Keywords

1 Introduction

Microbes form an important component of ecosystems and carry out various functions essential for the biosphere including nutrient cycles of nitrogen, phosphate, oxygen and carbon, in bioremediation of pollutants, soil building and even in the life of plants and animals. They are an essential part of food chains and food webs and are responsible for the decomposition of waste materials and dead organic matter of plants and animals. The heterogeneity of biosphere has assisted in the creation of a wide array of microorganisms with potential to produce novel pharmaceuticals, enzymes and fine chemicals. Proper management and utilisation of microbial diversity has a supreme role in development of industrial and commercial applications. Even the pathogenic microbial stocks have to be maintained for ready availability in academic and industrial research. Advancements in the field of biotechnology have paved way for the isolation, culturing and characterisation of new microorganisms (Smith et al. 2008).

Microbial culture collections or repositories are crucial resource centres for preserving and supplying microbial specimens. These microbial resource centres (MRCs) are living libraries of microorganisms which are indispensable for scientific research in life science. They serve as repositories of living microbial cells, genomes and information relating to heredity and the functions of biological systems. They are also responsible for the supply of microorganisms in primary and applied scientific research, including those for commercial application in fields such as pharmaceuticals, cosmetics and industrial enzymes. Microorganisms are distributed ubiquitously on earth, and this large number of microbes has to be identified, classified and preserved. Microbial repositories contain collections of cultivable organisms of algae, bacteria, fungi (including yeasts), protozoa and viruses and their replicable molecules like genomes, plasmids, viruses, cDNAs and also uncultivable microorganisms (as metagenome libraries).

The demand for bioprospecting of microbial resources has escalated in recent time which has contributed to an increase in the number and quality of microbial repositories. World’s microbial diversity represents 50% of total biodiversity in terms of species numbers. The microbial world is the biggest unexplored biodiversity reservoir in the world with only <1% of the bacterial species and <5% of fungal species currently known. A large fraction of the world’s microbial wealth remains untapped because of difficulties in sample collection from extreme locations and difficulties in laboratory culturing (Staley et al. 1997). A large numbers of microbial species are uncultivable and require novel techniques of isolation and identification. The available microbial resources need to be conserved and used in a sustainable way along with due protection for intellectual property, which can be done through ex situ safeguarding through microbial culture collections. In addition to their role in scientific, industrial, agricultural, environmental and medical research, microbial repositories also serve as an interface between governments, industry and the public, assisting policymakers and public in understanding the value of microbial conservation.

Despite the ecological and economic value of microbial diversity, its conservation and management has been largely ignored. One of the reasons for this is the unawareness regarding the role of microbial diversity to the economy and in improving quality of life. Hence there is a compelling need to convince policymakers to undertake microbial conservation and management.

2 Microbial Diversity

Microorganisms were discovered by Antonie van Leeuwenhoek in 1677. Louis Pasteur in 1857 described their properties and uses in his study on lactic acid fermentation. The role of microbes as causative agents of infections was observed by Robert Koch in 1876 as part of his study of anthrax. Microorganisms are distributed in the kingdoms of Monera, Protista, Fungi and a part of plants of the Whittaker’s five-kingdom system. Recent evolutionary studies based on ribosomal RNA sequences show that the evolutionary distance between bacteria to animals and plants is farther than the distance between plants and animals from each other. On the basis of this, Woese et al. (1990) proposed a new superior concept of domains over the kingdom classification, with three domains, Archaea, Bacteria and Eucarya. Microorganisms are now regarded as a collection of evolutionarily different organisms with bacteria, actinomycetes, cyanobacteria, etc. under domain Bacteria, while methanogens, extremely thermophilic organisms, extremely halophilic organisms, etc., are placed in the domain Archaea and moulds, yeasts, basidiomycetes, algae and protozoans, etc. are under Eucarya.

The diversity of microorganisms on Earth is truly astounding. About 1 billion bacteria are present in just a gram of soil of which only 1% can be cultured (Yandigeri et al. 2010). It is predicted that Earth is inhabited by as many as 1 trillion (1012) microbial species (Locey and Lennon 2016). This figure indicates that 99.999% of microbial taxa still remain undiscovered and stresses the need for continued investigation. The total number of species of virus, bacteria, algae, fungi and lichens recorded from India and the world are summed up in Table 17.1.

Table 17.1 Total number of microbial species of in India and World
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3 Conservation of Microbial Diversity

Biodiversity surveys rarely attempt at systematic study of microorganisms in different habitats. The reason for this is the labour intensiveness of sampling, culturing and identification of enormous numbers of microorganisms. Microbes make up about 50% of the living protoplasm on this planet, and hence microbial resources deserve greater attention (Yandigeri et al. 2010).

Microbial diversity includes the range of variability among all types of microorganisms in the natural world. Microbes are sources of new genes and organisms that are of value to biotechnology, in monitoring and predicting environmental changes, conservation and restoration of higher organisms and in understanding biological interactions and evolutionary history. In order to fully harness the benefits of microbial resources, a better understanding of their diversity is required. In addition to their ecological role, many microorganisms are also of immense scientific and economic benefits. Pathogenic microbes cause thousands of mortality every day. Medical research of epidemics requires pure culture of the causative organism.

