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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