This document summarizes a research study on the biodegradation of polystyrene foam by microorganisms found in a landfill in Thailand. Samples of microbes were taken from polystyrene foam and contaminated soil in the landfill. The microbes were cultured with polystyrene as the sole carbon source to identify those capable of degrading it. Analysis of the cultures over time found shifts in the dominant microbial species. Selected species were identified through molecular techniques as known aromatic compound degraders, including Herbaspirillum seropedicae and Ochrobactrum sp. Scanning electron microscopy was used to observe physical changes to polystyrene exposed to the cultures, indicating biodegradation

1. Biodegradation of Polystyrene Foam
by the Microorganisms from Landfill
Researcher : Pat Pataranutaporn1
Assistant prof. Savaporn Supaphol2
, prof. Amornrat Phongdara3
,Sureeporn Nualkaew3
1
PSU.Wittayanusorn school, 2
Kasetsart University, 3
Prince of Songkhla University

2. Pat Pataranutaporn
!
Assistant prof. Savaporn Supaphol
prof. Amornrat Phongdara
Sureeporn Nualkaew
Biodegradation of Polystyrene Foam
by the Microorganisms from Landfill

3. Hi, My name is Pat. I’m a
high school student from Thailand
with a weird hobby, doing research
project. This is one of my proudest
research. I would like to invite you
to take a look on this
!
Enjoy
Pat

4. !
• Non-biodegradable in the environment
• Made from non-renewable petroleum products
• Chronic, low-level exposure risks undetermined
“Styrofoam”
Polystyrene
Physical PropertiesDisadvantages
• chemical formula is (C8H8)n
• monomer styrene
• Thermoplastic
• blowing agents
Introduction !4

5. Bacteria nutritional requirements
!
‣ Energy source
‣ Carbon source
‣ Nitrogen source
‣ Minerals
‣ Water
‣ Growth factors
http://faculty.ccbcmd.edu/courses/bio141/
lecguide/unit6/metabolism/growth/factors.html
Polystyrene structure
Biodegradation
Introduction !5
Possibly work?

6. Aims of the research
‣To identify the microbe that able to growth in the
condition, which polystyrene is a sole carbon source
!
‣To study the changing of microbe community
structure in the selective culture which polystyrene is
a sole carbon source
!
‣To observe the biodegradability of polystyrene
Introduction !6

7. Methodology

8. Agarcultivation
Degradability
observation
(SEM)
Microbe
sampling
Community
fingerprint
16s Ribosomal RNA
identification
Molecular
cloning
Phylogenetic
tree
2months later
!8Methodology
Screening
Cultivation

9. Methodology !9
Degradability
observation
(SEM)
Agarcultivation
Phylogenetic
tree
2months laterScreening
Cultivation
Microbe
sampling
Community
fingerprint
16s Ribosomal RNA
identification
Molecular
cloning
Microbe sampling & cultivation

10. 2 aspects of samples
were collected from
the landfill that was
contaminated by
Polystyrene foam in
Pattani,Thailand
Styrofoam in the landfill Contaminated soil
Methodology !10Community structure analysis

11. Control
MSM broth
+ Sterile Polystyrene
!
!
S
MSM broth
+ Sterile Polystyrene
+ Landfill soil
F
MSM broth
+ Sterile Polystyrene
+ Landfill styrofoam
MSM broth
!
‣K2HPO4
‣KH2PO4
‣(NH4)2SO4
‣MgSO4
‣FeSO4.2HO2
‣MnCl2.4H2O
‣CoCl2.6H2O
‣CuCl2.2H2O
‣NiCl2.6H2O
‣Na2MoO4.2H2O
‣ZnSO4.7H2O
‣H3BO3
Sterile Polystyrene
Methodology !11Community structure analysis

12. Shake for 1 month then
inoculate to the new fresh
broth for sub culture.
Methodology !12Community structure analysis

13. Methodology !13Community structure analysis
Every week, The cell suspension in particular flask was taken
to the eppendorf then stored at 2C๐ for stop bacteria growth. This
solution used to monitor the changing of bacteria population.

