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 CultureBacteria 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 10.02.04.06.08.0

10.0

00.816666666666

667

2.955

5.26666666666667

7.96666666666667

Graph showing Cell Density of P. putida against Optical Density

Optical density/AU

Cell

dens

ity

/ 10^

8 ce

ll/m

l

0.44

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 10.02.04.06.08.0

01.6

3.6

6.1 6.4

Graph showing Cell Density of S. macrogoltabidus against Op-

tical Density

Optical density/AU

Cell

dens

ity

/ 10^

8 ce

ll/m

l Standard Curve for Bacteria Growth

0.46

Culturing Bacteria2ml 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 autoclav

ed 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

123456700.010.020.030.04

Effects of Different Bacterial Exposure on the Biodegradation

of HDPE PlasticsP. putida 120h UV

S. macro-goltabidus 120h UV

Control 120h UV

Time/week

Mas

s of

pla

stic

/gResults and Analysis

Mass showed increase then

decrease in samples exposed to bacteria, as compared to the

comparably constant graph of control samplesShows that bacterial

exposure causes HDPE degradation

P<0.05

1 2 3 4 5 6 700.010.020.030.04

Effects of Different Duration of UV Irradiation on the

Biodegradation of HDPE Plastics by P. putida 0h

72h96h120h

Time/weekMas

s of

pla

stic

/gResults 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 0h

72h96h120h

Time/weekMas

s of

pla

stic

/gResults 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 6 700.010.020.030.04

Effects of Different Duration of UV Irradiation on the

Biodegradation of HDPE Plastics by P. putida 0h

72h96h120h

Time/weekMas

s of

pla

stic

/gResults 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 0h

72h96h120h

Time/weekMas

s of

pla

stic

/gResults 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 PlasticsP. putida 72h UVS. macro-goltabidus 72h UV

Time/weekMas

s of

pla

stic

/gResults 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

123456700.010.020.030.04

Effects of Different Bacterial Exposure on the Biodegradation

of HDPE PlasticsP. putida 120h UV

S. macro-goltabidus 120h UV

Control 120h UV

Time/week

Mas

s of

pla

stic

/gResults and Analysis

Increase 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 0h

72h96h120h

Time/weekMas

s of

pla

stic

/gResults and Analysis

Increase 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

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

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