Transcript Presentation - IAC 2016, New Delhi

Microbial Conservation
Strategies and Methodologies:
Status and Challenges
Sushil K. Sharma
ICAR-National Bureau of Agriculturally Important Microorganisms
Kushmaur, Maunath Bhanjan-275 103, U.P., INDIA
Staphylococcus
Bacillus
Small is Immensely Beautiful
All Life in the Biosphere Depend
on
Microorganisms
Seme Important Biogeochemicals
Transformation Regulated by Microorganisms
140 million tons of nitrogen gas are removed
from atmosphere by microorganisms and made
available to the rest of the biosphere (Freiberg et
al., 1997).
 More than 40% of the CO2 drawn from the
atmosphere is accomplished by microorganisms
in marine environment, show the importance in
climate regulation and feeding to marine life in
deep ocean.
 Marine
microorganisms are responsible for
producing and degrading dimethyl sulphide
(DMS), a major climate cooling gas. Globally,
approx. 300 million tons of DMS per annum is
produced,
mainly
by
bacteria.

Why We Need to Conserve Microorganisms?
Instrumental value (Practical Uses)
Why We Need to Conserve Microorganisms?
Intrinsic Value
(means value independent of uses)
 Rights to exist
 They do not have wishes or hope but
they do have “Urges and Unconscious
drive”. Microbes do have ‘biological
Interest’.
 For example: (1) A halophilic that needs to
live in salt water may die if it is placed in
freshwater. (2) Acidophiles live in acidic
conditions and die in alkaline conditions.
‘silent and unseen majority of life’
Beijerinck (1913) maxim:
“Everything
is
Everywhere,
Environment Selects”
but,
the
• Microorganisms are ubiquitous and cosmopolitan and do
not exhibit any biogeographical structure, thus, they
unlikely to be at the risk of extinction.
However, this is not absolutely true
Many Microbes Face Real Threats of
their Continued Existence
Issues of the maxim
“Everything is Everywhere”
• Bacteria inhabiting leaf surface do indeed appear to be
ubiquitous but, many other exhibit localized endemism
and absence of panmixsis.
• Many pathogens have well-defined distribution in animals,
humans and crops.
“Environment Selects ”
• Many microbes need specific habitats and mutual
interdependence.
Examples of “Environments Selects”
1. Microbially-diverse endangered soil habitats.
2. Novel
anaerobic
fungi
associated
with
endangered Somali Ass, Red Kangroo and Indian
camel (Oontomyces anksri).
3. Buwchfawromyces eastonii gen. nov., sp. nov.
from buffalo faeces in west Wales.
4. Goodyera pubscens, an orchid, interacts with
same fungal partner in modest environment but
switch to different fungal partner under extreme
environment.
5. Occurrence of specific microbes in extreme
environment conditions such as desert soil, saline
areas, glaciers, hydrothermal vent, cryoconites,
Antarctic sediments etc.
Challenges in Conservation
 Difficult to conserve single species in natural
environments.
 Species-specific conservation is difficult to achieve
in the natural environment
 Large population size of most species and their
ubiquity further reduce need of protection
 The small scale/size of the organisms
 General lack of macroscopically visible qualities
 The protection of ecosystem diversity since they are
evolving very rapidly
Convention on Biological Diversity (CBD)
 Fungi are under Conservation programme of
Plants
 Changes in the microbial ecology
 New challenges of conservation biology
 Absence of descriptive data on prokaryotes
GW Griffith (2012) proposed development of
Global Strategies for Microbial Conservation
(GSMC)
The ‘game-changer’ in microbiology
Application of rRNA gene barcoding
16S rRNA gene,
D1/D2 26S rRNA gene
18S rRNA gene
Operational taxonomic units (OTUs)
Basic unit of microbial diversity
A strategy for identification
Next generation sequencing (NGS)
Strategies of Microbial Conservation
“in-situ” (‘on site’, ‘in place’) conservation
Microbes can be stored in the same place where they live and from
where they would be isolated
“ex- situ” ( 'off site', conservation)
Preserves and maintains the distinct wild cultivated species and their
genetic resources in artificial media and taxonomically well described
“in-factory” form of conservation
Intermediate form of conservation and mainly used by the agro-industrial
sector
“in-situ” (‘on site’, ‘in place’) conservation
Root nodules having millions of
Rhizobium sp.
