Results
One method to assess the activity of our designed mutants is a using the pNP-Butyrate degradation assay. pNP-B is similar in structure to the PET monomer, therefore an increase in its degradation could suggest an increase in the degradation of PET. Upon degradation, pNP-B undergoes a color reaction measurable in a wavelength of 405nm. We performed testing sessions with 96 well plates, testing each variant many times and using the pNP-B, bacteria transformed with an empty vector and without a vector as controls.
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 M9 minimal medium with glucose and hanged a sheet of graphite-felt half inside the medium and half outside.
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(see Protocols).
As seen from the results we achieved growth on PE, as expected, and on PET, which the bacteria is not known to utilize as a carbon source. We wanted to examine the PE and PET degradation by R. ruber as we didn't have a method for measuring the amount of PE/PET degraded. In order to do so we washed the pellets from any trace of bacteria and examined the samples through a Scanning Electron Microscope:
As seen from the images above, the bacteria are growing on the surface of the PE, suggesting they are utilizing it as a nutrient.
Similar to the PE pellets, we can see the bacteria are growing on the PET, but here we can see a clear difference between the control and the digested PET - there are breaks in the pellet and there is a rod shaped fiber that is more exposed, suggesting it was digested by the bacteria. Moreover, we can see bacteria growing on the edges of the fiber suggesting they are responsible for exposing it.
Our experiments with the R. ruber bacterium show a clear degradation of PET by it, a fact not known before.
