Biodegradation of Polystyrene Foam by the Microorganisms from Landfill Pat Pataranutaporn ! Assistant prof. Savaporn Supaphol prof. Amornrat Phongdara Sureeporn Nualkaew Hi, I would like to invite you to take a look on my research Pat Introduction !3 “Styrofoam” Polystyrene Disadvantage Physical Properties ! • chemical formula is (C8H8)n • Non-biodegradable in the environment • monomer styrene • Made from non-renewable petroleum products • Thermoplastic • Chronic, low-level exposure risks undetermined • blowing agents Introduction !4 Bacteria nutritional requirements ! ‣ Energy source Biodegradation ‣ Carbon source Possibly work? ‣ Nitrogen source ‣ Minerals ‣ Water ‣ Growth factors Polystyrene structure http://faculty.ccbcmd.edu/courses/bio141/ lecguide/unit6/metabolism/growth/factors.html Introduction !5 Aims of the research ‣To identify the microbe that able to growth in the condition that 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 To analyse the by product of polystyrene after degradation Methodology Methodology !7 Agar cultivation Community fingerprint 2 16s Ribosomal RNA Microbe Screening months later identification sampling Cultivation Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Microbe sampling & cultivation !8 Agar cultivation Community fingerprint 2 16s Ribosomal RNA Microbe Screening months later identification sampling Cultivation Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Community structure analysis !9 landfill that was contaminated by Polystyrene foam in Pattani,Thailand Styrofoam in the landfill Contaminated soil Methodology Community structure analysis !10 Sterile Polystyrene MSM broth ! ‣K2HPO4 ‣KH2PO4 ‣(NH4)2SO4 ‣MgSO4 ! ‣ FeSO4.2HO2 ‣MnCl2.4H2O ! ‣CoCl2.6H2O F S Control ‣CuCl2.2H2O ‣NiCl2.6H2O MSM broth MSM broth MSM broth ‣Na2MoO4.2H2O + Sterile Polystyrene + Sterile Polystyrene + Sterile Polystyrene ‣ZnSO4.7H2O + Landfill styrofoam + Landfill soil ‣H3BO3 Methodology Community structure analysis !11 Shake in shaker for 1 month then inoculate to new fresh broth for sub culture. Methodology Community structure analysis !12 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. Methodology Community structure analysis !13 400 "l. 0 11 2 3 4 5 6 77 8 9 time(week) transfer culture Methodology Community structure analysis !14 Agar cultivation Community fingerprint 2 16s Ribosomal RNA Microbe Screening months later identification sampling Cultivation Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Community structure analysis !15 Cell suspensions collected from each week of cultivation. DNA Extraction (Methode : QIAamp Protocol) 16s Ribosomal RNA Polymerase Chain Reaction (PCR) identification 16S rRNA gene Amplification Primer VFC &VR Denature Gradient Gel Electrophoresis (DGGE) Community Community structure analysis fingerprint Result Community structure analysis !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) Methodology Community structure analysis !17 Community structure trend time(week) Microbe diversity Dominant species Each DNA band represent 1 microbe DGGE Denature Gradient Gel Electrophoresis Looking for survivor! Result Community structure analysis !18 Bacteria from soil Bacteria from styrofoam Control DGGE 26/04/55 Marker Marker Negative Soil week 5 Soil week Soil week 6 Control week 6 Soil week 7 Soil week 8 Control week 7 Control week 8 Foam week 6 Foam week 7 Foam week 8 Foam week 5 week Foam Foam week 20 Soil week 1 Soil week Foam week 1 week Foam Marker Soil week 20 Running 300 minute from PCR product 24/04/55 template use 8 µl. Continuing band & found in control Continuing band Non-continuing band Molecular cloning & !19 Methodology identification Selected DNA Bacteria from soil Bacteria from styrofoam Control Marker Marker Marker Negative Soil week 5 Soil week Soil week 6 Control week 6 Soil week 7 Soil week 8 Control week 7 Control week 8 Foam week 6 Foam week 7 Foam week 8 Soil week 1 Soil week Foam week 5 week Foam Foam week 20 Foam week 1 week Foam Soil week 20 Selected for cloning Molecular cloning & !20 Methodology identification 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 Ligate with pGEM T-Easy Plasmid Transfer Plasmid to the competent cell (E.Coli) + Propagate extracted Plasmid + cutcheck with EcoR1 Molecular