Conserving microorganisms

- Search and Discovery Strategies for Biotechnology: the Paradigm Shift

The value of microorganisms in both direct and indirect terms has been stressed on many occasions during the debate on biodiversity and its conservation (58, 83, 438). Because of their direct value as a major resource for biotechnology development, the conservation of microbial gene pools is a crucial issue. In the past this issue has been addressed almost entirely from the standpoint of ex situ conservation. However, it has become increasingly obvious that this strategy on its own is quite inadequate for ensuring conservation in anything approaching a meaningful way. In this section, therefore, we argue for a complementary ex situ-in situ strategy for microbial conservation and urge that a concerted program for in situ conservation be a priority.

An answer to this question is entirely dependent on our knowledge of microbial diversity and the threats to its existence. If we do not know the extent of microbial diversity, it becomes axiomatic that we will not know what to conserve. The situation has been stated unequivocally by Jim Staley: “Until microbiologists can provide meaningful estimates of global diversity from studies of selected habitats and a better understanding of the importance of biogeography, it will be fruitless to estimate the degree to which microbial species on Earth are threatened” (420). But as Foissner (138) affirms, “microorganisms…present the greatest challenge to any serious attempt at assessing the overall scale of global species richness.”

Some sense of the magnitude of the problem facing microbiologists can be appreciated by focusing on fungi. If we accept the working figures of 72,000 and 1.5 million for known and total estimated species, respectively, Hawksworth (201) concluded that at the present rate of description, it will take another 888 years for the global inventory of fungi to be completed. Even if we accept Hammond's “moderate” accuracy rating, i.e., within a factor of 5, for fungi (198), the inventorying task would continue until 2188. The situation is likely to be similar with respect to other microbial groups particularly, where the number of taxonomists is known to be low (see, for example, Foissner's comments on soil ciliate diversity [138, 140]). The foregoing, of course, takes no account of infraspecific or genetic diversity, the importance of which for biotechnology has been stressed already.

All-taxa biodiversity inventories (ATBIs) have been proposed such that a selection of habitats are subject to intensive investigation in order to make as nearly complete an inventory of species as possible. ATBIs aim to describe all taxa at the species level and the locations where they can be found on subsequent sampling of the habitat site. Such an accounting system may be a reality for the best-known groups of macroorganisms, but is it feasible for hyperdiverse taxa and microorganisms? Even for the former, ATBIs likely will necessitate interpolation between sample sites within an ecosystem.

Tiedje (441) has advocated microbial ATBIs for the following reasons. (i) Finding new species: for the purposes of biotechnology search and discovery, it would be very useful to uncover the precise relationships between environmental difference and genotypic differences, i.e., what is the nature of the area-species curve for selected environments? (The epibiotic bacteria associated with nematodes referred to above is a good illustration of this type of analysis [372].) (ii) Determining the distribution and abundance of uncultured microorganisms. (iii) Categorizing rare microorganisms. (iv) Synthesizing genotypic, phenotypic, and ecological information in order to produce a greater understanding of microbial distribution. “For example, can an Arthrobacter landscape be predicted?” (441). However, the task of making a microbial ATBI is formidable and would almost certainly necessitate a degree of selectivity. For example, Tiedje (441) proposes a sampling strategy that could be driven by specific questions, such as how a host reflects microbial diversity, which in turn might direct sampling along a vegetation transect or through a phylogenetic line of insects at the selected site. To our knowledge such microbial ATBIs have not yet been attempted, a situation reflecting the major methodological and logistic resources that would be required.