Vigna Spp., Pink flower, rhizosphere soil
Sunderban Mangroove Ecosystem
In situ conservation: an ecosystemic approach for a
dynamic and effective conservation
Some famous hot springs in India
Gaurikund
Uttara Khnad
Manikaran, Himanchal
Pradesh
Barkeshwar
West Bengal
Rajgir, Bihar
Kipti, Arunchal Pradesh
Leh to Tso Moriri, Ladakh
Pali, Maharashtra
Unai, Gujrat
Lahaul-spiti valley, HP
Nelang valley, Uttarakhand
Yumesamdong, Sikkim
Hot Spring, Manikaran, HP
https://www.google.co.in/search?q=lahaul+spiti&biw
Cryoconite holes at Himalayan Glacier
Explored Area of Diu Island
Sea beach near Diu Fort
Hokka (Hyhaene indica):
Branched Palm in Diu
Marshy Vegetations of Diu Islands
Unique Microbial Diversity in Unexplored Niches
84.44% metagenomic reads of
bacterial diversity of Murlen
National Park located in IndoBurman Biodiversity hotspot
region were not identified at the
genus level which suggested that
the uniqueness and unidentified
bacterial community structure
might lead to reveal novel
bacterial populations with some
unique properties.
Murlen National Park, Mizoram
Metagenomic Analysis of Deulajhari Hot
Spring Odisha
Sampling site
Total genera
Identified (%)
Unidentified (%)
Cluster 1 (S1)
292
38.6
61.1
Cluster 2 (S2)
314
40.7
59.2
DH-1
171
42
57.3
DH-2
375
41
58.6
Two Novel Taxa from Manikaran
(a)
(b)
(a)Fictibacillus halophilus AS8 on LB Agar plate
(b) Transmission electron micrograph of strain AS8
(a)
(b)
(a)Lampropedia cohaerens CT6 on LB Agar plate
(b) Transmission electron micrograph of strain CT6
Fictibacillus halophilus
AS8 T
Lampropedia cohaerens
CT6 T
(Sharma et al., 2016, Int J Syst Evol
Microbiol
doi: 10.1099/ijsem.0.001051
(Tripathi et al., 2016, Int J Syst Evol
Microbiol 66: 1156-1162
Methods employed for the
preservation and maintenance of
microorganisms
Culture Collections Information
Worldwide (CCINFO)
World Directory Of Culture Collections (Sixth Version, 2014)

Culture Collections: 712

Countries and Regions: 73

Microorganisms: 25,499,69
o
Bacteria: 10,451,59
o
Fungi: 7,777,86
o
Virus: 37,787
o
Cell Line: 31,829
Microbial Preservation Status at
Global Level
Distribution map of number of registered
culture collections in different countries and
regions
Source: WDCM World Data Centre for Microorganisms (World Directory Of Culture Collections, Sixth
Version, 2014) (webpage: http://www.wfcc.info/ccinfo/index.php/home/statistics/#m1/).
Global Distribution of Culture Collections
Distribution and the quantity of culture
collections in different countries and regions all
over the world
Source: WDCM World data centre for microorganisms (World Directory Of Culture Collections, Sixth Version,
2014) (webpage: http://www.wfcc.info/ccinfo/index.php/home/statistics/#m1/).
Top 10 strain holders of World
Rank
1
2
3
4
5
6
7
8
9
10
Countries and Regions Total hold
U.S.A.