Microbial diversity offers numerous benefits. In agriculture, asymbiotic nitrogen fixers, mycorrhizal microbes and plant growth-promoting rhizobacteria (PGPR) like Pseudomonas fluorescens, Bacillus subtilis, Bradyrhizobium japonicum, etc. can be used as biofertilizers for improving crop nutrition. Some microbes assist in solubilisation of insoluble nutrient sources. Entomopathogenic fungus can be used as biocontrol agents against insect pests, and antagonistic microbes are involved in the suppression of growth and activity of plant pathogens. Microbial alkaloids, toxins and antibiotics can control plant pests. Some microbes like Trichurus spiralis, Paecilomyces fusisporus and Trichoderma viridae can act as compost accelerators at low C: P and C: N ratios, which is achieved through supplementation with rock phosphate and urea. Microbes are essential for degradation of complex plant and animal wastes and in nutrient cycles. Cellulose and lignin degrading microorganisms like Trichoderma viridae, Scytalidium thermophilum, Fusarium moniliformae and Phaenerochaete chrysosporium can be used for the management of agro-waste materials and bioconversion of distillery wastes and dyes (Singh and Nain 2014). They can also be used in bioremediation of oil spills, industrial waste and pesticides by oil/hydrocarbon degrading microbes and in bio-mineralisation of heavy metals and reclamation of degraded soils.

In industries, microbes are used in brewing of wine and beer, in the preparation of dairy products like cheese, curd and yoghurt. They also serve as sources of antibiotics, clinically important molecules and industrial enzymes. For humankind to benefit from biodiversity, we must understand and utilise its potential through proper identification and maintenance of biological resources (Yandigeri et al. 2010).

The importance of microbial culture banks has been recognised by industrial countries, which is the reason why majority of public collections are present in the Northern Hemisphere. Europe and America hold 56% of collections and 71% of the identified microorganisms. In contrast, the number of microbial culture collections in biodiversity rich regions, where approximately 20–30% of total novel isolations occur, is very low. The large numbers of novel bacterial and fungal species in the diversity hot spots are untapped reservoirs that hold bioeconomic potential (Komagata 1998).

India is one among the 12 countries of megadiversity with 1,27,000 species of listed plants, animals and microbes. Even though we are expected to have a rich microbial diversity, lack of skilled experts and adequate support restricts their discovery. India has 14,833 species of fungi, 2401 lichens, 17,000 flowering plants with an estimated 6 fungi per plant; therefore, it is anticipated that there are approximately 102,000 fungi (Arora et al. 2005).

Human interferences and natural calamities have resulted in the extinction of many organisms including plants, animals and microbes. All organisms serve as a reservoir for numerous other organisms, majority of which are microbes. The root microbiome, a dynamic community of microorganisms associated with plant roots, is unique for each plant species. The microbial communities inside the root and in the rhizosphere are distinct from each other and differ from the microbial communities of bulk soil (Gottel et al. 2011). Thus any loss of higher plants might result in the extinction of associated microorganisms and cause irreversible damage to biodiversity. While plants can be conserved as such in nurseries, botanical gardens or in/as cryobanks, seed banks and gene banks, microbes have to be preserved as pure cultures under laboratory conditions, followed by validation and preservation. Thus microbial culture collections serve not only in the preservation of microbes but also help in retrieving if they are completely lost from natural system. In situ microbial conservation method is inadequate because the organisms are prone to rapid evolution and are sensitive to environmental factors that can lead to their elimination.

The Convention on Biological Diversity (CBD) has recognised the importance of conservation of microorganisms and the significance of ex situ collections and benefit sharing. Molecular biology techniques and non-culture tools can be employed for the identification and characterisation of a vast diversity of microorganisms (Davison et al. 1998). A large fraction of microbes are unculturable, and hence unconventional isolation and identification strategies need to be developed (Smith et al. 2008). Metagenomic analysis of microbial communities does not require prior culturing and can be used for the identification of a vast majority of microbial biodiversity that can be missed by cultivation-based methods. It involves either shotgun or PCR directed sequencing of all genes from all the members of the sampled communities (Fig. 17.1).

Fig. 17.1
figure 1

Schematic representation of isolation of cultivable microorganisms by conventional dilution plating method and direct isolation of DNA from soil for construction of metagenome library

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The conservation and exploitation of microbial biodiversity still remains a challenge. Ex situ conservation strategies should be developed to conserve critical biodiversity elements, and the approaches to discover and understand microbial communities should be coordinated. The economic applications of microbial biodiversity should be identified, and the income received should be used to fund research, collection maintenance and biosystematics (Smith et al. 2008).

