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Biodegradation of Polystyrene Foam
by the Microorganisms from Landfill
Researcher : Pat Pataranutaporn1
Assistant prof. Sa...
Pat Pataranutaporn
!
Assistant prof. Savaporn Supaphol
prof. Amornrat Phongdara
Sureeporn Nualkaew
Biodegradation of Polys...
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 ...
!
• Non-biodegradable in the environment
• Made from non-renewable petroleum products
• Chronic, low-level exposure risks ...
Bacteria nutritional requirements
!
‣ Energy source
‣ Carbon source
‣ Nitrogen source
‣ Minerals
‣ Water
‣ Growth factors
...
Aims of the research
‣To identify the microbe that able to growth in the
condition, which polystyrene is a sole carbon sou...
Methodology
Agarcultivation
Degradability
observation
(SEM)
Microbe
sampling
Community
fingerprint
16s Ribosomal RNA
identification
Mo...
Methodology !9
Degradability
observation
(SEM)
Agarcultivation
Phylogenetic
tree
2months laterScreening
Cultivation
Microb...
2 aspects of samples
were collected from
the landfill that was
contaminated by
Polystyrene foam in
Pattani,Thailand
Styrof...
Control
MSM broth
+ Sterile Polystyrene
!
!
S
MSM broth
+ Sterile Polystyrene
+ Landfill soil
F
MSM broth
+ Sterile Polyst...
Shake for 1 month then
inoculate to the new fresh
broth for sub culture.
Methodology !12Community structure analysis
Methodology !13Community structure analysis
Every week, The cell suspension in particular flask was taken
to the eppendorf...
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 sc...
Methodology !15
Screening
Cultivation
Degradability
observation
(SEM)
Agarcultivation
Microbe
sampling
Community
fingerpri...
DNA Extraction
(Methode : QIAamp Protocol)
Polymerase Chain Reaction (PCR)
16S rRNA gene Amplification
Primer VFC &VR
Dena...
Result !17
DNA Replication : PCR(TopTaq Master Mix Kit)
16S rRNA gene Amplification
by using Primer VR (Medlin et al., 199...
Community structure trend
time(week)
Microbe diversity
Dominant species
DGGE
Denature Gradient
Gel Electrophoresis
Each DN...
Marker
Soilweek1
Soilweek5
Soilweek6
Soilweek7
Soilweek8
Soilweek20
Foamweek1
Foamweek5
Foamweek6
Foamweek7
Foamweek8
Foam...
Methodology !20
Marker
Soilweek1
Soilweek5
Soilweek6
Soilweek7
Soilweek8
Soilweek20
Foamweek1
Foamweek5
Foamweek6
Foamweek...
Methodology !21
DNA from S week7, F week 7 and con week 7
Polymerase Chain Reaction (PCR)
16S rRNA gene Amplification
Prim...
Result !22Molecular cloning &
identification
extracted Plasmid + cutcheck with EcoR1
Purify Plasmid + cutcheck with EcoR1
...
Result !23Molecular cloning &
identification
Sequence report - Electropherogram
F3 F10 S7
Control 4 Control 7 F5
Result !24Molecular cloning &
identification
Sequence blasting
Sample
Len
gth
(bp)
Similar sequence
Ma
x
ide
n
Ma
x
sco
re...
Result !25Molecular cloning &
identification
Herbaspirillum chlorophenolicum
Herbaspirillum frisingense
Herbaspirillum ser...
Result !26Molecular cloning &
identification
Herbaspirillum chlorophenolicum
Herbaspirillum frisingense
Herbaspirillum ser...
Result !27Molecular cloning &
identification
!
information
Found in soil
culture(S)
Found in foam culture(F) Found in cont...
Degradability
observation
(SEM)
Agarcultivation
Phylogenetic
tree
Community
fingerprint
16s Ribosomal RNA
identification
M...
Methodology !29Degradability Observation
The microscopic techniques
!
‣Test Method Used : In house method refer to
WI-RES-...
Methodology !30Degradability Observation
Control : Polystyrene in MSM broth without bacterial source.
Regular polystyrene ...
Methodology !31Degradability Observation
Polystyrene in Medium with bacteria from Styrofoam in the landfill.
100x 200x 500...
Polystyrene in Medium with bacteria from soil in the landfill.
Methodology !32Degradability Observation
100x 200x 500x
Reg...
Methodology !33Degradability Observation
Polystyrene in Medium with bacteria from soil in the landfill.
Agarcultivation
Degradability
observation
(SEM)
Phylogenetic
tree
Community
fingerprint
16s Ribosomal RNA
identification
M...
Methodology !36Agar Cultivation
MSM broth
!
‣K2HPO4
‣KH2PO4
‣(NH4)2SO4
‣MgSO4
‣FeSO4.2HO2
‣MnCl2.4H2O
‣CoCl2.6H2O
‣CuCl2.2...
Methodology !37Agar Cultivation
‣The purpose is to isolate the single colony of the
bacteria prior culture in the liquid b...
Methodology !38Agar Cultivation
‣No bacteria colony grow on the thin filter
‣Bacteria colony not separate well on the plat...
Conclusion & Discussion !39
Soilweek1
Soilweek5
Soilweek6
Soilweek7
Soilweek8
Soilweek20
Foamweek1
Foamweek5
Foamweek6
Foa...
Conclusion & Discussion !40
!
information
Found in soil
culture(S)
Found in foam culture(F) Found in control
Caulobacter s...
Conclusion & Discussion !41
‣The highest degradation trade was made by the
bacteria from the styrofoam in the landfill, re...
Research
Achievements
13th
NCSC, Jaipur India 2011
Youth summit 2012, Dubai UAE
JSTP Scholarship
STT 36
BYEE, Leverkuzen G...
present to HRH princess of Thailand
!44
A. M. Warhurst and C. A. Fewson. 1994. A review microbial metabolism and
biotransformations of styrene.Journal of Appl...
!45Scholarships
!46
Mentor
Dr.Opas(Tun,thagoon( Dr.Ampai,p(Sookhom( Asst.Prof
(C
s(Tun,thagoon( Dr.Ampai,p(Sookhom( Asst.Prof(Dr.Savaporn(...
!47Assistant Mentor
PSUWIT TEACHER SUT
KMUTT
PSU KU
If I have seen further it is by
standing on the shoulders of giants.
-...
RIPMy brave Bacteria
The biodegradation of Polystyrene
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The biodegradation of Polystyrene

