Community Structure Analysis �9

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 µl.

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

Marker

Soil week 1 Bacteria fromsoil Soil week 5 Soil week 6 Soil week 7 Soil week 8 Soil week 20

Marker Bacteria fromstyro Foam week 1 Foam week 5 Foam week 6 Foam week 7

Foam week 8 foam Community structureanalysis Foam week 20 Control week 6

Control week 7 Control Control week 8 Marker Negative DGGE 26/04/55 from PCRproduct 24/04/55 Non-continuing band Continuing band Continuing band&found incontrol Running 300minute template use8µl. 18 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 0.00390 -0.00053 Herbaspirillum seropedicae 0.01006 0.00883 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 0.00260 0.03951 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 Microbe Screening months later identification sampling Cultivation

Molecular cloning Phylogenetic tree Degradability observation (SEM) Methodology Agar Cultivation 33

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

MSM broth Methodology Agar Cultivation 34

‣The purpose is to isolate the single colony of the bacteria prior culture in the liquid broth ! ‣Problem : Agar is also the carbon source for bacteria result in unable to created selective condition ! ‣Using filter paper for bacteria attachment surface Methodology Agar Cultivation 35

‣No bacteria colony grow on the filter paper ‣Bacteria colony not separate well on the plate ‣Bacteria density in the plate with PS is more than plate with out PS Conclusion & Discussion 36 Control Soil source Styrofoam source

Soil week 7 Soil week 8

Soil week 5 Soil week Soil week 6 Soil week 1 Soil week

Soil week 20

Foam week 6 Foam week 7 Foam week 8 Foam week 1 week Foam 5 week Foam

Foam week 20

Control week 6 Control week 7 Control week 8 Conclusion & Discussion 37

Found in soil Found in foam culture(F) Found in control ! culture(S) information Herbaspirillum Caulobacter segnis Massilia aerilata Ochrobactrum sp. Azohydromonas seropedicae

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 Conclusion & Discussion 38

‣The highest degradation trade was made by the bacteria from the styrofoam in the landfill, relate ti the dominance species that were present in the continuos bold DNA band in the DGGE gel ‣The DNA sequence reveals that the bacteria that were in the consortium, some have a metabolism to degrade styrene and aromatic- hydrocarbon STT 36

BYEE Poster Prize from india JSTP Scholarship

Research 13th NCSC, Jaipur India 2011 Achievements

Youth summit 2012, Dubai UAE

BYEE, Leverkuzen Germany present to the princess of Thailand Reference 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. Scholarships 42 Advisor( Mentor Advisor(43 Advisor( Advisor(

Dr.Opas(Tun,thagoon( Dr.Ampai,p(Sookhom( Asst.Prof(Dr.Savaporn(Supaphol( (Current(advisor)( Dr.Opas(Tun,thagoon( Dr.Ampai,p(Sookhom( Asst.Prof(Dr.Savaporn(Supaphol( (Current(advisor)( Dr.Opas(Tun,thagoon( Dr.Ampai,p(Sookhom( Asst.Prof(Dr.Savaporn(Supaphol( Dr.Opas(Tun,thagoon( Dr.Ampai,p(Sookhom(Current(advisor)(( Asst.Prof(Dr.Savaporn(Supaphol( (Current(advisor)(

Miss(Apinya(Boonkhum( Mrs.RaCanawan(Inpang(

Miss(Apinya(Boonkhum( Mrs.RaCanawan(Inpang( Miss(Apinya(Boonkhum( Mrs.RaCanawan(Inpang( Assistant Mentor 44

If I have seen further it is by standing on the shoulders of giants. - Isaac Newton -

PSUWIT TEACHER SUT

KMUTT

PSU KU RIP My brave Bacteria