4 History of Microbial Repositories

Prof. Frantisek Král (1846–1911) established the first microbial culture collection in 1890 at the German University of Prague, Czech Republic, where fungal cultures were made commercially available (Smith et al. 2014). He had 30 years’ experience in manufacturing laboratory glass products, after which he worked as a technician at the Institute of Hygiene of the German University of Prague. His expertise in glass manufacturing was the reason for his appointment as the director of bacterial collection by Prof. Soyka. Later he held the post of associate Professor of Bacteriology. Král published the first catalogue of microorganisms from a culture collection in 1900. After his death in 1911, his culture collection was acquired by Professor Ernst Pribham, who transferred it to the University of Vienna and issued several catalogues listing the microbes in the collection. A portion of this collection was taken to Loyola University in Chicago by Prof. Pribham in the 1930s. Some of the cultures in the collection still remains at Loyola University while others were transferred to the American Type Culture Collection (ATCC, USA) after Pribham’s death (Uruburu 2003; Çaktu and Turkoglu 2011). After Král’s collection, many other culture collections were established; however only few collections established in the early era of microbiology survived the politically and scientifically turbulent twentieth century.

The oldest culture collections that are still in use are the Mycothèque de l’Université catholique de Louvain (MUCL) in Louvain-la-Neuve, Belgium, established in 1892, and the Collection of the Centraal bureau voor Schimmel cultures (CBS) founded in 1906, in Utrecht, the Netherlands (Uruburu 2003). MUCL is one among the earliest fungal collections which currently holds over 25,000 strains of filamentous and yeast-like fungi, representing over 3300 species of Ascomycetes, Basidiomycetes, Hyphomycetes and Zygomycetes. The CBS Fungal Biodiversity Centre, founded as an institute of the Royal Netherlands Academy of Arts and Sciences, was the first independent microbial centre to undertake the preservation and supply of a wide range of fungi. Now it holds a world-renowned collection of living filamentous fungi, yeasts and bacteria (Hawksworth 1985; Smith et al. 2014). The well-known American Type Culture Collection (ATCC) was established in 1925 at Washington. Now ATCC is located in Manassas, Virginia.

There is an international connection between recognised microbial repositories lead by World Federation of Culture Collections (WFCC). It was in 1946, Prof. P. Houduroy established a centralised information facility at the University of Lausanne, Switzerland. Information about the strains maintained in different collections was provided by this facility. This centre became associated with International Association of Microbiological Societies (IAMS) and is now renamed as International Union of Microbiological Societies (IUMS). At a conference on culture collections held in Canada in 1962, the IAMS was asked to form a section on Culture Collections under the chairmanship of Prof. Skerman. This was reorganised in 1970 and the section became the World Federation of Culture Collections (WFCC). Through the World Data Centre of Microorganisms (WDCM) established in Mishima, Japan, WFCC has collected and stored information on several hundreds of collections throughout the world (Uruburu 2003). There are at present 708 culture collections in 72 countries registered in World Data Centre for Microorganisms (WDCM) over the world which together they hold almost 2.5 million strains of a wide range of microorganisms (http://www.wfcc.info/ccinfo/, accessed August 25, 2016) (Fig. 17.2 and Table 17.2).

Fig.17.2
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The charts showing the number of holdings of different type of microbial cultures in the culture collections (a) and region-wise distribution of Culture Collections (b), prepared based on database available with Culture Collections Information Worldwide (CCINFO). CCINFO is a world directory of all registered culture collections maintained by World Data Centre for Microorganisms (WDCM) (http://www.wfcc.info/ccinfo/statistics)

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Table 17.2 Some important microbial culture collections in the world
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5 Types of Microbial Culture Collections

Culture collections are classified into four categories, namely, Private or In-house Culture Collections, Specialised Collections, Research Collections and Public Collection or Service Collections (Mahilum-Tapay 2009).

5.1 Private or In-House Culture Collections

These collections are established and maintained by particular organizations, institutions, laboratories, hospitals or individual companies to meet their requirements. Collections of this type may hold cultures ranging from small to large, but catalogues are not generally available. The cultures are supplied on a discretionary basis and are not usually open to the public.

5.2 Specialised Collection

They are collections of specific group of microorganisms in specialised fields such as brewing, antibiotic production, plant or animal pathology, etc. in which a large number of strains have been isolated and characterised and detailed taxonomic study carried out. Examples include UTEX Culture Collection of Algae at the University of Texas, USA, and Culture Collection of Algae and Protozoa (CCAP), UK.

5.3 Research Collections

Research collections are created by individual researchers or teams as a part of their research programmes. These collections often include novel and peculiar strains of scientific importance, but they lack long-term storage facilities, and hence the cultures could be lost when researchers change positions, retire or pursue different lines of research.

5.4 Public Collection or Service Collections

These collections are directly or indirectly supported by government funds and are established for the purpose of public service. All details of the cultures contained are generally listed in publicly available catalogues, and authenticated microbial cultures can be obtained from them on request at less than full economic cost. They are large collections and the number of strains contained varies from collection to collection. Microbial Type Culture Collection (MTCC), Chandigarh, India, is a public culture collection funded jointly by the Department of Biotechnology (DBT) and the Council of Scientific and Industrial Research (CSIR), Government of India.