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The biodegradation of Polystyrene is Pat's project under Assist.Prof Savaporn, and Prof. Amornrat.

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The biodegradation of Polystyrene

  1. 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. 2. Pat Pataranutaporn ! Assistant prof. Savaporn Supaphol prof. Amornrat Phongdara Sureeporn Nualkaew Biodegradation of Polystyrene Foam by the Microorganisms from Landfill
  3. 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. 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. 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. 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. 7. Methodology
  8. 8. Agarcultivation Degradability observation (SEM) Microbe sampling Community fingerprint 16s Ribosomal RNA identification Molecular cloning Phylogenetic tree 2months later !8Methodology Screening Cultivation
  9. 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. 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. 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. 12. Shake for 1 month then inoculate to the new fresh broth for sub culture. Methodology !12Community structure analysis
  13. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 23. Result !23Molecular cloning & identification Sequence report - Electropherogram F3 F10 S7 Control 4 Control 7 F5
  24. 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. 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. 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. 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. 28. Degradability observation (SEM) Agarcultivation Phylogenetic tree Community fingerprint 16s Ribosomal RNA identification Molecular cloning Methodology !28Degradability Observation 2months laterScreening Cultivation Microbe sampling
  29. 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. 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. 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. 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. 33. Methodology !33Degradability Observation Polystyrene in Medium with bacteria from soil in the landfill.
  34. 34. Agarcultivation Degradability observation (SEM) Phylogenetic tree Community fingerprint 16s Ribosomal RNA identification Molecular cloning Methodology !35Agar Cultivation 2months laterScreening Cultivation Microbe sampling
  35. 35. 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
  36. 36. 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
  37. 37. 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
  38. 38. Conclusion & Discussion !39 Soilweek1 Soilweek5 Soilweek6 Soilweek7 Soilweek8 Soilweek20 Foamweek1 Foamweek5 Foamweek6 Foamweek7 Foamweek8 Foamweek20 Controlweek6 Controlweek7 Controlweek8 Styrofoam sourceControl Soil source
  39. 39. 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
  40. 40. 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.
  41. 41. Research Achievements 13th NCSC, Jaipur India 2011 Youth summit 2012, Dubai UAE JSTP Scholarship STT 36 BYEE, Leverkuzen Germany BYEE Poster Prize from india
  42. 42. present to HRH princess of Thailand
  43. 43. !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
  44. 44. !45Scholarships
  45. 45. !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(
  46. 46. !47Assistant Mentor PSUWIT TEACHER SUT KMUTT PSU KU If I have seen further it is by standing on the shoulders of giants. - Isaac Newton -
  47. 47. RIPMy brave Bacteria

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