A new bacteria that able to breakdown and assimilates PET. It was a great discovery. We made a powerpoint presentation on that research paper. It was great challenge for us...

1. A bacterium that degrades and assimilates
poly(ethylene terephthalate)
Authors: Shosuke Yoshida1,2,*,
Kazumi Hiraga1,
Toshihiko Takehana3,
Ikuo Taniguchi4,
Hironao Yamaji1,
Yasuhito Maeda5,
Kiyotsuna Toyohara5,
Kenji Miyamoto2,†,
Yoshiharu Kimura4,
Kohei Oda1,†
Author Affiliations
1Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
2Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
3Life Science Materials Laboratory, ADEKA, 7-2-34 Higashiogu, Arakawa-ku, Tokyo 116-8553, Japan.
4Department of Polymer Science, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
5Ecology-Related Material Group Innovation Research Institute, Teijin, Hinode-cho 2-1, Iwakuni, Yamaguchi 740-8511, Japan.
Corresponding author. E-mail: kmiyamoto@bio.keio.ac.jp (K.M.); bika@kit.ac.jp (K.O.)
* Present address: Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8530,
Japan.
Science 11 Mar 2016
Vol. 351, Issue 6278, pp. 1196-1199
DOI: 10.1126/science.aad6359

2. A bacterium that degrades and assimilates
poly(ethylene terephthalate)
Course: Basic Microbiology
Course code: GEB104
Course Instructor: Md. Tofazzal Islam,
Ph D (Adjunct)
Professor, Department of Biotechnology
Bangabandhu Sheikh Mujibur Rahman Agricultural University
Gazipur-1706
E-mail: tofazzalislam@yahoo.com
Presented By: [Group-2]
Md. Naowaz Sharif
Bitali Islam
Arafaty Beanty Kyum
Md. Shabab Mehebub

4. Keywords
Bacteria:
• Prokaryotes.
• single cell with a simple internal structure.
• microscopic
• can live anywhere
Structure of Bacteria

5. Keywords
Degradation- the condition or process of Breakdown of any molecule
Assimilates- absorb/take in
Biodegradation- is known as the chemical dissolution of materials back into natural
elements by microorganisms.
PET- Poly(ethylene terephthalate) - PET containers are popular for packaging
sodas, water, juices, salad dressings, cooking oil, peanut butter, shampoo, liquid
hand soap, mouthwash, pharmaceuticals, even tennis balls.

6. Background of the research?
 PET Poly(ethylene terephthalate)- is used extensively worldwide in plastic
products.
• Its accumulation in the environment has become a global concern.
 There are very few reports on the biological degradation of PET or its utilization
to support microbial growth.
 Few fungal species can degrade PET but it is not yet a viable remediation.

7. Introduction
250 PET debris-contaminated environmental samples ( sediment, soil,
waste water, activated sludge) collected from PET bottle recycling site.
One sediment sample contained a distinct microbial consortium on the
PET film.
Microscopy revealed that the consortium on the film[ Fusarium
oxysporum and F. solani ] contained a mixture of bacteria.
Using limiting dilution of consortium [Fusarium oxysporum and F.
solani ] successfully isolated a bacterium that capable of degrading and
assimilating PET.
Attempted to determined how the metabolism works and metabolism of
PET evolved.

8. Objective
• Their objective of this research was to isolate
a novel microorganism which can degrade the
PET.

9. Method and Materials
250 PET [contaminated
environment sample]
SCREENING
MLE medium
Fusarium oxysporum
and F. solani grew

10. Method and Materials
Quantification of CO₂ Generation
Cultivated Fusarium oxysporum and F.solani
Presence of Fusarium oxysporum and F. solani with PET
Absence of PET
Absence of Fusarium oxysporum and F. solani
Pure medium

11. Method and Materials
H-MNR SPECTROSCOPY
ISOLATION OF IDONELLA SAKAIENSIS
GROWTH OF IDEONELLA SAKAIENSIS ON PET FILM in YSV medium
SCANNING ELECTRON MICROSCOPY
REVERSE-PHASE HPL ANALYSIS
ETHYL ACETATE EXTRACTION
WHOLE GENOME ANALYSIS
RNA SEQUENCE ANALYSIS

12. Method and Materials
PROTEIN PREPARATION
ENZYMES ASSAYS FOR PET FILM
ENZYMES ASSAYS FOR (hCPET)
ENZYMES ASSAYS FOR (BHET)
ENZYMATIC PREPARATION OF(MHET)
KINETIC ANALYSIS OF MHETase

13. Method and Materials
ENZYMATIC ACTIVITIES OF MHETase for the small aromatic esters
Enzyme activities for p-NP-aliphatic esters
Phylogenic analysis
Transcription start site (TSS) identification for ISF6_4831 &
ISF6_0224 GENES
PROTEIN SEARCH ON THE FULLY SEQUENCED GENOMES
16S rRNA analysis

14. Result and Discussion
 PET film degradation by Fusarium
oxysporum and F. solani .
• 10 mL of MLE medium at 30°C.
• (A) Growth of Fusarium oxysporum and F. solani
on PET film after 20 days.
Finding: These fungus are able to grow in PET film.