6 Services Offered by Culture Collections

Microbial culture collections provide authentic biological material for high-quality research and teaching in the form of reference strains and also play a vital role in the conservation, prospecting and sustainable use of microbial resources (Sharma and Shouche 2014). The primary functions of microbial repositories involve collection, accession maintenance, preservation, identification, documentation, patent deposition, cataloguing, safe keeping, screening and distribution of microbial cultures on request, preparation of printed or online catalogues of collect culture data and making them accessible to the researchers. Thus researchers and taxonomists should be able to easily identify a strain of particular interest from the database provided by culture collection centres. In addition, microbial repositories also provide training courses and workshops related to the identification and maintenance of microorganisms to personnel from medical, environmental, industry or government laboratories who are involved in isolating and identifying microorganisms, diagnosing disease, quality control, fermentation, culture management, etc. Research initiatives related to taxonomic studies and microbiological preservation are also carried at microbial culture collections. The services offered by a culture collection depend upon the size and interests of the collection.

6.1 Culture Deposition

In a culture collection, new depositions or accessions can be made by researchers. They will be made available for the depositors and other researchers on request. On deposition of a culture, a unique accession number will be allotted by the centre. According to International Code of Bacteriological Nomenclature, for any valid publication of new species, its type strain should be designated and deposited in one or more established culture collections. Microbial repositories will have curators for each specific group of microorganisms, who conduct research and are responsible for the development and maintenance of their collections and new acquisitions. Most journals today require deposition of nucleic acid or protein sequences in public repositories along with the deposition of cultures from which the sequences are derived. All specific information like the source of isolation, taxonomic status, published data, special properties, reason for deposit, growth medium, growth temperature, pH and optimum procedure for long-term maintenance of a strain have to be provided while making a deposition (Malik and Claus 1987).

Microbial cultures that are usually considered for deposition are:

  1. (a)

    Published strains of newly named taxa

  2. (b)

    Type, neotype, selected reference strains, specific and unique biotypes

  3. (c)

    Strains with special properties and applications (bioassay, quality control, etc.)

  4. (d)

    Strains with applications in agriculture, biotechnology, medicine, education, etc.

  5. (e)

    Strains mentioned in patent applications

  6. (f)

    Genetically manipulated strains, plasmid carriers, special mutants, etc.

6.2 Distribution of Cultures

An important service of culture collections is to supply of authentic and viable strains for research, teaching or industrial applications. Supply of culture will require a fee, but the supply of cultures between Culture Collections is on an exchange basis. Microbial repositories usually provide guarantee for viability, for purity and, to a certain extent, to the properties cited in publications or their catalogues. There is a chance for alteration of characteristics of certain mutated strains, phage hosts and strains with plasmids, and hence Culture Collections often insist on reporting of discrepancies about a strain received from them. Microbial repositories can import cultures from abroad, but licences or import permits are required for this. The details on export and import restrictions of microorganisms are published in the News Letters of WFCC. The distribution of patent cultures is usually restricted to only authorized persons, subject to the approval of the patentee (Crespi 1985). Patent cultures are accepted on payment of a fee for their maintenance of the cultures.

6.3 Microbial Identification

Bacteria and fungi have been conventionally identified by isolation in culture followed by enzymatic reactions and morphological analyses. The identification of microorganisms, however, remains a challenge because of tedious and days-long biochemical and staining protocols and are prone to errors (Braga et al. 2013). Molecular techniques based on DNA amplification and sequencing provide more secure molecular identification of microorganisms. The 16S rRNA gene homology analysis has provided a tool for estimating bacterial phylogeny, which has led to rapid changes in bacterial taxonomy. Sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) regions of rRNA, followed by comparative sequence analysis, has been the ‘gold standard’ for molecular identification of most fungi (Tsui et al. 2011). This molecular technique is fast and accurate but is dependent on sequence quality in existing reference databases. Recently, identification based on mass spectrometry (MS), especially MALDI-TOF-MS, has been shown to be an alternative accurate and fast method to identify unknown bacteria on the genus, species and even subspecies level, based on profiles of proteins and peptides derived from whole bacterial cells (Braga et al. 2013).

6.4 Patent Cultures

Microbial repositories accept and maintain cultures which are subject to patent application, mostly at the payment of specific fee. Such cultures will be catalogued and given an accession number, but their distribution will be subjected to the approval of the patentee while the patent is pending. In 1977, an International Treaty was signed by 78 countries at Budapest and came into force in 1980 and is administered by World Intellectual Property Organization (WIPO), Geneva. As per provisions of the treaty, cultures deposited at any of the International Depositary Authority (IDA) are recognised by the member states for the patent purpose. A patent becomes valid only if the microorganisms cited in the patent application are deposited with an IDA recognised culture collection (Malik and Claus 1987). As on June, 2016, there are 45 culture collections recognised by IDA. In India there are two depositories, namely, Microbial Culture Collection (MCC), Pune and Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, that have acquired the status of International Depositary Authority under the Budapest Treaty.

6.5 Data Documentation and Catalogue Preparation

All major microbial repositories collect and document data regarding the specimens in their collection. Detailed record on strain characteristics, nutritional requirements (growth medium, temperature, pH, biochemical characters) along with literature references, application and history are documented. These data are published in the form of catalogues and catalogue supplements, which serve as a source of information for microbiologists. An International Centre for Information on Culture Collections has been established at Lausanne, Switzerland, for regular exchange of information between different collections.

