1. Ong Kim Yao (4P3) Poh Yong Rui (4O3) Group 1-121

2.  Microbes can degrade HDPE plastic by using the polymer as a carbon source (Arutchelvi et al., 2008)  Exposure to UV radiation accelerates chemical degradation of HDPE plastic (Albano et al., 2005)

3.  Thermal exposure of HDPE plastics to accelerates light-induced degradation (Andrady, 1999)  Sierra et al. (2003) suggested that biodegradation of polychlorinated biphenyls (PCBs) occurs faster in soil conditions

4. To determine the optimum conditions for maximum biodegradaton of plastics To study the effects of different environmental conditions on the biodegradation of plastics To study the effects of varying exposure time to UV irradiation on the biodegradation of plastics

5.  The following factors affect the rate of biodegradation of HDPE plastic:  Exposure time to UV radiation  Environmental conditions for biodegradation  Types of microbe culture  Types of plastic

6. Constant Variables Amount of bacterial culture used Amount of culture medium for bacteria/fungus Amount of plastic used Independent Variables Types of bacterial culture Environmental conditions for biodegradation Exposure time to UV radiation Types of plastic Dependent Variables Change in dry mass of plastic samples Amount of dissolved O 2 gas present in test container Tensile strength and elongation at break

7.  Sterile vials  Inoculating loop  Alcohol burner  Incubator  Forceps  Oven (up to150°C)  UV lamp (for 365 nm UV radiation)  Electronic balance  Autoclave  Vernier dissolved O 2 probe  Datalogger  Spatula  Thermometer  Spectrophotometer  Rotary shaker

8.  Paper towels  HDPE plastic  Deionised water  Bleach  M63 minimal media  Cornware  Petri dishes  Nutrient agar powder  Bacterial cultures (Pseudomonas putida and Sphingomonas macrogoltabidus)  Nutrient broth  Loamy soil  Wire mesh  Ethanol  Aluminium foil

9. Culture Bacteria Prepare Environmental Conditions Pre-treatment of Plastics Exposing plastic to Bacteria Measure Dependent Variables

10. Bacterial culture Heat-treated HDPE plastic which is not UV-irradiated Purpose: To show that UV irradiation has an effect on plastic degradation No bacterial culture First set-up  UV-irradiated HDPE plastic only Second set-up  Heat-treated HDPE plastic only Purpose: To show that bacterial cultures have an effect on plastic degradation

11. For soil conditions, the best conditions concluded from plastic biodegradation in liquid medium was used, as shown in the above diagram.

12. Bacteria was cultured in sterile centrifuge tubes with nutrient broth. The cell density of the bacteria culture was adjusted to approximately 4 × 10 7 cells/ml.

13. 0.44

14. 0.46

15. 2ml of bacterial culture prepared earlier was inoculated in a sterile vial with 13.5ml of M63 minimal media and 4.5ml of NB. The control vial contained 15ml of M63 minimal media and 5ml of NB.

16. Soil was autoclaved to remove microbes. Sterile vial was filled with 20cm 3 of loamy soil. Soil was adjusted to 50% of maximum water capacity. 2ml of bacterial culture was added to the soil each.

17. Exposed to 365nm UV radiation for 72, 96, 120 hours Cut up into small pieces HDPE Plastic Grocery Bags Exposed to thermal radiation in the oven at 115°C for 48 hours Mass recorded weekly The entire experiment was repeated with cornware in place of HDPE plastics

18. Plastic samples were placed in vials containing liquid medium or soil. Liquid medium and bacterial culture were changed every week to remove waste material and dead cells.

19. Used to measure (every 7 days):  Change in dry mass of plastic samples  Amount of dissolved O 2 present Analytical Balance Dissolved O 2 Probe

20.  Scaled-up set-ups to measure  Tensile strength  Elongation at break  Focus of set-up  effects of different UV irradiation duration on degradation  Same methodology as normal set-ups except for the following changes

21. 80ml of liquid medium (with same ratio of NB and M63 medium). Plastic cut into rectangular shape. 100ml reagent bottles used.

22. Purpose of control: To show that different UV irradiation durations have an effect on plastic degradation

23. Mass showed increase then decrease in samples exposed to bacteria, as compared to the comparably constant graph of control samples Shows that bacterial exposure causes HDPE degradation P<0.05

24. Greater changes in mass of HDPE samples exposed to UV when compared to samples without exposure P<0.05

25. Similarly, greater changes in mass of HDPE samples exposed to UV Shows that UV irradiation increases rate of HDPE degradation P<0.05

26. Change in mass: 96h>120h>72h>0h

27. Change in mass: 72h>120h>96h>0h Shows that varying UV irradiation changes rate of HDPE degradation P<0.05

28.  T-test: p>0.05 for P. putida  Probably due to early stage of degradation  HDPE mass starting to fall  Change in mass was not significant initially  Expecting to see more changes in the following 3-4 weeks of exposure

29. Mass of HDPE sample exposed to P. putida was lower Suggests that P. putida is more efficient in degradation P<0.05

30.  p>0.05 for other UV exposure times  Probably due to early stage of degradation as elaborated earlier  Expecting P. putida to be more efficient in degradation

31. Increase in mass of samples exposed to bacteria

32. Increase is due to biofilm formation

33. Biofilm

34.  Formation of biofilm “is a pre- requisite for biodegradation” to occur (Arutchelvi et al., 2008)  Research showed a rise in density of biofilm attached to HDPE exposed to Pseudomonas sp., and density remained constant for 30 days (Balasubramanian et al., 2010)  Initial increase in mass might be attributed to the formation of biofilm

35.  Dissolved oxygen readings not significant  Readings fluctuated greatly  Probably because oxygen tends to escape and re-dissolve in the medium in order to achieve dynamic equilibrium with the atmosphere  Hence unable to reflect the degradation activities of the bacteria

36.  Growth of mold in some samples  Spores entered since containers cannot be air- tight  Try to prevent by keeping environment as sterile as possible

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