261637
Japan
254830
India
194174
China
187661
Korea (Rep. of)
167090
Brazil
114494
Denmark
102066
Thailand
99376
Germany
95593
Belgium
93421
INDIA
Number of Culture Collections
30
Number of Cultures Held
194174
Number of Service of Patent deposits 4
Number of Service of Storage deposits 0
Number of Service of Distribution
12
Number of Service of Identification 16
Number of Service of Training
70
Number of Service of Consult
27
Acronym
WDCM Number
Collection
ABRC
WDCM 912
Anaerobic Bacterial Resource Centre
AYL
WDCM 934
WHYLABS RESOURCE CENTRE FOR MICROORGANISMS
BAB
WDCM 1058
Bank A Bug
BDU
WDCM 976
National Facility for Marine Cyanobacteria
BT
WDCM 1036
Bacillus thuringiensis
CCDMBI
WDCM 119
Culture Collection, Department of Microbiology
CIPDE
WDCM 462
Collection of Insect Pathogens, Dept. of Entomology
CM
WDCM 1033
Chroococcus minor
DBV
WDCM 173
Division of Standardisation
DMSRDE
WDCM 166
DMSRDE Culture Collection
DUM
WDCM 40
Delhi University Mycological Herbarium
EntoPatho
WDCM 1013
Entomopathogens
GFCC
WDCM 946
Goa University Fungus Culture Collection and Research Unit
ITCC
WDCM 430
Indian Type Culture Collection
MCC
WDCM 930
Microbial Culture Collection
Acronym
WDCM Number
Collection
MCM
WDCM 561
MACS Collection of Microorganisms
MPKV
WDCM 448
Biological Nitrogen Fixation Project College of Agriculture
MRCJ
WDCM 1117
Col. Sir R. N. Chopra, Microbial Resource Center Jammu
MTCC
WDCM 773
Microbial Type Culture Collection & Gene Bank
NAIMCC
WDCM 1060
National Agriculturally Important Microbial Culture Collection
NCCPF
WDCM 1118
National Culture Collection of Pathogenic fungi
NCDC
WDCM 775
National Collection of Dairy Cultures
NCIM
WDCM 3
National Collection of Industrial Microorganisms
NFCCI
WDCM 932
National Fungal Culture Collection of India
NIICC
WDCM 961
NII Microbial Culture Collection
NMCC
WDCM 972
North Maharashtra Microbial Culture Collection Centre
NTCCI
WDCM 107
Culture Collection, Microbiology and Cell Biology Laboratory
UMFFTD
WDCM 562
Food and Fermentation Technology Division, University of
Mumbai
VBCCA
WDCM 931
VISVA-BHARATI CULTURE COLLECTION OF ALGAE
VPCI
WDCM 497
Fungal Culture Collection
Culture Collections of Specific Nature
Culture Collection
Country
Swiss Collection of Arbuscular
Mycorrhizal Fungi (SAF)
Switzerland
International Culture Collection of
Arbuscular Mycorrhizal Fungi
(INVAM)
Industrial Yeast Collection
Collection for Extremophile
Microorganisms and Type Cultures
(EMTC)
Marine Culture Collection of China
USA
Italy
Russian
Federation
China
Agriculturally Important Culture Collections
Culture Collection
Country
Agriculture Culture Collection of China
(ACCC)
China
Korean Agricultural Culture Collection
(KACC)
Korea
National Collection of Agricultural and
Industrial Microorganisms
Hungary
Culture Collection Beijing Agricultural
University (CCBAU)
China
International Collection of Microrganisms
from Plants
New Zealand
Russian Collection of Agricultural
Microorganisms (RCAM)
Russian
Federation
United States Department for Agriculture
(USDA)
USA
NAIMCC
(National Agriculturally Important Microbial Culture Collection)
(A Designated Repository under NBA, Govt of
India)
Established in 2001
An Unit of ICAR-NBAIM, Mau (U.P.)
