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Microbial Degradation Activities

Microbial Degradation Activities. Ong Kim Yao (4P3) Poh Yong Rui (4O3). Group 1-121. Background. Microbes can degrade HDPE plastic by using the polymer as a carbon source ( Arutchelvi et al. , 2008 )

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Microbial Degradation Activities

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

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

  3. Background • 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. Objectives

  5. Hypothesis • 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. Variables

  7. Apparatus • Electronic balance • Autoclave • Vernier dissolved O2 probe • Datalogger • Spatula • Thermometer • Spectrophotometer • Rotary shaker • Sterile vials • Inoculating loop • Alcohol burner • Incubator • Forceps • Oven (up to150°C) • UV lamp (for 365 nm UV radiation)

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

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

  10. Set-up • 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 • Bacterial culture • Heat-treated HDPE plastic which is not UV-irradiated • Purpose: To show that UV irradiation has an effect on plastic degradation

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

  12. Microorganism Culture

  13. Standard Curve for Bacteria Growth 0.44

  14. Standard Curve for Bacteria Growth 0.46

  15. Culturing Bacteria

  16. Preparing Soil Conditions

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

  18. Exposing Plastic to Bacteria

  19. Measure Dependent Variables Used to measure (every 7 days): • Change in dry mass of plastic samples • Amount of dissolved O2 present Analytical Balance Dissolved O2 Probe

  20. Measure Dependent Variables • 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. Measure Dependent Variables

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

  23. Results and Analysis 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. Results and Analysis Greater changes in mass of HDPE samples exposed to UV when compared to samples without exposure P<0.05

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

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

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

  28. Results and Analysis • 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. Results and Analysis Mass of HDPE sample exposed to P. putidawas lower Suggests that P. putidais more efficient in degradation P<0.05

  30. Results and Analysis • 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. Results and Analysis Increase in mass of samples exposed to bacteria

  32. Results and Analysis Increase in mass of samples exposed to bacteria Increase is due to biofilm formation

  33. Results and Analysis Biofilm

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

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

  37. References • Aamer Ali Shah (2007). Role of Microorganism in Biodegradation of Plastics. Retrieved October 30, 2011 from http://eprints.hec.gov.pk/2361/1/2216.htm • Albano, C., Karam, A., Gonzalez, G., Dominguez, N., Sanchez, Y., Manzo, F. & Guzman-Garcia, C. (2005). Effect of gamma irradiation on HDPE/HA (80:20) composites.  Polymers for Advanced Technologies, 16, 283–285. Retrieved October 25, 2011 from http://onlinelibrary.wiley.com/doi/10.1002/pat.580/pdf • Anthony L. Andrady (1999). Environmental Degradation of Plastics under Land and Marine Exposure Conditions. Retrived October 30, 2011 from http://www.5gyres.org/media/Environmental_Degradation%20of%20Plastics_by_Andrady.pdf • Arutchelvi, J., Sudhakar, M., Arkatkar, Ambika, Doble, Mukesh, Bhaduri, Sumit & Uppara, ParasuVeera (2008). Biodegradation of polyethylene and polypropylene.  Indian Journal of Biotechnology, 7, 9–22. Retrieved October 25, 2011 from http://nopr.niscair.res.in/bitstream/123456789/7326/4/IJBT%207%281%29%209-22.pdf • Balasubramanian, V., Natarajan, K., Hemambika, B., Ramesh, N., Sumathi, C.S., Kottaimuthu, R., Rajesh Kannan, V. (2010). High-density polyethylene (HDPE)-degrading potential bacteria from marine ecosystem of Gulf of Mannar, India.  Letters in Applied Microbiology, 51, 205–211. Retrieved June 27, 2012 from http://onlinelibrary.wiley.com/doi/10.1111/j.1472-765X.2010.02883.x/abstract;jsessionid=764380A26A30D96287C29066129DD8FD.d03t01?deniedAccessCustomisedMessage=&userIsAuthenticated=false • Borghei, Mehdi, Karbassi, Abdolreza, Khoramnejadian, Shahrzad, Oromiehie, Abdolrasoul & Javid, Amir hossein (2010). Microbial biodegradable potato starch based low density polyethylene.  African Journal of Biotechnology, 9, 4075–4080. Retrieved December 9, 2011 from http://www.academicjournals.org/AJB/PDF/pdf2010/28Jun/Borghei%20et%20al.pdf

