the biodegradation of polystyrene

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

Pat Pataranutaporn !Assistant prof. Savaporn Supaphol prof. Amornrat Phongdara Sureeporn Nualkaew

Biodegradation of Polystyrene Foam by the Microorganisms from Landfill

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

!

• 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

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?

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

Methodology

Agar cultivation

Degradability observation

(SEM)

Microbe sampling

Community

fingerprint

16s Ribosomal RNA

identification

Molecular cloning

Phylogenetic tree

2 months later

!8Methodology

Screening Cultivation

Methodology !9


(SEM)

Agar cultivation

Phylogenetic tree

2 months later Screening

CultivationMicrobe sampling

Community

fingerprint

16s Ribosomal RNA

identification

Molecular cloning

Microbe sampling & cultivation

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

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


Shake for 1 month then inoculate to the new fresh broth for sub culture.



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.

0 1 2 3 4 5 6 7 8 96 7 8time(week)

1

400 µl.

5 9

transfer culture


Sampling schedule

Methodology !15

Screening Cultivation


(SEM)

Agar cultivation

Microbe sampling

Community

fingerprint

16s Ribosomal RNA

identification

Molecular cloning

Phylogenetic tree

2 months later

Community structure analysis

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.


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

Community structure trend

time(week)

Microbe diversity

Dominant species

DGGE Denature Gradient Gel Electrophoresis

Each DNA band represent 1 microbe

Looking for survivor!


Mar

ker

Soil

wee

k 1

Soil

wee

k 5

Soil

wee

k 6

Soil

wee

k 7

Soil

wee

k 8

Soil

wee

k 20

Foam

wee

k 1

Foam

wee

k 5

Foam

wee

k 6

Foam

wee

k 7

Foam

wee

k 8

Foam

wee

k 20

Con

trol

wee

k 6

Con

trol

wee

k 7

Con

trol

wee

k 8

Mar

ker

Mar

ker

Neg

ativ

e

DGGE 26/04/55

Running 300 minutefrom PCR product 24/04/55

template use 8 µl.

Bacteria from styrofoam Bacteria from soil Control

Continuing band & found in control

Continuing band

Non-continuing band

Result !19Community structure analysis

Methodology !20

Mar

ker

Soil

wee

k 1

Soil

wee

k 5

Soil

wee

k 6

Soil

wee

k 7

Soil

wee

k 8

Soil

wee

k 20

Foam

wee

k 1

Foam

wee

k 5

Foam

wee

k 6

Foam

wee

k 7

Foam

wee

k 8

Foam

wee

k 20

Con

trol

wee

k 6

Con

trol

wee

k 7

Con

trol

wee

k 8

Mar

ker

Mar

ker

Neg

ativ

e

Bacteria from styrofoam Bacteria from soil Control

Selected DNA

Molecular cloning & identification

Selected for cloning

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

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


Sequence report - Electropherogram

F3 F10 S7

Control 4 Control 7 F5


Sequence blasting

SampleLength (bp)

Similar sequenceMax

iden

Max

score

E.Value

F10 444 Herbasprillium.sp 98% 753 0.0F3 504 Massialia aerilata 97% 830 0.0S7 485 Caulobacter segnis

ATCC 2175698% 830 0.0

Control4 1,055 Azohydromonas australica

83%1297 0.0

Control7 1,006 Ochrobactrum rhizosphaerea

82%1193 0.0


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

0.024280.00260

0.00219

0.013570.00561

-0.00301

0.00130

0.00616

0.00883

-0.00053

Neighbourhood joining tree contract from the

Specimen DNA sequence


!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;ProteobacteriaBetaProteobacteri

a Burkholderiales Oxalobacteraceae

Massilia


Betaproteobacteria; Burkholderiales;

Oxalobacteraceae; Herbaspirillum


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


(SEM)

Agar cultivation

Phylogenetic tree

Community

fingerprint

16s Ribosomal RNA

identification

Molecular cloning

Methodology !28Degradability 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


Control : Polystyrene in MSM broth without bacterial source.

Regular polystyrene foam that didn’t use in experiment.

100x 200x 500x


Polystyrene in Medium with bacteria from Styrofoam in the landfill.

100x 200x 500x


Polystyrene in Medium with bacteria from soil in the landfill.


100x 200x 500x



Polystyrene in Medium with bacteria from soil in the landfill.

Agar cultivation


(SEM)

Phylogenetic tree

Community

fingerprint

16s Ribosomal RNA

identification

Molecular cloning

Methodology !35Agar 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


‣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


‣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

Conclusion & Discussion !39

Soil

wee

k 1

Soil

wee

k 5

Soil

wee

k 6

Soil

wee

k 7

Soil

wee

k 8

Soil

wee

k 20

Foam

wee

k 1

Foam

wee

k 5

Foam

wee

k 6

Foam

wee

k 7

Foam

wee

k 8

Foam

wee

k 20

Con

trol

wee

k 6

Con

trol

wee

k 7

Con

trol

wee

k 8

Styrofoam sourceControl Soil source


!information

Found in soil culture(S) Found in foam culture(F) Found in control

Caulobacter segnis Massilia aerilata Herbaspirillum seropedicae Ochrobactrum sp. Azohydromonas

Taxonomy


Alphaproteobacteria; Caulobacterales;

Caulobacteraceae; Caulobacter

Bacteria;ProteobacteriaBetaProteobacteri

a Burkholderiales Oxalobacteraceae

Massilia


Betaproteobacteria; Burkholderiales;

Oxalobacteraceae; Herbaspirillum


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


‣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.

Research Achievements

13th NCSC, Jaipur India 2011

Youth summit 2012, Dubai UAE

JSTP Scholarship

STT 36

BYEE, Leverkuzen Germany

BYEE Poster Prize from india

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 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

!45Scholarships

!46Mentor

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

Advisor(

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


Advisor(



Advisor(



Advisor(


!47Assistant Mentor

PSUWIT TEACHER SUT

KMUTT

PSU KU

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

- Isaac Newton -

RIPMy brave Bacteria

the biodegradation of polystyrene

Science