To Conserve the Agriculturally Important Microbial Wealth of India
Annual collection of microbes at NAIMCC
6017
7000
5243
6000
Total No. of Cultures
6157
4448
5000
4690
3842
4000
3000
2495
2753
2009
2010
2000
1000
0
2011
2012
2013
Year
2014
2015
2016
Bacterial Colony under Stereomicroscope
Bacillus aquimaris SNL18
Brevibacterium sp. L11
Bacillus marisflavi IARIS14
NAIMCC-B-01609
NAIMCC-B-01134
NAIMCC-B-01562
Bacillus circulans NCIM 2107
NAIMCC-B-00081
Bacillus licheniformis AMAAS353 Lactobacillus fermentum 138
NAIMCC-B-00953
NAIMCC-B-01446
Repatriated Fungal Cultures from CABI-UK
Fusarium illudens
Gymnascella dankaliensis
Colletotrichum curcumae
CABI-256214
CABI-119450
CABI-288937
NAIMCC-F-00779
NAIMCC-F-01219
NAIMCC-F-00647
Achaetomium sp.
CABI-205029
Chaetomium longicolleum
Gliomastix
NAIMCC-F-02412
CABI-256285
CABI-354106
NAIMCC-F-00518
NAIMCC-F-01170
Cyanobacterial strains in NAIMCC
Microbial Resources from Different Niches
Available in NAIMCC
 Soil
 Rhizospheric
Soil
 Water
 Water Sediments
 Cold
deserts
 Cold
Springs
 Mangroves
Soil
 High Altitude
 Hot Water Springs

Hot Desert
 Saline
Environment
Soil
Microbes with Major Traits of
Agricultural Importance
Biofertilization
Biocontrol
Biofortification
Bioremediation
Bioconversion
Abiotic stress management
Preservation Methods
Preservation of AIMs
Long Term
Short Term
Sub-culturing
Glycerol
Stocks
Mineral
Oil- storage
Lyophilization
Cryopreservation
Challenges in Ex-situ Microbial Conservation
Priority to conservation rather than to full
characterization
Assessment of microbiological purity
Maintaining the culture under conditions that
minimize change
Reliable identification
Perform de-replication to limit redundancy of
collections
Registration of all the information gathered in a
special electronic database
“in-factory” form of conservation
Conservation Priority: 1
Microbial Communities/Ecosystems involved in
important Global-scale Biogeochemical Cycles
Examples
 Role of microorganisms is in the production and
decomposition of DMS, climate cooling gas
 Viruses influence on microbial ecosystems,
particularly regulating microbial populations.
Thus, viruses must be included in the
conservation programme.
Conservation Priorities: 2
Microbial Communities/Ecosystems involved in
Regional and Local-scale Cycle
Pollution can cause declines of changes in microbial
population both at regional and local scale.
Examples:
 Coral reefs which harbour great microbial
diversity ( Great Barrier Reefs, Australia).
 Microbial community shift caused by the heavy
metal/hydrocarbon contamination in soil.
Conservation Priorities: 3
Microbial Communities/Ecosystems involved in
that have Immediate or Potential Uses
Examples
 Thermus acquaticus isolated from Yellow National
Park, USA.
 Unusual physiological traits: Cryoendolithic
communities of the Antarctic Dry Valley, provide
opportunity
to
understand
community
development in extreme polar region.
 Giant sulphur bacteria (600µm) found in
Namibian Shelf can provide insight in sulphur
cycle.
Conservation Priorities: 4
Microorganisms with Intrinsic Value
Decision they have intrinsic value could potentially
lead to protection for all the microorganisms, which
impose considerable restriction on many activities
Practical steps and policy change required
for implementation microbial conservation
There are 7 major steps
1. Microbial Ecology Research
2.Incorporation of Microorganisms into Conservation
Efforts
3.Protection of Microbial Communities in their Own
Right
4. Change of Legislation to Protect Microorganisms
5. Improved Public Outreach and Education
6. Ex-situ Conservation
7. Institutional Changes
Summary and concluding Remarks
“Microbes orchestrate life
on earth”
Tom Curtis said
‘If the last blue whale choked to death on the last
panda, it would be disastrous but not the end of
the world. But if we accidentally poisoned the
last two species of ammonia oxidizers, that
would be another matter. It could be happening
now and we wouldn’t even know’.
no life can exist without the contribution of microbes
‘all nitrifiers’
Anammox
Thank You