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Dr. Duong Van Hop
A CASE STUDY ON
Collaboration project on taxonomic and ecological studies of
microbes in Vietnam and utilization under NP on ABS
BETWEEN
Institute of Microbiology& Biotechnology (IMBT)
Vietnam National University, Hanoi (VNU)
AND
Japan National Institute of Technology and Evaluation (NITE)
Vietnam National University, Hanoi (VNU)
Institute of Microbiology and Biotechnology (IMBT)
= Objectives =
1) Conservation of biological diversity,
2) Sustainable use of its components, and
3) Fair and equitable sharing of the
benefits arising out of the utilization of
genetic resources.
Convention on Biological Diversity: CBD
The Nagoya Protocol was adopted at 1:29 a.m.
30 October 2010.
NAGOYA PROTOCOL ON ACCESS TO GENETIC RESOURCES AND THE FAIR
AND EQUITABLE SHARING OF BENEFITS ARISING FROM THEIR
UTILIZATION TO THE CONVENTION ON BIOLOGICAL DIVERSITY
Microbial world
(1) Almost microbes are invisible need various techniques in studying them
(2) Much Difficul in managing microbial GR in comparing to animal and plant
(3) Amount 1-5% microbes are reported from the world so there is no traditional
knowlege to many microbes.
Microbial cullures is starting GR for biotechnology and bioindustry (good values in medicine , pharmacy, comestic, food products)
1. Isolation 2.Screening 3. Genetical
improvement
4. Pilot
fermentation
5. Production
fermentation
6. Product
development and
comercialization
National Bioresources Centers
(NBRCs)
Universities and Institutions Factories & Companies
Asean Consortium for the conservation and sustainable use Microbial
resources (ACM).
Members (ACM) are 23 Organizations from 13 Countries
- 11 Culture Collections Included -
-LIPI
-
MARD
I
-Univ.
Philippines
-Univ.
St.Thomas
-NITE
-RIKEN-
BRC
-Institute of Microbiology
Chinese Academy of
Sciences
-KRIBB-BRC
-NAAS
-KNRRC
-Institute of Biology
Mongolian Academy of Sciences
-Vietnam National
Univ.
-Ministry of
Environment
BIOTE
C
-Research Institute of Science
-Pathein
Univ.
-IMTECH.
ACM established in 2004, at Kisarazu, Chiba, Japan
Aim of the ACM • Framework for international cooperation to encourage
microbiological researches
• Construction of the mechanism for academia and industry to
utilize microbial resources
• Establishment of Microbial Resource Centre network
• Establishment of international standards for biological
material transfer and benefit-sharing
• Improvement and share of standardized techniques
Task Forces in ACM
1. Asian BRC Network (ABRCN) Chair: Ken-ichiro Suzuki (Japan)
2. Human Resource Development (HRD) Chair: Rosario G. Monsalud (Philippines)
3. Management of Material Transfer (MMT) Chair: Katsuhiko Ando (Japan)
The ACM members expect the expansion of the activities to reach the international
standardized scheme to encourage microbiologists to study biological diversity to
solve the global problems on environments and human welfare.
Case study of
IMBT, VNU - DOB, NITE, Japan
on collaboration project on taxonomic and
ecological studies of microbes in Vietnam
and utilization
under NP ABS
Mutual interest
VTCC,IMBT, VNU-Vietnam
• Establishing Vietnam Culture Collection and strengthening capacity in studying microbial diversity and management
DOB,NITE, Japan
• Enrichment culture
collection and studying
tropical microbial diversity
in Vietnam
The project divided by 2 phase:
Phase 1: From 2004- 2015 focussing on studying microbial
diversity in Vietnam and culture collection management.
Phase 2: from 2016 -2021 for utilization of the microbial GR
Signing of Memorandum of Understanding (MOU)
between the Ministry of Science and Technology (MOST) of Viet Nam
and the National Institute of Technology and Evaluation of Japan
for the Joint Research Program on Conservation and Sustainable Use of
Biological Resources
Signing for MOU
between
Dr. Le Minh Sat, Vice
Director General of
Department of Science
and Technology, MOST,
and Mr. Masahiro
Miyazaki, Director
General of Department
of Biotechnology, NITE,
on 15 March 2004,
Kisarazu, Chiba, Japan
Main outcomes from phase 1
• Technology tranfering
• Enrichment of Vietnam Type Cultures Collection (VTCC)
• Providing qualified data for VTCC
• Building up E-catalogue of VTCC
• Finding new taxa of microbe in Vietnam
• Manpower development
• Publication in domestic & Internatinal Journals
Antibiotic Screening from Actinomycetes T. Suemoto and S. Miyadoh (NITE/NBRC)
Collect soil samples as
sources for actinomycete
isolation.
