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)
 Exposure to UV radiation accelerates
chemical degradation of HDPE plastic
(Albano et al., 2005)
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
Objectives
• 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
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

Variables
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 O2 gas
present in test
container
• Tensile strength
and elongation
at break
Apparatus







Sterile vials
Inoculating loop
Alcohol burner
Incubator
Forceps
Oven (up to150°C)
UV lamp (for
365 nm UV
radiation)








Electronic balance
Autoclave
Vernier dissolved O2
probe
Datalogger
Spatula
Thermometer
Spectrophotometer
Rotary shaker
Materials






Bacterial cultures
(Pseudomonas putida and
Sphingomonas
macrogoltabidus)
Nutrient broth
Loamy soil
Wire mesh
Ethanol
Aluminium foil








Paper towels
HDPE plastic
Deionised water
Bleach
M63 minimal media
Cornware
Petri dishes
Nutrient agar powder
Procedure
Culture
Bacteria
Pre-treatment
of Plastics
Prepare
Environmental
Conditions
Measure
Dependent
Variables
Exposing plastic
to Bacteria
Set-up
•No bacterial culture
•Bacterial
First set-upculture
 UV-irradiated HDPE plastic only
•Heat-treated
plastic which
is not
UV-irradiated
Second set-up HDPE
 Heat-treated
HDPE
plastic
only
•Purpose:
irradiation
hashave
an effect
on plastic
•Purpose: To
To show
show that
that UV
bacterial
cultures
an effect
on
degradation
plastic degradation
Set-up
For soil conditions, the best conditions concluded from plastic
biodegradation in liquid medium was used, as shown in the above
diagram.
Microorganism Culture
Bacteria was
cultured in
sterile
centrifuge tubes
with nutrient
broth.
The cell density
of the bacteria
culture was
adjusted to
approximately 4
× 107 cells/ml.
Standard Curve for Bacteria Growth
Cell density / 10^8 cell/ml
Graph showing Cell Density of P. putida
against Optical Density
10.0
8.0
7.966666667
6.0
5.266666667
4.0
2.955
2.0
0.816666667
0.0
0
0
0.2
0.44 0.6
0.4
Optical density/AU
0.8
1
Standard Curve for Bacteria Growth
Cell density / 10^8 cell/ml
Graph showing Cell Density of
S. macrogoltabidus against Optical Density
8.0
6.0
6.1
4.0
6.4
3.6
2.0
1.6
0.0
0
0
0.2
0.40.46
0.6
Optical density/AU
0.8
1
Culturing Bacteria
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.
Preparing Soil Conditions
Soil was
autoclaved
to remove
microbes.
Sterile vial
was filled
with 20cm3
of loamy
soil.
Soil was
adjusted to
50% of
maximum
water
capacity.
2ml of
bacterial
culture was
added to
the soil
each.
Pre-treatment of Plastics
HDPE
Plastic
Grocery
Bags
The entire experiment
was repeated with
cornware in place of
Exposed
HDPE plastics
Exposed
to 365nm to thermal
radiation
UV
in the
radiation
oven at
for 72, 96,
120 hours 115°C for
48 hours
Cut up
into
small
pieces
Mass
recorded
weekly
Exposing Plastic to Bacteria
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.
Measure Dependent Variables
Analytical Balance
Dissolved O2 Probe
Used to measure (every 7 days):


Change in dry mass of plastic samples
Amount of dissolved O2 present
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