6.6 Research and Training

All major culture collections offer training and consultation services in the fields of microbial cultivation, enrichment, isolation, identification and modern preservation techniques.

6.7 Safety and Security

It is mandatory to carry out safety operations in the collections including biosafety, chemical and physical safety, etc. and also to ensure good laboratory practice. Risk assessments must be carried out before cultures are brought into the collection, and specific procedures are applied. Adequate controls should be implemented to manage risk, to all who may come into contact with cultures, products and services provided. Particular attention needs to be given to the containment and bio-security aspects of strains which are potentially harmful to man, animals or crops.

7 Preservation of Microbial Cultures

Culture collections have a need to maintain healthy, viable microbial cultures for many months or years. Microorganisms occur as communities of mixed populations in nature. Once a strain or species is isolated and cultured in laboratory conditions, they become unstable, and their characteristics change on continuous maintenance in artificial media and conditions. Genetically engineered microbes and those containing plasmids may undergo changes with time when cultured. Isolation, selection, research and development of microorganisms with industrial applications required huge investment and effort. The authenticity and production efficiency of such cultures are also essential for patent application, and the loss of stability in such cultures may cause serious disruption to industrial processes (Malik and Claus 1987). Once a strain is selected and preserved, a distribution and seed stock should be maintained and stored separately. An original culture should also be preserved without subculturing. A preserved culture should be checked for its viability, purity and identity before making it available outside the collection.

Different methods are used for short-term and long-term microbial preservation of bacteria and fungi (Prakash et al. 2013). These include repeated subculturing, preservation on agar beads, slant-grown cultures overlaid with oil, using silica gel and other sterile supports, cryopreservation and lyophilisation. The techniques used in preservation of microorganisms can be divided in two groups. The first group aims at minimum detention of the cell vitality through hypobiosis, a state known as ‘cells at rest’. The second group of techniques that include drying, freezing at low temperatures and lyophilisation involves maintenance of microorganisms in an anabiotic state.

Anabiosis, the state or ability of an organism to reversibly decrease or stop its vital activities, is a form of adaptation to survive and preserve under adverse conditions. Many microorganisms are able to survive under varying environmental stresses like warmth, cold, freezing and thawing, drying and moisturising. Sporulating bacteria and fungi are the most resistant to drying, chilling and heating. Many microbes that do not form spores also remain viable even after prolonged refrigeration. The properties of the state of anabiosis are:

  • Absence or drastic reduction of metabolism

  • Preservation of cellular structure for a long time

  • Survival in the absence of free water

  • Increased resistance to extreme conditions

  • Ability to recover the vital activity under optimal conditions

These properties can be applied for long-term preservation of cultures (Uzunova-Doneva and Donev 2005).

The technique applied should cause only minimal damage to cells and must be able to stabilise genotypic, morphological and physiological features as well as viability until it is required for future use. A single universal preservation technique cannot be used for all microorganisms due to variation in susceptibility among them to different preservation techniques (Malik and Claus 1987). Among the different methods used, cryopreservation and lyophilisation are the most widely used in culture collections. Cryopreservation is the storage of cultures at temperatures that retard chemical reactions of around −70 °C and below. Freeze drying or lyophilisation involves the sublimation of ice from frozen material at reduced pressure directly from the solid phase to the gas phase. It requires storage either under vacuum or at atmospheric pressure in an inert gas.

7.1 Pre-preservation Conditions

The factors to consider prior to preservation of a microbiological culture are cultivation method, temperature, composition and pH of medium, aeration, age, physiological condition and concentration of the culture at the time of conservation (Uzunova-Doneva and Donev 2005).

7.1.1 Nutrient Media

Microorganisms can be cultured either in liquid broth or solid agar nutrient media for cell culture preparation before conservation. The composition of nutrient medium affects the cell resistance. Researchers have two opposing views regarding the choice of growth media. Culturing on rich nutrient media is recommended by some for obtaining a high percentage of viable cells. The opposing view is the use of nutrient poor media which induces cell metabolism reorganisation towards energy storage. Any media that increases the accumulation of proteins, carbohydrates and lipids in the cells increases its resistance lyophilisation. Tween 80 and oleic acid are usually added to medium to increase cell viability during preservation.

The acidity of medium used is another factor that influences cell resistance during conservation. Each organism has an optimal range of pH, under which it shows maximum growth. The media used to grow cells prior to preservation should be at its optimal pH for maximum survival. Similarly, optimal incubation temperature and aeration should be provided for growth.

In yeasts, medium level of aeration provides oxygen necessary for yeast metabolism, mediates proper mixing of nutrients and removes carbon dioxide released. Saccharomyces cerevisiae cultivated in aerobic conditions shows more resistant to hypo- and hypertonic shock compared to anaerobically grown cultures.

7.1.2 Culture Age

Choice of culture age is another important factor that influences percentage vitality of preserved microbial cultures. The optimum duration of culture varies with different organisms. Microorganisms are more resistant to freezing and dehydration at the end of the logarithmic growth phase or at the beginning of the stationary phase. For example, 48 hours culture of S. cerevisiae is most appropriate for conservation.