Transfer the soils to glass Petri dishes,
and put them, opening the lids, at
room temperature or higher for 5-
7days.
This process promotes actinomycete
spore formation and decreases
bacterial cell number.
Heat at 100℃ for 30min.
(Close the lids of Petri dishes)
Non-spore forming bacteria
are killed. Most actinomycete
spores do not die
in these conditions.
Sprinkle (or spread) the treated soil
samples on HV-agar*1, directly.
Pick up actinomycete colonies
by toothpick and transfer them to
new HV-agar.
(4 strains / Petri dish)
Transfer the isolates from HV-agar to YS-
agar*2 in order to purify the colonies. (1
strain / Petri dish)
Drying
30℃,
5days
Incubation
28℃,
7days
Incubation
28℃,
6days
2nd Screening (to select the strain producing antibiotics in liquid medium)
1st Screening (to select the strains producing antibiotics)
3rd Screening (to select the strain producing organic solvent-soluble antibiotics*4)
Isolation of Actinomycetes
Cultivate the selected strains
in over 400 ml of liquid medium.
Extraction, Purification and Identification of Antibiotics
1st day 6th day 13rd day 19th day
41st day
44th~59th day 60th day 61st day
酢酸エチル抽出
Concentration
Analyze the active fraction by LC/MS (retention time, molecular weight and UV absorption).
Elucidate the structure by LC/MS data, biological activity and producing strain based on database.
22nd day
Incubation
28℃,
3days
Transfer purified and
selected isolates to slant
culture (YS-medium).
Remove duplicate strains by
morphological observation.
Spreading the
treated samples Pick up
colonies
Purification of
the isolates
Preservation of
the isolates
*1 Selective isolation medium for actinomycetes.
HV stands for “Humic acids-Vitamin”.
Actinomycetes play a role as decomposers of humic
acids, difficult-to-degrade organic matters.
LC/MS analysis Database search
Identification of antibiotics
Large scale
production
Sampling of
soils Drying of
soil samples
Heating of
the samples
Incubation
30℃,
100rpm
3days
Packed column chromatography
Extract with organic solvent (ethyl acetate) and purify the antibiotic by using some
chromatographic (silica-gel column, gel-filtration, TLC, LC etc.) methods. (Bioassay for antibiotic activity by paper disk method)
Anthracyclines (antitumor) Ethyl acetate extraction
*2 YS-agar medium is often used for purification
and subculture. YS stands for “Yeast extract and
Starch”. Because of its nutrient richness, it is suitable
for the growth of many kinds of bacteria.
*3 The agar medium in which test organisms have inoculated. Four kinds of organisms used in this case: Gram positive bacteria (Bacillus subtilis & Kocuria rhizophila), Gram negative bacteria (Escherichia coli), yeast (Candida albicans)
Extraction and purification of antibiotics
Inoculate the selected
actinomycetes (1 strain /
Petri dish) on antibiotic-
producing agar medium
(soybean meal agar).
Inoculate the strains identified as
antibiotic producers in the 1st
screening into the antibiotic-
producing liquid medium (80ml).
Mix ethyl acetate with same volume
of any culture filtrate which passed at
the 2nd screening, and then centrifuge
for separation into solvent layer and
water layer.
Incubation
30℃,
100rpm
3days
22nd day
Incubation
30℃,
13days
36th day 36th day 39th day
40th day 40th day
41st day
Hollow 4 pieces out of the agar medium with
a straw, and put one of them on each bioassay
plate*3. On the next day, if a growth-inhibition zone is formed
around the agar piece, the actinomycete produces
antibiotics against assay organisms.
Put the paper disks dipped in the culture
filtrates on bioassay plates*3. On the next day, if a growth-inhibition zone is
formed around the paper disk, the strain can
produce antibiotics in the liquid medium.
Put paper disks dipped in the solvent layer
on bioassay plates*3. On the next day, if a growth-inhibition zone is
formed, the strain is considered to produce organic
solvent-soluble antibiotics.
Bioassay for
antibiotic activity
(agar-piece method)
Antibiotic production
on agar medium
Production
in liquid medium
Bioassay for
activity of solvent layer Antibiotic extraction
with organic solvent
Bioassay for
antibiotic activity
(paper disk method)
35th day
O
HO
O
R1O O
O
R2
OH
OH
NH2
H3C
OH
*4 Organic solvent-soluble antibiotics were targeted in this case,
because of experimental easiness, e.g. concentration of solvent.
