Ong Kim Yao (4P3) Poh Yong Rui (4O3) Group 1-121.
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Transcript of Ong Kim Yao (4P3) Poh Yong Rui (4O3) Group 1-121.
Microbial Degradation ActivitiesOng 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
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
Hypothesis
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
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
Apparatus
Paper towels HDPE plastic Deionised water Bleach M63 minimal media Cornware Petri dishes Nutrient agar
powder
Materials
Bacterial cultures (Pseudomonas putida and Sphingomonas macrogoltabidus)
Nutrient broth Loamy soil Wire mesh Ethanol Aluminium foil
Procedure
Culture Bacteria
Prepare Environment
al Conditions
Pre-treatment of Plastics
Exposing plastic to Bacteria
Measure Dependent Variables
•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 cultureFirst set-up UV-irradiated HDPE plastic onlySecond set-up Heat-treated HDPE plastic only•Purpose: To show that bacterial cultures have an effect on plastic degradation
Set-up
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
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 10.0
2.0
4.0
6.0
8.0
10.0
0
0.816666666666667
2.955
5.26666666666667
7.96666666666667
Graph showing Cell Density of P. putida against Optical Density
Optical density/AU
Cell d
en
sity
/ 1
0^
8
cell/m
l
0.44
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 10.0
2.0
4.0
6.0
8.0
0
1.6
3.6
6.1 6.4
Graph showing Cell Density of S. macrogoltabidus against Op-
tical Density
Optical density/AU
Cell d
en
sit
y /
10^
8
cell/m
l
Standard Curve for Bacteria Growth
0.46
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 autoclave
d 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
Exposed to
365nm UV
radiation for 72, 96, 120 hours
Cut up
into small piece
s
HDPE Plastic
Grocery Bags
Exposed to
thermal radiation in the oven at 115°C for 48 hours
Mass recorde
d weekly
The entire experiment was repeated with cornware in place of HDPE plastics
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.
Used to measure (every 7 days): Change in dry mass of plastic samples Amount of dissolved O2 present
Measure Dependent Variables
Analytical Balance
Dissolved O2 Probe
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
Measure Dependent Variables
80ml of liquid medium (with
same ratio of NB and M63 medium).
Plastic cut into
rectangular shape.
100ml reagent bottles used.
Measure Dependent Variables
•Purpose of control: To show that different UV irradiation durations have an effect on plastic degradation
12345670
0.01
0.02
0.03
0.04
Effects of Different Bacterial Exposure on the Biodegradation
of HDPE Plastics
P. putida 120h UV
S. macro-goltabidus 120h UV
Control 120h UV
Time/week
Mass o
f p
lasti
c/g
Results and AnalysisMass 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
1 2 3 4 5 6 70
0.010.020.030.04
Effects of Different Duration of UV Irradiation on the
Biodegradation of HDPE Plastics by P. putida
0h72h96h120h
Time/weekMass o
f p
lasti
c/g
Results and AnalysisGreater changes in mass of HDPE samples exposed
to UV when compared to
samples without exposure
P<0.05
1 2 3 4 5 6 70
0.02
0.04
Effects of Different Duration of UV Irradiation on the
Biodegradation of HDPE Plastics by S. macrogoltabidus
0h72h96h120h
Time/weekMass o
f p
lasti
c/g
Results and AnalysisSimilarly, greater changes in mass of HDPE samples exposed to UV
Shows that UV irradiation increases rate of HDPE
degradation
P<0.05
1 2 3 4 5 6 70
0.010.020.030.04
Effects of Different Duration of UV Irradiation on the
Biodegradation of HDPE Plastics by P. putida
0h72h96h120h
Time/weekMass o
f p
lasti
c/g
Results and AnalysisChange in mass: 96h>120h>72h>0h
1 2 3 4 5 6 70
0.02
0.04
Effects of Different Duration of UV Irradiation on the
Biodegradation of HDPE Plastics by S. macrogoltabidus
0h72h96h120h
Time/weekMass o
f p
lasti
c/g
Results and AnalysisChange in mass: 72h>120h>96h>0h
Shows that varying UV irradiation changes rate
of HDPE degradation
P<0.05
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
1 2 3 4 5 6 70
0.02
0.04
Effects of Different Bacterial Exposure on the Biodegradation
of HDPE Plastics
P. putida 72h UVS. macro-goltabidus 72h UV
Time/weekMass o
f p
lasti
c/g
Results and AnalysisMass of HDPE sample exposed to P. putida was
lower
Suggests that P. putida is more efficient in
degradation
P<0.05
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
12345670
0.01
0.02
0.03
0.04
Effects of Different Bacterial Exposure on the Biodegradation
of HDPE Plastics
P. putida 120h UV
S. macro-goltabidus 120h UV
Control 120h UV
Time/week
Mass o
f p
lasti
c/g
Results and AnalysisIncrease in mass
of samples exposed to
bacteria
1 2 3 4 5 6 70
0.02
0.04
Effects of Different Duration of UV Irradiation on the
Biodegradation of HDPE Plastics by S. macrogoltabidus
0h72h96h120h
Time/weekMass o
f p
lasti
c/g
Results and AnalysisIncrease in mass
of samples exposed to
bacteria
Increase is due to biofilm formation
Results and Analysis
Biofilm
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
Results and Analysis
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 air-tight
Try to prevent by keeping environment as sterile as possible
Problems
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