7.1.3 Cell Concentration

Studies on the correlation between population density and survival after lyophilisation show that the percentage of damaged cells increases at extreme conditions with decrease in cell concentration, but the increase in concentration leads to higher intercellular contacts, toxic metabolite production, etc. that reduces the vitality. Most culture collections recommend an initial concentration 108–1010 cells/ml.

7.1.4 Equilibration

It involves preparation of microbial cells to stabilise the cellular structure before conservation. The physical parameters are optimised osmotically using a cryoprotectant before cooling in a controlled way to protect cells from freezing injury. Various biochemical changes occur during equilibration causing either positive effect or negative effect on the metabolic processes before preservation. The cells undergo a transfer stage that prepares them to withstand the extreme conditions like osmotic and temperature shock used of conservation. Capacity for resistance varies among different microbes. Spore forming organisms retain high viability under most conservation methods. This is because spores are naturally resistant with low water content. Non-spore forming microorganisms show relatively low resistance to cryogenic and lyophilic treatment. Generally speaking, prokaryotes are more resistant than the eukaryotes, of which Gram-positive bacteria have higher resistance than Gram-negative ones.

7.2 Cryoprotectants

Preservation at ultra-lower temperatures causes injury to cells in two ways. Direct damage to cells from ice crystals formed and secondary damage caused by the increase in concentration of solutes resulting in osmotic imbalance during ice formation. Cryoprotectants are used to protect cells from cryo-injuries during cryopreservation (Prakash et al. 2013). The cryoprotectants may be of endocellular or extracellular type. The important properties of cryoprotectants are:

  • Ability to maintain viability, morphological, biochemical, taxonomical and genetic properties of the microorganism during preservation

  • It should be non-toxic and have high water solubility

  • Should show colligative properties, which is the collective properties that a solution attains in the presence of the compound

  • Low eutectic temperature, i.e. the temperature at which a eutectic mixture freezes or melts, preventing any structure forming after the solvent has been removed. A eutectic mixture is a mixture of two or more components that do not interact chemically but, which at certain ratios, inhibit the crystallization process of one another resulting in a system having a lower melting point than either of the components

  • Prevent hyper concentration of salts in the suspension

  • Stabilisation of hydrogen bonds in the crystal lattice to prevent formation of large crystals

  • Good penetration into cells in the case of endocellular protectants

  • Non-reactive and should not precipitate at high concentrations

7.2.1 Endocellular Cryoprotectants

These compounds penetrate into cells through cell membrane and create conditions for reducing water content inside cells at low temperatures to reduce the damaging effect of the concentrated solutes. They lower the freezing point of water, promote hydrogen bond formation and vitrification of solvents and prevent ice crystal formation inside the cells. The overcooling of the suspension before freezing results in small crystal formation, which reduces mechanical damage to cells during the cryogenic treatment. Endocellular cryoprotectants are considered ideal for microbial preservation. The most commonly used endocellular protectants are glycerol and dimethyl sulphoxide (DMSO) (Fuller 2004).

7.2.1.1 Glycerol

Addition of glycerol increases the stability of microorganisms during dehydration, lowers active water (aw) and preserves the cell vitality during freezing. Usually 10–15% glycerol is used prior to cryopreservation. An increase in synthetic processes before the anabiotic transition is observed in some microorganisms after the addition of extra glycerol to nutrient media. Even though glycerol shows good penetration at physiological temperatures, its penetration capability lowers at low temperatures and consequently provides less protection.

7.2.1.2 Dimethyl Sulphoxide (DMSO)

The cryoprotective activity of DMSO is concentration dependent. Ten percent concentration is used by most of the world culture collections. DMSO has a better penetrating ability than glycerol but it is toxic at higher concentrations.

7.2.2 Extracellular Cryoprotectants

Extracellular cryoprotectants do not penetrate the membrane of a eukaryotic cell or the bacterial cell wall. For this reason, they are incapable of preventing cell membrane rupture that occurs as a result of intracellular water-crystal formation. Examples of these non-penetrating molecules are dextran, hydroxyethyl starch (HES) and polyvinylpyrrolidone (PVP). These molecules function to reduce hyperosmotic gradients that form as the samples transition through the freezing process. As less water is available to function as a solute outside the cell, salt concentrations drastically increase. The increased osmotic pressure on cells cause alterations in cell shape, cellular interactions, subcellular distribution of molecules and ultimately cell viability.

7.2.2.1 Dextran

Being chemically inert, it is either used alone or in combination with other cryoprotectants. It has been successfully used for the preservation of viruses and microorganisms.

7.2.2.2 Hydroxyethyl Starch (HES)

It is a modified natural polymer of amylopectin that acts as a protectant that is non-toxic and biologically inert.

7.2.2.3 Polyvinylpyrrolidone (PVP)

Many hypotheses have been put forward regarding the cryoprotection mechanism of PVP. One is that PVP protect cells with its ability of cell penetration by pinocytosis. According to another hypothesis, the protective mechanism of PVP is by its ability to bind with the cell membrane and form an envelope around it.

Combined media for microbial preservation containing different sugar concentrations (sucrose, glucose, trehalose), colloids (gelatin, agar, peptone, milk and sera) and salts (sodium glutamate) are also used in cryopreservation. They result in a higher percentage of cell viability when compared to single component protective media.