Laboratory 1
Laboratory 2
Advance
Manpower development
• 16 visits (Scientists and researchers) from VTCC to
NITE (1-2 months ):
• 10 visits of scientists to attended annual ACM
meeting (I.II, III,IV,V,VI,VII.VIII,IX)
• Conducting 18 Technical training workshops at IMBT
for a number of 150 Vietnam participants
• Annual project workshops (2004-2015)
Exchange of Researchers
Ms. Le Thi Hoang Yen and Ms. Nguyen Thi Van
From 11 January to 11 March 2011
Nhóm vi sinh vật Số chủng VSV
được quản lý
trong ACCESS
Số chủng VSV được quản lý trong cataloge
online
Số lượng
chủng
Số lượng
loài
Số lượng
chi
Vi khuẩn hiếu khí 2029 749 162 49
Vi khuẩn kỵ khí 50 - - -
Xạ khuẩn 3250 667 332 39
Nấm sợi 2750 1024 203 48
Nấm men 1403 960 189 56
Tổng số 9482 3400 886 192
website: http://www.imbt.vnu.edu.vn/vtcc/
Building up E-catalogue of VTCC >5000 sequence (18s, 23s, 16s ITS) were provided
from DOB, NITE for the catalogue
Documentation
• Using Access software for culture management
• VTCC is member of WFCC in 2008
(http://wdcm.nig.ac.jp/hpcc.html)
• Establishing microbial catalogue online for 3400
cultures (http://biotechvnu.edu.vn/vtcc)
• Register data of 2082 culture to global catalogue
online (http://gcm.wfcc.info) in 2013
Registeration of 2082 microbial gene source data to
global catalogue of microbe in 2013
(http://gcm.wfcc.info)
Summary ABS from Joint Project MAT based on equality and mutual benefit
Non-monetary benefits • Technology transfering
• Enrichment of Vietnam Type Cultures Collection (VTCC)
• Providing qualified data for VTCC
• Building up E-catalogue of VTCC
• Finding new taxa of microbe in Vietnam
• Manpower development
• Publication in domestic & Internatinal Journals
Monetary benefits
• 16 visits (Scientists and researchers) from VTCC to NITE (1-2 months ):
• 10 visits of VTCC scientists to attended annual ACM meeting (I.II, III,IV,V,VI,VII.VIII,IX)
• >5000 sequence (18s, 23s, 16s ITS)
• Conducting 18 Technical training workshops at IMBT for a number of 150 Vietnam participants
• Chemicals glassware for research activities at VTCC
Phase 2: from 2016 -2021 for utilization of the
microbial GR
President of NITE, Prof. Dr. Takashi Tatsumi signing at the whorkshop
Prof. Dr. Katsuhiko Ando presenting on access Microbial GR based on NP
Outline of MOU and PA in phase 2
• MOU: the Parties(NITE and IMBT) recognize the
importance of international scientific and technological
cooperation between Japan and Vietnam on conservation and
sustainable use of microbial resources in compliance with the
Convention on Biological Diversity (the ”CBD”) and relevant
laws and regulations of Japan and Vietnam;
• The objective of activities under this MOU is to promote
conservation and sustainable use of biological resources in
Japan and Vietnam especially with a view to use biological
resources in scope of academic and industry
Outline of MOU and PA in phase 2
(cont.) • PA: PROJECT AGREEMENT between Biological Resource Center,
National Institute of Technology and Evaluation of Japan (NITE)
And Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi of Vietnam (IMBT) on Transfer and Benefit-Sharing of Microbial Resources
• This Project Agreement (the “PA”) is entered into effective on April 1, 2016 (the “Effective Date”) between Biological Resource Center, National Institute of Technology and Evaluation (“NBRC“) of Japan and Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi (“VNUH-IMBT“) of Vietnam (collectively the “Parties” or individually a “Party”).
• the projects between NBRC and VNUH-IMBT have continued for 12 years, and over 13,000 microbial strains have been collected through corporation between the Parties. The activities resulted in publication of scientific papers and technical transfer from NBRC to VNUH-IMBT. Especially, the actual practice of material transfer based on the principle of the CBD and in view of mutual benefit is unique, and it contributes to the promotion of bio-industry in Japan and Vietnam.
Outline of MOU and PA in phase 2
(cont.) • MTA1 (Matterial Transfering Agreement) : the PROVIDER agrees
to distribute the strain(s) to the RECIPIENT , The RECIPIENT shall not use the MATERIAL for COMMERCIAL USE
• MTA2 : for distribution of the MATERIAL to third party:
On bebefit sharing
-For academic activities: none-moneytery bebefits
-For comercial activities: shall be larger than 0 % (zero percent) and not be more than 1% (one percent) of the net sales. Each party shall pay to the other Party 50% (fifty percent) of amount of money paid by the third parties.
For the Recipient who has the nationality of the country of origin of the Material, the Providers Should not to claim any payment with regard to the Condition of the Benefit Sharing from such Recipient.