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Biodegradation of Polystyrene foam by the Microorganism from Landfill

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Biodegradation of Polystyrene foam by the Microorganism from Landfill

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Biodegradation of Polystyrene foam by the Microorganism from Landfill

  1. 1. Pat Pataranutaporn ! Assistant prof. Savaporn Supaphol prof. Amornrat Phongdara Sureeporn Nualkaew Biodegradation of Polystyrene Foam by the Microorganisms from Landfill
  2. 2. 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
  3. 3. ! • 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 !3
  4. 4. 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 !4 Possibly work?
  5. 5. 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 !5
  6. 6. Methodology
  7. 7. Agarcultivation Degradability observation (SEM) Microbe sampling Community fingerprint 16s Ribosomal RNA identification Molecular cloning Phylogenetic tree 2months later !7Methodology Screening Cultivation
  8. 8. Methodology !8 Degradability observation (SEM) Agarcultivation Phylogenetic tree 2months laterScreening Cultivation Microbe sampling Community fingerprint 16s Ribosomal RNA identification Molecular cloning Microbe sampling & cultivation
  9. 9. 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 !9Community structure analysis
  10. 10. 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 !10Community structure analysis
  11. 11. Shake for 1 month then inoculate to the new fresh broth for sub culture. Methodology !11Community structure analysis
  12. 12. Methodology !12Community 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.
  13. 13. 0 1 2 3 4 5 6 7 8 96 7 8 time(week) 1 400 µl. 5 9 transfer culture Methodology !13Community structure analysis Sampling schedule
  14. 14. Methodology !14 Screening Cultivation Degradability observation (SEM) Agarcultivation Microbe sampling Community fingerprint 16s Ribosomal RNA identification Molecular cloning Phylogenetic tree 2months later Community structure analysis
  15. 15. 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 !15Community structure analysis
  16. 16. Result !16 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
  17. 17. Community structure trend time(week) Microbe diversity Dominant species DGGE Denature Gradient Gel Electrophoresis Each DNA band represent 1 microbe Looking for survivor! Methodology !17Community structure analysis
  18. 18. 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 !18Community structure analysis
  19. 19. Methodology !19 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
  20. 20. Methodology !20 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
  21. 21. Result !21Molecular cloning & identification extracted Plasmid + cutcheck with EcoR1 Purify Plasmid + cutcheck with EcoR1 PCR Product 16S rRNA gene Amplification Primer AF1 & 1541R Gel electrophoresis
  22. 22. Result !22Molecular cloning & identification Sequence report - Electropherogram F3 F10 S7 Control 4 Control 7 F5
  23. 23. Result !23Molecular cloning & identification Sequence blasting Sample Length (bp) Similar sequence Max iden Max score E.Value 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
  24. 24. Result !24Molecular 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
  25. 25. Result !25Molecular 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
  26. 26. Degradability observation (SEM) Agarcultivation Phylogenetic tree Community fingerprint 16s Ribosomal RNA identification Molecular cloning Methodology !26Degradability Observation 2months laterScreening Cultivation Microbe sampling
  27. 27. Methodology !27Degradability 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
  28. 28. Methodology !28Degradability Observation Control : Polystyrene in MSM broth without bacterial source. Regular polystyrene foam that didn’t use in experiment. 100x 200x 500x
  29. 29. Methodology !29Degradability Observation Polystyrene in Medium with bacteria from Styrofoam in the landfill. 100x 200x 500x Regular polystyrene foam that didn’t use in experiment.
  30. 30. Polystyrene in Medium with bacteria from soil in the landfill. Methodology !30Degradability Observation 100x 200x 500x Regular polystyrene foam that didn’t use in experiment.
  31. 31. Methodology !31Degradability Observation Polystyrene in Medium with bacteria from soil in the landfill.
  32. 32. Agarcultivation Degradability observation (SEM) Phylogenetic tree Community fingerprint 16s Ribosomal RNA identification Molecular cloning Methodology !32Agar Cultivation 2months laterScreening Cultivation Microbe sampling
  33. 33. Methodology !33Agar 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
  34. 34. Methodology !34Agar 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
  35. 35. Methodology !35Agar 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
  36. 36. Conclusion & Discussion !36 Soilweek1 Soilweek5 Soilweek6 Soilweek7 Soilweek8 Soilweek20 Foamweek1 Foamweek5 Foamweek6 Foamweek7 Foamweek8 Foamweek20 Controlweek6 Controlweek7 Controlweek8 Styrofoam sourceControl Soil source
  37. 37. Conclusion & Discussion !37 ! 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
  38. 38. Conclusion & Discussion !38 ‣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.
  39. 39. Research Achievements 13th NCSC, Jaipur India 2011 Youth summit 2012, Dubai UAE JSTP Scholarship STT 36 BYEE, Leverkuzen Germany BYEE Poster Prize from india
  40. 40. present to HRH princess of Thailand
  41. 41. !41 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
  42. 42. !42Scholarships
  43. 43. !43 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(
  44. 44. !44Assistant Mentor PSUWIT TEACHER SUT KMUTT PSU KU If I have seen further it is by standing on the shoulders of giants. - Isaac Newton -
  45. 45. RIPMy brave Bacteria

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