7.3 Preservation Techniques

7.3.1 Continuous Subcultivation

This method involves frequent subculture of an organism from a depleted to fresh agar nutrient medium. It is one of the oldest and simplest preservation techniques and is used for short-term maintenance and preservation in laboratories and industries. The initial inoculum used should be from multiple colonies because use of a single colony will increase the probability of unwanted selection. The main demerit of this technique is the possibility of loss of innate strain properties and activity.

7.3.2 Cryopreservation

Cryopreservation refers to the preservation of biological materials at cryogenic temperatures, using deep freezers (−80 to −150 °C), dry ice (−80 °C) or liquid nitrogen (−196 °C). Storage at ultra-low temperatures at or below liquid nitrogen is the least damaging preservation technique for bacteria. Low temperature halts metabolism, protects proteins and DNA from denaturation and slows the mobility of cellular water and helps protect cells for long periods of time. Preservation at −196 °C is ideal because there is no chance of DNA mutation at this temperature. Microbial cells can also be stored at −80 °C for short-term preservation. During cryopreservation, the microbial culture is mixed with a cryoprotectant like glycerol or DMSO in cryovials and stored either immersed in liquid nitrogen (−196 °C) or in its vapour phase (−135 to −150 °C). Storage in liquid phase can result in entry of liquid nitrogen into the cryovials and its bursting during rapid thawing, and hence storage in vapour phase is safer (Prakash et al. 2013). Much research has been carried out on the optimum rate of cooling. Controlled cooling at the rate of −1 to −5 °C min−1 and rapid thawing at 37 °C water bath have proved most successful in maintaining cell viability (Smith et al. 2008). Slow warming can result in recrystallization of ice causing damage to cells. Dumont et al. reported high cell recovery at low and high cooling rates, while intermediate cooling rates were found to be detrimental to cell viability. They also found that the cellular response to cooling is not only dependent on cooling rate but also on the cell size, water permeability and presence of a cell wall (Dumont et al. 2004).

7.3.3 Lyophilisation

Lyophilisation or freeze drying is a technique in which water molecules are directly removed from frozen material by sublimation under vacuum-reduced pressure. It has been successfully used in the preservation of bacteria, yeasts and the spores of filamentous fungi. Freeze-drying process prevents cellular shrinkage or other structural changes and helps retain viability. Most microbial repositories prefer this technique for long-term preservation because of low cost of maintenance and ease of transportation of lyophilized cultures. Since the ampoules are completely sealed, they are protected from infection and infestation and hence stay viability for many years. Storage should be done out of direct sunlight; chilled storage reduces the rate of deterioration and extends shelf life.

It has been observed that most non-sporulating fungi, some species of yeast and certain bacteria lose viability following lyophilisation. Lyo-injury can occur to cells during the cooling or drying stages. During the phase change during freeze drying, the liquid crystalline structure of cell membranes can degenerate to gel phase, disrupting its fluid-mosaic structure and causing leakage of the membrane, ultimately resulting cellular damage. The optimisation of lyoprotectants and suspension media is essential for successful lyophilisation of certain microorganisms. A mixture of lyoprotectants and matrix materials or excipients should be used along with the cells for lyophilisation. Maximum cell viability and stability of lyophilised cultures can be obtained with stationary-phase cultures preserved in borosilicate ampules with a 1–2% final moisture content stored at 4 °C in the dark (Morgan et al. 2006; Smith et al. 2008). Skimmed milk as such or in combination with inositol is a suitable protectant for fungi. Another protectant is trehalose, which protect membranes by attaching to the phospholipids, replacing water and lowering the transition temperature. After lyophilisation, the ampoules or vials must be sealed, which can be done either using heat sealed glass or butyl rubber bungs in glass vials. The former is preferred as or butyl rubber bungs may leak over long-term storage.

7.3.4 Silica Gel Storage

Vegetative cells, propagules or dormant stages like spores, cysts or sclerotia of an organism can be preserved by inoculating onto cold silica gel and dehydrating by air drying to enable storage without growth or metabolism. It has been found to be successful with sporulating fungi, with cultures maintaining morphological stability for 7–20 years. The technique is relatively simple and cheap and does not require expensive apparatus. The main disadvantage is that it is limited to sporulating fungi and is unsuitable for non-sporulating fungi or fungi with delicate or complex spores (Smith et al. 2008). The recovery rate of cells is often low, and there is a possibility of introducing contaminants during repeated retrievals.

7.3.5 Liquid Drying

Liquid drying (L-drying) is used as an alternative to lyophilisation for preserving bacteria that are sensitive to freezing like Spirilla and Azomonas insignis. In this procedure, drying occurs directly from the liquid phase.

7.3.6 Gelatine Discs

Bacterial cultures are suspended in melted nutrient gelatine, poured as drops into Petri dishes and allowed to solidify. These drops are then freeze dried, or dried over a desiccant, and the resultant flat discs are stored over silica gel. Revival of the culture is done by placing a single disc in warmed broth, and the resulting suspension is plated onto a suitable growth medium. This technique is most suitable for short-term storage of limited number of cultures. The main advantages are ease of handling and storage.