cloning Purify Plasmid + cutcheck with EcoR1 Nucleotide sequencing Blasting + Neighbourhood joining tree contracting Phylogenetic tree Molecular cloning & !21 Result identification PCR Product 16S rRNA gene Amplification extracted Plasmid + cutcheck with EcoR1 Primer AF1 & 1541R Gel electrophoresis Purify Plasmid + cutcheck with EcoR1 Molecular cloning & !22 Result identification Sequence report - Electropherogram F3 F10 S7 Control 4 Control 7 F5 Molecular cloning & !23 Result identification Sequence blasting Length Max Max Sample Similar sequence E.Value (bp) iden score 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 Molecular cloning & !24 Result identification 0.00260 0.00130 Herbaspirillum chlorophenolicum 0.00260 0.00616 Herbaspirillum frisingense -0.00053 0.00390 Herbaspirillum seropedicae 0.00883 0.01006 F10 0.00952 -0.00301 Collimonas arenae 0.00479 Herminiimonas glaciei 0.00260 Janthinobacterium lividum 0.00561 0.01357 0.00000 0.00219 Janthinobacterium agaricidamnosum 0.00000 0.00260 Janthinobacterium agaricidamnosum(2) 0.02428 0.01535 Massilia brevitalea 0.00700 Naxibacter varians 0.00655 Naxibacter haematophilus 0.01396 0.05697 0.00523 0.00045 F3 0.00523 0.00132 Massilia aerilata 0.01596 Methylibium petroleiphilum PM1 Neighbourhood joining 0.01541 Schlegelella thermodepolymerans 0.02928 0.00521 tree contract from the 0.00511 Azohydromonas lata 0.00521 0.00055 Rubrivivax gelatinosus IL144 Specimen DNA sequence 0.00922 0.00509 Aquincola tertiaricarbonis 0.00391 0.51242 0.00110 Control4 0.00391 0.00531 Azohydromonas australica 0.00787 0.00541 Brevundimonas nasdae 0.00787 0.00023 Streptomyces longisporoflavus 0.01328 0.01434 Mycoplana bullata 0.00653 S7 0.02719 0.00653 0.00697 Caulobacter segnis ATCC 21756 0.02785 Phenylobacterium koreense 0.01553 0.04718 Rhizobium alamii 0.00260 0.01231 Ensifer adhaerens 0.03951 0.00260 Sinorhizobium fredii NGR234 0.01099 0.00061 Brucella ovis ATCC 25840 0.01259 Ochrobactrum haematophilum 0.00393 0.00783 -0.00160 Control7 0.00783 0.00476 Ochrobactrum rhizosphaerae out group 0.61463 Molecular cloning & !25 Result identification Found in soil Found in foam culture(F) Found in control ! culture(S) information Herbaspirillum Caulobacter segnis Massilia aerilata Ochrobactrum sp. Azohydromonas seropedicae Bacteria; Bacteria; Bacteria; Bacteria Bacteria;Proteobacte Proteobacteria; Proteobacteria; Proteobacteria; Proteobacteria riaBetaProteobacteri Alphaproteobacteria; Betaproteobacteria; Alphaproteobacteria; Betaproteobacteria Taxonomy a Burkholderiales Caulobacterales; Burkholderiales; Rhizobiales; Burkholderiales Oxalobacteraceae Caulobacteraceae; Oxalobacteraceae; Brucellaceae; Alcaligenaceae Massilia Caulobacter Herbaspirillum Ochrobactrum Azohydromonas Morphology & Negative, Bacilli, Negative, Bacilli, Negative, Spirilla, Negative, Bacilli Negative, Bacilli classification Aerobic, Mesophilic Aerobic Aerobic, Mesophilic Styrene ✓ Na ✓ ✓ Na degradation Aromatic ✓ compound ✓ Na ✓ Na degradation Carbon fixation - Na - ✓ Na Polycyclic aromatic ✓ Na - ✓ Na degradation Chlorophenol ✓ Na ✓ ✓ Na degradation Nitogen ✓ ✓ ✓ ✓ ✓ metabolism polyhydroxybutyrate (PHB) production Cellulose Other pathway ! degradation pathway Polyhydroxybutyrate fermentation Methodology Degradability Observation !26 Agar cultivation Community fingerprint 2 16s Ribosomal RNA Microbe Screening months later identification sampling Cultivation Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Degradability Observation !27 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 Methodology Degradability Observation !28 100x 200x 500x Control : Polystyrene in MSM broth without bacterial source. Regular polystyrene foam that didn’t use in experiment. Methodology Degradability Observation !29 100x 200x 500x Polystyrene in Medium with bacteria from Styrofoam in the landfill. Regular polystyrene foam that didn’t use in experiment. Methodology Degradability Observation !30 100x 200x 500x Polystyrene in Medium with bacteria from soil in the landfill. Regular polystyrene foam that didn’t use in experiment. Methodology Degradability Observation !31 Polystyrene in Medium with bacteria from soil in the landfill. Methodology Agar Cultivation !32 Agar cultivation Community fingerprint 2 16s Ribosomal RNA