbone, we see a clear difference in the texture of the PET. We assume that this is the result of the PET degradation activity of the LC-Cutinase enzyme.

As part of our work with the P. putida KT2440 strain we wanted to test its antibiotic resistances, so we can select the appropriate cloning vectors.

Here are our results:

Analysis of these results showed that P. putida KT2440 is sensitive to Kanamycin, Streptomycin and Spectinomycin.

These results led us to choose the pSEVA224 and pSEVA434 vectors - with Kanamycin and Spectinomycin resistances respectively.

In order to test P. putida's ability to utilize TPA and PCA as a carbon source we measured O.D values for a period of ~100 hours with P. putida in liquid M9 media with either TPA or PCA as a carbon source. To prevent contamination we used Ampicillin and Chloramphenicol (at 100μg/ml and 34.1μg/ml respectively) as selection markers.

From these results we conclude that P. putida can utilize PCA as a sole carbon source, as previously proposed. In contrast, our results show no growth with TPA, which leads us to the conclusion that P. putida cannot utilize it as a sole carbon source.

Reviewing the results leads us to the conclusion that the TPA degradation pathway must be cloned into the P. putida bacterium for efficient PET degradation.

Here are the results of E. coli strain BL21's growth with liquid M9 media containing ethylene glycol as a sole carbon source:

We can conclude from these results that our E. coli strain BL21, like other E. coli strains, can utilize EG as a sole carbon source, with an optimal concentration of 30mM.

In order to test the PET degradation ability of the LC-Cutinase proteins, all variants were grown on M9 minimal medium plates with shredded PET pellets as a sole carbon source to test their ability to degrade PET. (for detailed instructions on the preparation of the media see Protocols).

As seen in the images above, all LC-Cutinase variants were able to grow on the plates containing shredded PET, suggesting that the PET is degraded and E. coli is utilizing one of its products (ethylene glycol).
We cannot conclude which variant degrades the PET with the highest efficiency, as the PET in the plates is not evenly spread and the amount of PET degraded cannot be measured in this assay.

However, we have clearly shown that our bacteria can utilize PET as a carbon source using the LC-Cutinase protein, which is a proof-of-concept for one part of our project.

To verify our hypothesis that PCA (Protocatechuic acid) is a suitable electron mediator for our fuel cell we measured its potential using cyclic voltammetry (CV).

Calculating the values from the CV measurements we found that the substance had a Potential of 174 mV. We concluded that the potential was low enough to function as an electron mediator in the system.

In order to test the ability of P. putida to grow in an electrochemical fuel cell we had to assess its ability to product a biofilm on Graphite-felt sheets like those used in the fuel cell.
We incubated the bacteria in a liquid LB medium and hanged a sheet of graphite-felt half inside the medium and half outside.

As seen from the photo above, our bacterium is able to form a biofilm on the graphite electrode.

After testing the compatability of each component of our fuel cell, we ran several tests to assess its efficiency:

We can see a big rise in power output induced in the sample containing bacteria in the 250-500 mV range, compared to the control. In the second day of measurements, there was a decline in power output probably due to cell death. The last measurement is not shown due to contamination in the control cell.

The inner resistance was measured in the first day with values of 60 Ω for the cell containing bacteria and 1000 Ω for the control.

Our test results may suggest that there is potential in constructing a fuel cell based on the electrochemical activity of P. putida KT 2440 with protocatechuic acid as an electron mediator. That being said, more experiments are required in order to confirm our hypothesis and determine the proper concentrations of the protocatechuic acid and characterize the mechanisms with which it mediates P. putida’s electron transfer. It should be mentioned that protocatechuic acid polymerizes in the presence of amine groups, a reaction that could interfere with the electron transfer. Future research should examine the prevention of such reactions, perhaps by reducing concentrations, replacing mediators etc.