14. 0 1 2 3 4 5 6 7 8 96 7 8
time(week)
1
400 µl.
5 9
transfer culture
Methodology !14Community structure analysis
Sampling schedule

15. Methodology !15
Screening
Cultivation
Degradability
observation
(SEM)
Agarcultivation
Microbe
sampling
Community
fingerprint
16s Ribosomal RNA
identification
Molecular
cloning
Phylogenetic
tree
2months later
Community structure analysis

16. DNA Extraction
(Methode : QIAamp Protocol)
Polymerase Chain Reaction (PCR)
16S rRNA gene Amplification
Primer VFC &VR
Denature Gradient Gel
Electrophoresis (DGGE)
Community structure analysis
16s Ribosomal RNA
identification
Community
fingerprint
Cell suspensions collected
from each week of cultivation.
Methodology !16Community structure analysis

17. Result !17
DNA Replication : PCR(TopTaq Master Mix Kit)
16S rRNA gene Amplification
by using Primer VR (Medlin et al., 1998) & VFC (Muyzer et al., 1993)
Community structure analysis

18. Community structure trend
time(week)
Microbe diversity
Dominant species
DGGE
Denature Gradient
Gel Electrophoresis
Each DNA band represent 1 microbe
Looking for survivor!
Methodology !18Community structure analysis

19. Marker
Soilweek1
Soilweek5
Soilweek6
Soilweek7
Soilweek8
Soilweek20
Foamweek1
Foamweek5
Foamweek6
Foamweek7
Foamweek8
Foamweek20
Controlweek6
Controlweek7
Controlweek8
Marker
Marker
Negative
DGGE 26/04/55
Running 300 minute
from PCR product 24/04/55
template use 8 µl.
Bacteria from styrofoamBacteria from soil Control
Continuing band & found in control
Continuing band
Non-continuing band
Result !19Community structure analysis

20. Methodology !20
Marker
Soilweek1
Soilweek5
Soilweek6
Soilweek7
Soilweek8
Soilweek20
Foamweek1
Foamweek5
Foamweek6
Foamweek7
Foamweek8
Foamweek20
Controlweek6
Controlweek7
Controlweek8
Marker
Marker
Negative
Bacteria from styrofoamBacteria from soil Control
Selected DNA
Molecular cloning &
identification
Selected for cloning

21. Methodology !21
DNA from S week7, F week 7 and con week 7
Polymerase Chain Reaction (PCR)
16S rRNA gene Amplification
Primer AF1 & 1541R
16s Ribosomal RNA
identification
Molecular cloning &
identification
Molecular
cloning
Ligate with pGEM T-Easy Plasmid
Transfer Plasmid to the competent cell (E.Coli) + Propagate
extracted Plasmid + cutcheck with EcoR1
Nucleotide sequencing
Phylogenetic
tree
Blasting + Neighbourhood
joining tree contracting
Purify Plasmid + cutcheck with EcoR1

22. Result !22Molecular cloning &
identification
extracted Plasmid + cutcheck with EcoR1
Purify Plasmid + cutcheck with EcoR1
PCR Product
16S rRNA gene Amplification
Primer AF1 & 1541R
Gel electrophoresis

23. Result !23Molecular cloning &
identification
Sequence report - Electropherogram
F3 F10 S7
Control 4 Control 7 F5

24. Result !24Molecular cloning &
identification
Sequence blasting
Sample
Len
gth
(bp)
Similar sequence
Ma
x
ide
n
Ma
x
sco
re
E.Va
lue
F10 444 Herbasprillium.sp 98% 753 0.0
F3 504 Massialia aerilata 97% 830 0.0
S7 485 Caulobacter segnis
ATCC 21756
98% 830 0.0
Control4 1,055 Azohydromonas
australica
83%1297 0.0
Control7 1,006 Ochrobactrum
rhizosphaerea
82%1193 0.0

