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Team:ITB Indonesia/Data
From 2014.igem.org
Data
- Ethylene Glycol Assay using Chromic Acid
Poly(ethylene terephthalate) (PET) is a plastic that is a polymer of ethylene terephthalate (C10H8O4) units (Sarker et al., 2010). Polyethylene terephthalate (PET) can be degrade via limited enzymatic hydrolysis of the ester bond of the polymer backbone, one enzyme that have been assesed for this purpose is cutinase (Ribitsch et al., 2012). When polyethylene terephthalate degraded, it will produce terephthalic acid and ethylene glycol (Venkatachalam et al., 2012). Ethylene glycol is one of alcohol compound. Oxidation of alcohols by strong oxidants such as chromic acid (K2Cr2O7) in suphuric acid(H2SO4) is possible, but differs depending on the degree of alcohol. If a reaction has occurred using chromic acid in sulphuric acid, there is a color change from orange to green. In our project we use chromic acid to detect ethylene glycol as product of the PET degradation by LC cutinase enzyme.figure 1 shown the result of ethylene glycol assay using chromic acid.
Figure 1. Ethylene Glycol Concentration LC ITB and LC UC Davis
Each sample used for measure the absorbance contain a fixed amount of chromic acid. The absorbance of the chromic acid will represent the total amount of ethylene glycol inversely. It means that if the absorbance is high, the amount of ethylene glycol in solution is low, and if the absorbance is low, the amount of ethylene glycol in solution is high. If the absorbance is high, the amount of chromic acid is high, which means that the amount of ethylene glycol being oxidized by chromic acid is low and vice versa. Both of LC Cutinase UC Davis and OmpA-LC Cutinase ITB_Indonesia shown the degrading activity of PET become ethylene glycol as once of the product.The optimum time to produce ethylene glycol is on the fourth hour after inoculation.
Reference
Ribitsch D, Enrique HA, Katrin G, Anita D, Sabine Z, Annemarie M, Rosario DR, Georg S, Karl G, Helmut S and Georg MG. 2012. A New Esterase from Thermobifida halotolerans Hydrolyses Polyethylene Terephthalate (PET) and Polylactic Acid (PLA). Polymers. 4: 617-629
Sarker M, Aminul K, Mohammad MR, Mohammed M and ASMD Mohammad. 2011. Waste Polyethylene Terephthalate (PETE-1) Conversion into Liquid Fuel. Journal of Fundamentals of Renewable Energy and Applications. 1
Venkatachalam S, Shilpa GN, Jayprakash VL, Prashant RG, Krishna R and Anil KK. 2012. Degradation and Recyclability of Poly (Ethylene Terephthalate). Intech
Scaning Electron Microscope (SEM) Result Analysis
For biodegrading experiments, we used 3x5 cm2 bottle plastics with average weight 0.05 g. Then, we washed it several times with water and ethanol.
Plastic sample was applying just before we inoculated the bacteria on the medium. Bacterial culture were grown at 37C in Luria-Broth medium. After 3 days of incubation, we washed the plastic with water and ethanol then dried for measurement of the weight loss.
Meanwhile, for UC Davis bacterial culture, we inoculated the bacteria in Luria-Broth medium supplemented with 170 ppm cloramphenicol. After an absorbance A600 nm of 0.6 was reached, we added 1% of arabinose and plastic sample. After 2 days of incubation, we washed the plastic with water and ethanol then dried for measurement of the weight loss.
We used medium supplemented with antibiotics and plastic sample as control on this experiment. The scaning electron microsccopy analysis of fractured surface of PET film was carried out using Scaning electron microscope. The surface of the treated PET samples were coated with conductive heavy metals such as gold/palladium.
SEM image shows that cracks were observed at the surface of a plastic sample (PET) after incubation on the bacterial culture. But, there is no significant weight decreased of the plastics samples. From this SEM study we conclude that both our LC Cut UC Davis and OmpA-LC-Cut Fussion ITB 2014 able to degrade PET plastics samples. Figure 1. shown the control sampel that we could not observe any cracks. Figure 2 and 3 shown the PET degradation by Lc cutinase from UC davis and from ITB_Indonesia.