The enormity of environmental degradation as a consequence of human intervention is well known through the effects of land and water pollution, oil and mineral extraction, land management disturbance, deforestation, urbanization, and global warming. To this list might be added large-scale introduction of alien species. However, the question which is of interest here concerns the effects of such environmental degradation on microbial abundance and species richness. Staley (420) examined this question from the points of view of symbiotic and free-living microbiota and, while citing several cases of the loss or significant reduction of symbiotic microorganisms, could provide few examples of free-living species. However, the available data on environmental degradation of soils point to disturbances of free-living microbial communities causing reduced diversity (163). Reduced diversity may reflect environmental perturbations that are continuous or long-lasting rather than punctuated. Reports are starting to appear that reveal the extent of microbial population changes in the face of habitat destruction. Of 100 prokaryote rDNA clones recovered from a mature Brazilian rainforest soil and an adjacent pasture soil, none had been described previously, while several appear to represent members of new bacterial divisions (43). Moreover, the greatest number of unclassified bacteria were found in the forest soil. rRNA intergenic spacer analysis confirmed this result, and eventually it might provide a means of evaluating the ecological health of particular ecosystems. A dramatic shift in the bacterial communities of a Hawaiian rainforest soil and a pasture soil resulting from forest clearing about 80 years ago has been reported from Tiedje's laboratory (351). Culture-independent analysis showed that the change from rainforest to pasture effected a 50% change in bacterial composition and that the change was not merely seasonal. None of the dominant forest phylotypes (which appear to represent new taxa) were detected in the pasture soil. Whether the microbial diversity of degraded environments of these types returns to its original state is unknown. Similarly, we have no knowledge of the colonization of isolated environments by additional species transported from other regions (Broady [54] discusses this point in relation to the algal diversity of Antarctica).

 

The lack of even preliminary databases for the most part makes this a difficult question to address. Guidance may be provided from various sources. Biogeographers, for example, recognize a number of geographic locations distinguished by their exceptional levels of biodiversity and endemism; such locations are defined as hot spots on the basis of their floral and faunal diversity (342). These hot spots, for example, contain about 20% of the world flora in only 0.5% of its land area. Of the 18 hot spots, 14 are in tropical forests and the remainder are in Mediterranean biomes; 5 of the hot spots have already lost 90% or more of their original integrity, and the rest are under considerable threat (319). Undoubtedly other hot spots occur, among them coral ecosystems. Thus, one approach for prioritizing in situ protection of microbial diversity is to establish research stations in hot spot areas; by the same token, sites selected for ATBI action could also be prioritized. Such arguments reinforce the need for microbiologists to take a serious view on biogeographic distribution and to lobby for the protection of unusual, pristine, and threatened habitats. Terrestrial and marine geothermal sites, deep ocean trenches, and polar regions must be included in the latter category. Examples of microbial diversity protection actions include the Yellowstone Thermophiles Conservation Project (456), and the caution being exercised in penetrating and sampling Lake Vostoc in East Antarctica (249, 466). It may appear paradoxical, but a case can also be made for the preservation of a range of polluted sites in order to access organisms that have evolved novel metabolic abilities.

 

It has been estimated that the annual value of the earth's ecosystem services and natural capital is about U.S.$33 trillion (83). This value was computed on the basis of 17 ecosystem services (at least half of which rely directly if not totally on microorganisms) for 16 biomes and should be compared with the annual spending on conservation via nature reserves. Currently the latter is estimated to be $6 billion per year, and James et al. (235) claim that the additional cost to buy and manage a broadly representative system of reserves, equivalent to 15% of the earth's land area, would be approximately $17 billion per year. These authors maintain that the “cost of global conservation is well within our means—the obstacle to progress is the lack of political will.”

 

Culture collections have provided a service to the scientific community for over 50 years. In fact, the first service “culture collection” was established by Franticek Král in Prague during the latter part of the 19th century. In the decades that followed, several service culture collections were established around the world (66). The traditional service role of these collections was to provide authenticated cultures and expert advice on their cultivation and preservation to the scientific community. The ex situ conservation of microorganisms was seen to be essential for ensuring that a source of living cells was readily available for scientific and industrial purposes. This is still the case, especially since organisms isolated from environmental samples cannot always be found again, and even if they are, they may lack the desired properties exhibited by the earlier strains. The benefits that arise from the provision of well-characterized, quality-controlled biological material are measured not only in a financial sense but also in the benefit they confer on a global basis in terms of groundbreaking products of value in agriculture, industry, and health care. Many of the leading technological breakthroughs in recent years have been facilitated by the supply of such resources, as exemplified by the use of Taq polymerase in PCR. The economic value of biological resource centers (BRCs) (see below) was recently the subject of a workshop organized by the World Federation of Culture Collections (265).