Measure Dependent Variables
80ml of liquid
medium (with
same ratio of NB
and M63 medium).
100ml reagent
bottles used.
Plastic cut
into
rectangular
shape.
Measure Dependent Variables
•Purpose of control: To show that different UV
irradiation durations have an effect on plastic
degradation
Mass showed increase
then decrease in samples
exposed to bacteria, as
compared to the
Effects of Different Bacterial
Exposure
comparably
constant
graph of
samples
on the Biodegradation
ofcontrol
HDPE
Results and Analysis
Mass of plastic/g
Plastics
0.04
0.03
Shows that bacterial exposure
causes HDPE degradation
P. putida 120h UV
0.02
S. macrogoltabidus
120h UV
Control 120h UV
0.01
0
1
2
3
4
5
Time/week
6
7
P<0.05
Results and Analysis
Greater changes in
mass of HDPE samples
exposed to UV when
Effects of Different Duration
of UV
compared
to samples
without exposure
Irradiation on the Biodegradation
of
Mass of plastic/g
HDPE Plastics by P. putida
0.04
0.03
0h
72h
96h
120h
0.02
0.01
0
1
2
3
4
5
Time/week
6
7
P<0.05
Results and Analysis
Mass of plastic/g
Similarly, greater
changes in mass of
HDPE
samples
Duration
of UV
exposed to UV
0.05
Effects of Different
Irradiation on the Biodegradation of
HDPE
Plastics
S. irradiation
macrogoltabidus
Shows
thatbyUV
increases rate of HDPE
degradation
0.04
0.03
0h
72h
96h
120h
0.02
0.01
0
1
2
3
4
5
Time/week
6
7
P<0.05
Change in mass: 96h>120h>72h>0h
Results and Analysis
Mass of plastic/g
Effects of Different Duration of UV
Irradiation on the Biodegradation of
HDPE Plastics by P. putida
0.04
0.03
0h
72h
96h
120h
0.02
0.01
0
1
2
3
4
5
Time/week
6
7
Mass of plastic/g
Change in mass: 72h>120h>96h>0h
Results and Analysis
0.05
Effects of Different Duration of UV
Irradiation on the Biodegradation of
HDPE Plastics
macrogoltabidus
Shows by
thatS.varying
UV
irradiation changes rate of
HDPE degradation
0.04
0.03
0h
72h
96h
120h
0.02
0.01
0
1
2
3
4
5
Time/week
6
7
P<0.05
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
Results and Analysis
Mass of HDPE sample
exposed to P. putida
Bacterial Exposure
was lower
Mass of plastic/g
Effects of Different
on the Biodegradation of HDPE Plastics
0.05
Suggests that P. putida is more
efficient in degradation
0.04
0.03
P. putida 72h UV
0.02
S. macrogoltabidus
72h UV
0.01
0
1
2
3
4
5
Time/week
6
7
P<0.05
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

Results and Analysis
Increase in mass of
samples exposed
to
Bacterial
Exposure
bacteria
Mass of plastic/g
Effects of Different
on the Biodegradation of HDPE
Plastics
0.04
0.03
P. putida 120h UV
0.02
S. macrogoltabidus
120h UV
Control 120h UV
0.01
0
1
2
3
4
5
Time/week
6
7
Results and Analysis
Increase in mass of
samples exposed
to
Effects of Different Duration
of UV
bacteria
Irradiation on the Biodegradation
of
Mass of plastic/g
HDPE Plastics by S. macrogoltabidus
0.05
Increase is due to
biofilm formation
0.04
0.03
0.02
0.01
0
1
2
3
4
5
Time/week
6
7
0h
72h
96h
120h
Results and Analysis
Biofilm
Results and Analysis
Formation of biofilm “is a prerequisite 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

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
Problems
Growth of mold in some
samples
 Spores entered since
containers cannot be airtight
 Try to prevent by keeping
environment as sterile as
possible

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, Parasu Veera (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.1472765X.2010.02883.x/abstract;jsessionid=764380A26A30D96287C29066129DD8FD.d03t01?deniedAccessCust
omisedMessage=&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
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 polybutylene terephthalate. 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 StarchPolyethylene 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
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, Smiti Snigdha & 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. Acta Chimica Slovenica, 51, 579–588. Retrieved November 7, 2011 from http://acta.chemsoc.si/51/51-3-579.pdf
Pometto, Anthony L. III, Lee, Byungtae & Johnson, Kenneth E. (1992). Production of an Extracellular PolyethyleneDegrading 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
THANK YOU
FOR YOUR UNDIVIDED ATTENTION