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 Affiliations1Department 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: [email protected] (K.M.); [email protected] (K.O.)
* Present address: Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8530, Japan.
Science 11 Mar 2016Vol. 351, Issue 6278, pp. 1196-1199
DOI: 10.1126/science.aad6359
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: [email protected]
Presented By: [Group-2]
Md. Naowaz Sharif Bitali IslamArafaty Beanty KyumMd. Shabab Mehebub
KeywordsBacteria: • Prokaryotes.
• single cell with a simple internal structure.
• microscopic
• can live anywhere
Structure of Bacteria
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.
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.
Introduction250 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.
Objective
• Their objective of this research was to isolate a novel microorganism which can degrade the PET.
Method and Materials
250 PET [contaminated environment sample]
SCREENING
MLE medium
Fusarium oxysporum and F. solani grew
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
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
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
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
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.
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.
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
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
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.
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.
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.
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.
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.
Result and Discussion
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
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.
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.
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
ConclusionBy 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.
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
Thank You
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