  38. References • Farrell, A & Quilty, B (2002). Substrate-dependent autoaggregation of Pseudomonas putida CP1 during the degradation of mono-chlorophenols and phenol.  Journal of Industrial Microbiology & Biotechnology, 28, 316–324. Retrieved December 7, 2011 from http://www.springerlink.com/content/15htgedtjefxv22v/fulltext.pdf • Gijsman, Pieter, Meijers, Guido & Vitarelli, Giacomo (1999). Cornparison of the UV-degradation chemistry of polypropylene, polyethylene, polyamide 6 and polybutyleneterephthalate.  Polymer Degradation and Stability, 65, 433–441. Retrieved October 25, 2011 from http://cid.ispa.asso.fr/GEIDEFile/Degrad_0001.PDF?Archive=191929191910&File=Degrad+0001_PDF • Huang, Yi-Li, Li, Qing-Biao, Deng, Xu, Lu, Ying-Hua, Liao, Xin-Kai, Hong, Ming-Yuan & Wang, Yan (2004). Aerobic and anaerobic biodegradation of polyethylene glycols using sludge microbes.  Process Biochemistry, 40, 207–211. Retrieved October 25, 2011 from http://envismadrasuniv.org/Biodegradation/pdf/Glycols%20using%20sludge%20microbes.pdf • Johnson, Kenneth E., Pometto, Anthony L. III & Nikolov, Zivko L. (1993). Degradation of Degradable Starch-Polyethylene Plastics in a Compost Environment.  American Society for Microbiology, 59, 1155–1161. Retrieved November 8, 2011 from http://aem.asm.org/content/59/4/1155.full.pdf • Kaur, Inderjeet & Gautam, Neena (2010). Starch Grafted Polyethylene Evincing Biodegradation Behaviour.  Malaysian Polymer Journal, 5, 26–38. Retrieved December 9, 2011 from http://www.cheme.utm.my/mpj/images/100501_3nee1.pdf • Lanini, S., Houi, D., Aguilar, O. & Lefebvre, X. (2001). The role of aerobic activity on refuse temperature rise: II. Experimental and numerical modelling.  Waste Management & Research, 19, 58–69. Retrieved October 25, 2011 from http://wmr.sagepub.com/content/19/1/58.full.pdf • Morancho, J.M., Ramis, X., Fernández, X., Cadenato, A., Salla, J.M., Vallés, A., Contat, L. & Ribes, A. (2006). Calorimetric and thermogravimetric studies of UV-irradiated polypropylene/starch-based materials aged in soil.  Polymer Degradation and Stability, 91, 44–51. Retrieved November 8, 2011 from http://anvalllu.webs.upv.es/papers/2006_DSCTGAUVPPstarchsoil.pdf

  39. References • Mostafa, H. M., Sourell, H. & Bockisch, F. J. (2010). The Mechanical Properties of Some Bioplastics Under Different Soil Types for Use as a Biodegradable Drip Tubes.  Agricultural Engineering International: the CIGR Ejournal, 12, 1–16. Retrieved November 7, 2011 from http://www.cigrjournal.org/index.php/Ejounral/article/viewFile/1497/1270 • Nanda, Sonil, Sahu, SmitiSnigdha & Abraham, Jayanthi (2010). Studies on the biodegradation of natural and synthetic polyethylene by Pseudomonas spp.  Journal of Applied Sciences & Environmental Management, 14, 57–60. Retrieved October 29, 2011 from http://www.bioline.org.br/pdf?ja10027 • Olive Green Marketing (n.d.). Olive Green. Retrieved December 9, 2011 from http://www.olivegreen.com.sg/ • Orhan, Yüksel, Hrenovićb, Jasna & Büyükgüngöra, Hanife (2004). Biodegradation of plastic compost bags under controlled soil conditions. ActaChimicaSlovenica, 51, 579–588. Retrieved November 7, 2011 from http://acta.chem-soc.si/51/51-3-579.pdf • Pometto, Anthony L. III, Lee, Byungtae & Johnson, Kenneth E. (1992). Production of an Extracellular Polyethylene-Degrading Enzyme(s) by Streptomyces Species.  Applied and Environmental Microbiology, 58, 731–733. Retrieved December 9, 2011 from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC195314/pdf/aem00043-0307.pdf • Premraj, R & Doble, Mukesh (2005). Biodegradation of polymers.  Indian Journal of Biotechnology, 4, 186–193. Retrieved December 9, 2011 from http://nopr.niscair.res.in/bitstream/123456789/5718/1/IJBT 4(2) 186-193.pdf • Reynolds, Jackie & Farinha, Mark (2005). Counting Bacteria.  Richland College, 1–10. Retrieved December 7, 2011 from http://www.biotech.ug.edu.pl/odl/doc/numbers.pdf • Sierra, Isabel, Valera, José Luis, Marina, M. Luisa & Laborda, Fernando (2003). Study of the biodegradation process of polychlorinated biphenyls in liquid medium and soil by a new isolated aerobic bacterium (Janibacter sp.).  Chemosphere, 53, 609–618. Retrieved November 7, 2011 from http://infolib.hua.edu.vn/Fulltext/ChuyenDe2009/CD240/60.pdf • Verma, Shefali (2002). Anaerobic Digestion of Biodegradable Organics in Municipal Solid Wastes.  Retrieved October 25, 2011 from http://www.seas.columbia.edu/earth/wtert/sofos/Verma_thesis.pdf

  40. FOR YOUR UNDIVIDED ATTENTION Thank you

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