Team:PuiChing Macau/Proof Of Concept
Introduction:
In our project, our Food waste-PLA system is composed of Breaking down (glucose test), Fermentation (Acid tolerant) and PLA production system+phasin.
To confirm our concepts, we did food waste analyzing (pH tests) on the food waste, an acid challenge to our acid tolerant system.
Figure 1. Concept of our system
Acid Tolerant
We have known that e-coli cannot grow well in an acidic environment where food waste contributes to a low pH environment while we are generating lactic acid. (Lactic Acidosis Fermentation) That’s why we transformed the fabB gene to the e-coli to change fatty acids to unsaturated fatty acids to produce a persistent decrease in intracellular pH, eventually helping it grow better in a low pH environment.
Total colonies（CFU/mL) =
No. of colonies *Total dilution factor/Volume of the culture plate in mL
Figure 2a. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 4 (N=4, error bar: SEM)
Figure 2b. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 7 (N=4, error bar: SEM)
Figure 3. (1) flow chart of breaking down part
Figure 4. (2) flow chart of acid tolerant system
Figure 5. (3) flow chart of PLA-PHA production system.
PLA production system
For the final step, we transformed the PHAC and PCT gene to the bacteria to polymerase the lactic acid. We also added phasin, which could affect Phac on the polymer-elongating, similar to the prepolymerization PhaC. [1]
We did IR test and melting point testing on the PLA powder we made to ensure that it is the PLA we want.
Figure 6a. IR test result of PHA in and PHA un, to validate the molecules are PLA or PHA.
Figure 6b. IR test result of Phac in and Phac un
Figure 7c: IR test result of Phasin in and Phasin un
Figure 7d: IR test result of Control in and Control un
Conclusion
With the three parts validating our concept of our system, we are able to produce PLA and PHA. We made tests and experiments to improve our system and verify our products. However, our system is still not perfect, and we will make it better by doing more testing and studying more about PLA and PHA.
References
1. Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase, NCBI, 2014, (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993283/)