15. Result and Discussion
(B) SEM image of degraded PET film after
70 days. Inset, intact PET film. Bar, 0.5 mm.
Finding: Fusarium oxysporum and F. solani .
can degraded PET film.
(C) Time course of PET film degradation by
Fusarium oxysporum and F. solani .
Finding: after 80 days Fusarium oxysporum
and F. solani can loss only 50 mg weight of
PET film.

16. Result and Discussion
 PET film degradation by I.
sakaiensis 201-F6
• (D) SEM image of degradation of PET
film by I. sakaiensis 201-F6
• Arrow Indicate Contact points of cell
appendages and PET film surface.
• Finding: I. sakaiensis can adhere
with PET film surface.
• Magnification are shown in the right
panel.
D

17. Result and Discussion
• (E) Cell cell contact with
appendages.
• (F) Shorter appendages between the cell
and the PET film surface.
• Assist delivery of secreted enzymes

18. Result and Discussion
• (G) Degraded PET film surface. Inset, intact PET
film Bar, 1 µm.
• Finding: I. sakaiensis can degraded PET film.
• (H) Time Course of PET film Degradation by I.
sakaiensis.
• Finding: I. sakaiensis can loss around 60 mg
weight of PET film within only 40 days.
• Comparative to Fusarium oxysporum & F. solani,
I. sakaiensis is faster degraded PET film.

19. Result and Discussion
 (A) PET film surface degraded.
Inset, intact PET film. Bar, 5 µm.
Finding: PETase enzyme is
responsible for degradation of
PET film.
ISF6_4831 protein is a PET hydrolase (PETase ) Effect on PET film.

20. Result and Discussion
• HPLC[High-performance liquid
chromatography] spectrum of
the products released
from the PET film.
• Finding: PETase release
those products by
degradating PET film.

21. Result and Discussion
• (C) Unrooted phylogenetic
Tree of known PET
hydorlyting enzyme.
• Finding: The enzymes are
related to each other but
they have no root or
ancestor.

22. Result and Discussion
(E) Activity of PET hydrolytic enzyme for highly
crystallized PET.
Finding: PETase is more effective.
(F) Effect of temperature on enzymatic PET film
hydrolysis.
Finding: In environment friendly temperature
(20-40)C PETase secreted more.

23. Result and Discussion

24. Result and Discussion
PET degradation Pathway
PETase secreted
PETase Breakdown PET to MHET
MHETase secreted
I. Sakaiensis
adhere to PET
MHET uptake and converted to
TPA and EG
TPA converted to PCA
I. Sakaiensis use PCA for its growth

25. Result and Discussion
Responsible for
breakdown of PET
Responsible for
breakdown of MHET
In I. sakaiensis two most
effective gene ISF6_4831
and ISF6_0224 found.

26. Result and Discussion
 The MHETase, PETase, TPADO, Pca34
and Pca45 homologs were searched on
the Integrate fully sequenced genome
database. Unable to find similar
organism.
• Maximum likelihood phylogenetic tree
for the 16S rRNA.
• Two microorganism which are similar
in PETase homologs.
• But I. sakaiensis has PETase, MHETase,
TPADO, Pca34 gene but in
amycolatopsis mediterranei u32 TPADO
large subunit is absent.

27. A bacterium was Isolated from the
consortium on the PET film,
[Fusarium oxysporum and F.
solani.]
 It is a novel bacteria Ideonella
sakaiensis 201-F6
Result and Discussion

28. Conclusion
By screening natural microbial communities exposed to PET in the
environment, this research found
 A new novel bacterium I. sakaiensis .
 which is able to utilize PET as its major energy and carbon
source.
convert PET into two environmentally benign monomers
[ terepthalic acid and ethylene glycol]
This new isolated bacteria is very effective for bioremediation.
Because previously the ability to enzymatically degrade PET for
microbial growth has been limited to a few fungal species.

29. Acknowledgement
We are grateful to Yoshida et al. and also grateful to Md.
Tofazzal Islam.
We are thankful to : Effi Haque
Abid al Reza
Babry Fatema
Rayhan Imam Gazzali
Pictures are available in www.google.com

30. Thank You