25. Result !25Molecular cloning &
identification
Herbaspirillum chlorophenolicum
Herbaspirillum frisingense
Herbaspirillum seropedicae
F10
Collimonas arenae
Herminiimonas glaciei
Janthinobacterium lividum
Janthinobacterium agaricidamnosum
Janthinobacterium agaricidamnosum(2)
Massilia brevitalea
Naxibacter varians
Naxibacter haematophilus
F3
Massilia aerilata
Methylibium petroleiphilum PM1
Schlegelella thermodepolymerans
Azohydromonas lata
Rubrivivax gelatinosus IL144
Aquincola tertiaricarbonis
Control4
Azohydromonas australica
Brevundimonas nasdae
Streptomyces longisporoflavus
Mycoplana bullata
S7
Caulobacter segnis ATCC 21756
Phenylobacterium koreense
Rhizobium alamii
Ensifer adhaerens
Sinorhizobium fredii NGR234
Brucella ovis ATCC 25840
Ochrobactrum haematophilum
Control7
Ochrobactrum rhizosphaerae
out group
0.00783
0.00391
0.00391
0.00922
0.00521
0.00521
0.01596
0.01541
0.00783
0.01259
0.01099
0.00260
0.00260
0.01553
0.61463
0.00523
0.00700
0.00523
0.00655
0.00000
0.00260
0.01006
0.00390
0.00260
0.00952
0.00000
0.00479
0.00260
0.01535
0.00653
0.00653
0.00787
0.02785
0.00787
0.01328
0.00541
0.00023
0.00697
0.01434
0.00476
0.01231
-0.00160
0.00393
0.00061
0.02719
0.03951
0.04718
0.51242
0.00531
0.00110
0.00511
0.00509
0.00055
0.02928
0.05697
0.00132
0.00045
0.01396
0.02428
0.00260
0.00219
0.013570.00561
-0.00301
0.00130
0.00616
0.00883
-0.00053
Neighbourhood joining
tree contract from the
Specimen DNA sequence

26. Result !26Molecular cloning &
identification
Herbaspirillum chlorophenolicum
Herbaspirillum frisingense
Herbaspirillum seropedicae
F10
Collimonas arenae
Herminiimonas glaciei
Janthinobacterium lividum
Janthinobacterium agaricidamnosum
Janthinobacterium agaricidamnosum(2)
Massilia brevitalea
Naxibacter varians
Naxibacter haematophilus
F3
Massilia aerilata
Methylibium petroleiphilum PM1
Schlegelella thermodepolymerans
Azohydromonas lata
Rubrivivax gelatinosus IL144
Aquincola tertiaricarbonis
Control4
Azohydromonas australica
Brevundimonas nasdae
Streptomyces longisporoflavus
Mycoplana bullata
S7
Caulobacter segnis ATCC 21756
Phenylobacterium koreense
Rhizobium alamii
Ensifer adhaerens
Sinorhizobium fredii NGR234
Brucella ovis ATCC 25840
Ochrobactrum haematophilum
Control7
Ochrobactrum rhizosphaerae
out group
0.00783
0.00391
0.00391
0.00922
0.00521
0.00521
0.01596
0.01541
0.00783
0.01259
0.01099
0.00260
0.00260
0.01553
0.61463
0.00523
0.00700
0.00523
0.00655
0.00000
0.00260
0.01006
0.00390
0.00260
0.00952
0.00000
0.00479
0.00260
0.01535
0.00653
0.00653
0.00787
0.02785
0.00787
0.01328
0.00541
0.00023
0.00697
0.01434
0.00476
0.01231
-0.00160
0.00393
0.00061
0.02719
0.03951
0.04718
0.51242
0.00531
0.00110
0.00511
0.00509
0.00055
0.02928
0.05697
0.00132
0.00045
0.01396
0.02428
0.00260
0.00219
0.013570.00561
-0.00301
0.00130
0.00616
0.00883
-0.00053
Neighbourhood joining
tree contract from the
Specimen DNA sequence

27. Result !27Molecular cloning &
identification
!
information
Found in soil
culture(S)
Found in foam culture(F) Found in control
Caulobacter segnis Massilia aerilata
Herbaspirillum
seropedicae
Ochrobactrum sp. Azohydromonas
Taxonomy
Bacteria;
Proteobacteria;
Alphaproteobacteria;
Caulobacterales;
Caulobacteraceae;
Caulobacter
Bacteria;Proteobacte
riaBetaProteobacteri
a Burkholderiales
Oxalobacteraceae
Massilia
Bacteria;
Proteobacteria;
Betaproteobacteria;
Burkholderiales;
Oxalobacteraceae;
Herbaspirillum
Bacteria;
Proteobacteria;
Alphaproteobacteria;
Rhizobiales;
Brucellaceae;
Ochrobactrum
Bacteria
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Azohydromonas
Morphology &
classification
Negative, Bacilli,
Aerobic, Mesophilic
Negative, Bacilli,
Aerobic
Negative, Spirilla,
Aerobic, Mesophilic
Negative, Bacilli
Negative, Bacilli
Styrene
degradation
✓ Na ✓ ✓ Na
Aromatic
compound
degradation
✓ Na ✓
✓
Na
Carbon fixation - Na - ✓ Na
Polycyclic aromatic
degradation
✓
Na - ✓ Na
Chlorophenol
degradation
✓ Na ✓ ✓ Na
Nitogen
metabolism
✓ ✓ ✓ ✓ ✓
Other pathway
Cellulose
degradation pathway
polyhydroxybutyrate
(PHB) production
!
Polyhydroxybutyrate
fermentation

