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

Bonn guidelines on ABS

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

Tokyo. 23 Feb.2010

Working together=Technology transfer

Amylase CMCase Xylanase

Protease Phytase Dioxygenase

Screeing enzyme producers

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)

Visits of VTCC young microbiologists training

at NITE, japan

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

Cells of strain No. VTCC-Y-0826

Colony of strain No. VTCC-Y-0826

VTCC-Y- 0826

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.

Thank you for the kind attention

Vietnam National University, Hanoi (VNU)

Institute of Microbiology and Biotechnology (IMBT)