8 Quality Control

Microbial repositories are closely associated with academic and industrial research laboratories as providers of reference strains for microbiology and molecular biology studies, and hence the quality controls to be maintained are high and demanding. They are responsible for more than 60% of descriptions of prokaryotic species, which indicates the level of professional expertise offered by such facilities. Quality control checks include accurate inventory with backup facility, validation of storage temperature and quality, proper specimen handling, minimum subculturing, availability of sufficient distribution stocks and preservation. Characterisation of morphological, anatomical, physiological, immunological and molecular properties of strains is carried out before and after preservation (Broughton et al. 2012; Prakash et al. 2013). Techniques derived from various biological disciplines like biochemistry, bacteriology, mycology and ecology to provide anatomical, physiological and biochemical data are used to characterise and authenticate microorganisms. In addition to long-term preservation of microbial cultures, microbial culture collections should maintain the genotypic and phenotypic stability of its preserved cultures, which is essential for the authentication of previous findings. Molecular techniques like amplified fragment length polymorphism, multilocus sequence typing, pulsed-field gel electrophoresis, PCR binary typing (PBIT), real-time PCR and whole genome comparison can be used to monitor the genetic drift in bacteria, fungi and microalgae (Muller et al. 2007; Ragimbeau et al. 2008).

It has been made mandatory for all culture collections to implementation the international criteria in quality management to provide high-quality microbial cultures and authentic information. They are now required to provide access to genes and genetic elements of deposited strains with associated information. The use of bioinformatics tools has improved data handling, cross referencing and in the transformation of collection catalogues into digital format.

The use of next-generation sequencing techniques for elucidation of whole genome sequences of microbes and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in microbial typing have reduced the cost and time for comparison of genotypic and phenotypic integrity of microbes before and after preservation.

9 Importance of Microbial Repositories in Bioprospecting

Bioprospecting is the exploration and sustainable utilisation of biological resources for commercially valuable genetic and biochemical resources. Majority of the drugs in use today are derived from microorganisms. Of these, 45% are produced by actinomycetes, 38% by fungi and 17% by unicellular bacteria (Bérdy 2005). Since the beginning of industrial microbiology, enormous numbers of microorganisms have been isolated from various environments with applications in scientific research and industrial fermentation. A large numbers of these valuable isolates have become lost and are no longer available due to lack of facilities for preservation or because of change in field of study by the researcher. Once lost, it is difficult to isolate a species with exactly the same properties even if samples are collected at the same location. Microbial repositories can play an important role to prevent such loss by proper maintenance of cultures and their supply on demand. Microbial prospecting being a popular field of research, a large number of new microbial strains and species are expected to be isolated in near future, which will require the help of microbial repositories. Establishment of new culture collections and exchange of information among them are crucial for advancements in microbiology, microbial industry and biotechnology.

10 Culture Collections in India

India, being one among the 12 countries of megadiversity, is rich in floral, faunal and microbial diversity. Many novel species of fungi and bacteria, including actinomycetes, viruses and cyanobacteria, have been isolated by Indian researchers, and they form an invaluable gene pool. Indian Council of Agricultural Research (ICAR) pioneered microbial diversity studies in India with initial focus on crop improvement for food and economy. India’s rich microbial diversity has yet been effectively identified and catalogued owing to the lack of adequate support and expertise (Arora et al. 2005).

India endorsed the Biological Diversity Act (BDA) in 2002 after signing the Convention on Biological Diversity (CBD). The Biological Diversity Act gives effect to the provisions made in the CBD. It addresses access to biological resources and associated traditional knowledge to ensure equitable benefit sharing arising out of their use to the country and its people, thereby contributing to achieving objectives of the CBD. India is one of the first few countries to have enacted such legislation. There are 30 microbial repositories in India according to WFCC statistics. According to WDCM, India ranks third in holding of microbial cultures (194,174 accessions), after USA with 261,637 and Japan with 246,343 microbial accessions. The list of all WDCM registered culture collections in India are listed in Table 17.3.

Table 17.3 Indian culture collections registered with World Data Centre for Microorganisms (WDCM)
Full size table

11 Conclusions

Conservation and provision of microbial resources for research is now recognised as being an essential component in the advancement of the life sciences. The extent of diversity of the microbial world is largely unknown, and of that which is known, the genotypic and phenotypic diversity is enormous. Convention on Biological Diversity (CBD) is the most significant international agreement that ensures the need for the conservation of microbial diversity, sharing of microbial resources and their utilisation. In situ conservation strategies are practically not applicable to the conservation of microbial diversity, possible only through maintaining as pure cultures under laboratory conditions, followed by preservation. The main objectives of culture collections are to act as a depository, to supply authentic microbial cultures and to provide related services to the scientific community working in research institutions, universities and industries. Biotechnology has a pivotal role in pharmaceutical, industry and academia sectors. There is an increasing demand on culture collections for authenticated, reliable biological material and associated information that have paralleled with the growth of biotechnology. The full potential of microbial diversity is yet to be harnessed. A coordinated approach to resource provision is an urge to accelerate innovation and discovery. Further development and investment is needed in the microbial domain at global level.