28. Degradability
observation
(SEM)
Agarcultivation
Phylogenetic
tree
Community
fingerprint
16s Ribosomal RNA
identification
Molecular
cloning
Methodology !28Degradability Observation
2months laterScreening
Cultivation
Microbe
sampling

29. Methodology !29Degradability Observation
The microscopic techniques
!
‣Test Method Used : In house method refer to
WI-RES-SEM-Quanta-001 and WI-RES-SEM-001
‣Test Equipment : Scanning Electron Microscope,
Quanta40, FEI, Czech Republic
‣Test Technique : Electron micrograph
‣Test Condition
Mode : low vacuum
Detector : Large Field Detector()LFD
High Voltage : 15.00,20.00 kV

30. Methodology !30Degradability Observation
Control : Polystyrene in MSM broth without bacterial source.
Regular polystyrene foam that didn’t use in experiment.
100x 200x 500x

31. Methodology !31Degradability Observation
Polystyrene in Medium with bacteria from Styrofoam in the landfill.
100x 200x 500x
Regular polystyrene foam that didn’t use in experiment.

32. Polystyrene in Medium with bacteria from soil in the landfill.
Methodology !32Degradability Observation
100x 200x 500x
Regular polystyrene foam that didn’t use in experiment.

33. Methodology !33Degradability Observation
Polystyrene in Medium with bacteria from soil in the landfill.

35. Agarcultivation
Degradability
observation
(SEM)
Phylogenetic
tree
Community
fingerprint
16s Ribosomal RNA
identification
Molecular
cloning
Methodology !35Agar Cultivation
2months laterScreening
Cultivation
Microbe
sampling

36. Methodology !36Agar Cultivation
MSM broth
!
‣K2HPO4
‣KH2PO4
‣(NH4)2SO4
‣MgSO4
‣FeSO4.2HO2
‣MnCl2.4H2O
‣CoCl2.6H2O
‣CuCl2.2H2O
‣NiCl2.6H2O
‣Na2MoO4.2H2O
‣ZnSO4.7H2O
‣H3BO3
MSM + Agar
No carbon source
MSM broth
MSM + Agar
+ Polystyrene-coacrylic acid (PSA)
(particles diameter 500 nm)
Control

37. Methodology !37Agar Cultivation
‣The purpose is to isolate the single colony of the
bacteria prior culture in the liquid broth
!
x Problem : Agar is also the carbon source for
bacteria result in unable to created selective
condition
!
‣Using thin filter(which no carbon structure) for
bacteria attachment surface

38. Methodology !38Agar Cultivation
‣No bacteria colony grow on the thin filter
‣Bacteria colony not separate well on the plate
‣Bacteria density in the plate with PS is more than plate with out PS

39. Conclusion & Discussion !39
Soilweek1
Soilweek5
Soilweek6
Soilweek7
Soilweek8
Soilweek20
Foamweek1
Foamweek5
Foamweek6
Foamweek7
Foamweek8
Foamweek20
Controlweek6
Controlweek7
Controlweek8
Styrofoam sourceControl Soil source

40. Conclusion & Discussion !40
!
information
Found in soil
culture(S)
Found in foam culture(F) Found in control
Caulobacter segnis Massilia aerilata
Herbaspirillum
seropedicae
Ochrobactrum sp. Azohydromonas
Taxonomy
Bacteria;
Proteobacteria;
Alphaproteobacteria;
Caulobacterales;
Caulobacteraceae;
Caulobacter
Bacteria;Proteobacte
riaBetaProteobacteri
a Burkholderiales
Oxalobacteraceae
Massilia
Bacteria;
Proteobacteria;
Betaproteobacteria;
Burkholderiales;
Oxalobacteraceae;
Herbaspirillum
Bacteria;
Proteobacteria;
Alphaproteobacteria;
Rhizobiales;
Brucellaceae;
Ochrobactrum
Bacteria
Proteobacteria
Betaproteobacteria
Burkholderiales
Alcaligenaceae
Azohydromonas
Morphology &
classification
Negative, Bacilli,
Aerobic, Mesophilic
Negative, Bacilli,
Aerobic
Negative, Spirilla,
Aerobic, Mesophilic
Negative, Bacilli
Negative, Bacilli
Styrene
degradation
✓ Na ✓ ✓ Na
Aromatic
compound
degradation
✓ Na ✓
✓
Na
Carbon fixation - Na - ✓ Na
Polycyclic aromatic
degradation
✓
Na - ✓ Na
Chlorophenol
degradation
✓ Na ✓ ✓ Na
Nitogen
metabolism
✓ ✓ ✓ ✓ ✓
Other pathway
Cellulose
degradation pathway
polyhydroxybutyrate
(PHB) production
!
Polyhydroxybutyrate
fermentation

41. Conclusion & Discussion !41
‣The highest degradation trade was made by the
bacteria from the styrofoam in the landfill, relate to
the dominance species that were present in the
continuos bold DNA band in the DGGE gel.
!
‣The DNA sequence reveals that the bacteria in the
consortium, some have a metabolism to degrade
styrene and aromatic- hydrocarbon.
‣The next step of research should focus on the
metabolism & the by product of degradation of the
bacteria in the consortium that were discovered.

42. Research
Achievements
13th
NCSC, Jaipur India 2011
Youth summit 2012, Dubai UAE
JSTP Scholarship
STT 36
BYEE, Leverkuzen Germany
BYEE Poster
Prize from
india

43. present to HRH princess of Thailand

44. !44
A. M. Warhurst and C. A. Fewson. 1994. A review microbial metabolism and
biotransformations of styrene.Journal of Applied Bacteriology
!
G.C. Okpokwasili and C.O. Nweke. 2005. Microbial growth and substrate utilization kinetics.
African Journal of Biotechnology
!
Medlin, L., H.J. Elwood, S. Stickel and M.L. Sogin. 1998. The Characterization of amplifiled
eukaryote 16S like rRNA coding regions. Gen. 71: 491-499.
!
Muyzer G., E.C. De Waal and A.G. Uitterlinden. 1993. Profiling of complex microbial
populations by denaturing gradient gel electrophoresis analysis of polymerase chain
restriction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59: 695- 700.
!
QIAGEN. 2001. QIAGEN PCR Cloning Handbook.
!
Sielicki, M., Focht. D.D. and Martin, J.P. (1978) .Microbial transformations of styrene and
['4C]styrene in soil and enrichment cultures. Applied and Encironmental Microbiology
!
Supaphol, S. 2005. Intrinsic Bioremediation and The Molecular Analysis of Microorganisms in
Hydrocarbon Contaminated Thai Soil. Ph.D. Thesis, Kasetsart University.
!
Zhou, J., M.A. Bruns and M.T. James. 1995. DNA Recovery from Soils of Diverse Composition.
Amer. Soc. Micro. 62: 316-322.
Reference

45. !45Scholarships

46. !46
Mentor
Dr.Opas(Tun,thagoon( Dr.Ampai,p(Sookhom( Asst.Prof
(C
s(Tun,thagoon( Dr.Ampai,p(Sookhom( Asst.Prof(Dr.Savaporn(Su
(Current(advisor)(
Adv
agoon( Dr.Ampai,p(Sookhom( Asst.Prof(Dr.Savaporn(Supaphol(
(Current(advisor)(
Advisor(
ss(Apinya(Boonkhum( Mrs.RaCanawan(Inpang(
Ampai,p(Sookhom( Asst.Prof(Dr.Savaporn(Supaphol(
(Current(advisor)(
Advisor(
nkhum( Mrs.RaCanawan(Inpang(

47. !47Assistant Mentor
PSUWIT TEACHER SUT
KMUTT
PSU KU
If I have seen further it is by
standing on the shoulders of giants.
- Isaac Newton -

48. RIPMy brave Bacteria