It is not feasible to maintain an adequate representation of all known cultivated species of microorganism and cell lines in ex situ collections. The database of the World Data Centre for Microorganisms shows that over 800,000 living cultures are maintained in 484 culture collections distributed across the world (270). These holdings consist of 343,253 cultures of bacteria (42%), 372,304 cultures of filamentous fungi (46%), 14,374 cultures of viruses (2%), 5,156 cell lines (0.6%), and 80,485 cultures of other living microorganisms (10%). However, these holdings represent only about 10 to 15% of known species and a tiny fraction of the total estimated diversity of microbial species. It is clear that the task of providing adequate ex situ coverage of microorganisms is enormous. However, for both ecological understanding and biotechnological development to advance, ways need to be found to isolate, classify, and conserve a vastly greater array of microorganisms, including the more structurally complex and fastidious ones. Culture collections will need to be closely associated with these activities.

The ex situ conservation of microbial genetic resources requires skilled practitioners and incurs high maintenance costs. At present, the majority of the major service collections are in developed countries in the northern hemisphere (417). Glowka et al. (168) noted the irony in this situation: “In general, the countries richest in species are the ones where scientific knowledge on individual species is least.” This means that the ex situ conservation of microbial genetic resources in the countries of origin, as advocated by the Convention on Biological Diversity, is seriously compromised by the lack of expertise and funds for capacity building in many countries. This situation needs to be addressed if developing countries are to benefit from the exploitation of their biological resources (66).

In recent years, other services have been added to the custodial role of culture collections, such as the bulk supply of cultures for screening purposes, patent deposit facilities, the supply of cultures for quality control, safe deposit facilities for valuable cultures, and identification of cultures (146). Indeed, service culture collections have grown into BRCs, which are seen to be an integral part of the infrastructure that underpins the conservation of biological diversity, the successful development of the biotechnology industry, and ecological studies linked to the sustainability of life support systems (144, 421). The current role of BRCs is to provide the scientific community with access to properly maintained culturable material (e.g., animal, human, and plant cells, archaea, bacteria, and viruses), replicate parts of these (e.g., cDNA banks, genomes, and plasmids), and associated databases. These core activities provide a sound basis for maintaining and preserving the increasingly large amounts of biological material and associated information that are being generated by the application of novel selective isolation strategies and automated data acquisition systems (67). There is also an urgent need to update and improve quality control methods and simplify access to microbial databases, with particular reference to developments in genomics, proteomics, and phenomics. The application of genomics will help to promote industrial and economic advancement and will lead to new manufacturing processes, provided that the complexity of phenotypic interactions can be unraveled using assays to interrogate the transcriptome, the proteome, and the metabolome.

Acquisition and distribution of biomaterial. In their pivotal role in the conservation of biodiversity, BRCs need to provide a framework for the control and administration of the exchange of biological materials within the requirements of the Convention on Biological Diversity. This will require not only a need to increase the capacity of holdings but will also necessitate the development of new technologies to preserve and expand the range of those currently held by BRCs. These goals will not be easy to attain due to the need to handle organisms from extreme environments that cannot be cultivated or preserved using present methodologies. A future requirement may well be the provision of the DNA rather than the organisms themselves. The BRCs are in a unique position to adapt their current roles in order to fulfill new requirements arising from developments in molecular biology. This will undoubtably increase running costs, but the benefits of linking new technologies and the relevant biological resources will have a profound effect, especially in terms of exploitation. Similarly, the emergence of proteomics will lead to improved characterization and eventually to the successful exploitation of the biological resource.

The discovery of new biological material, especially novel microorganisms, can be expected to increase rapidly as improved selective isolation and characterization procedures are used to dissect out the vast pool of microbial diversity present in natural ecosystems. A coordinated national, regional, and global acquisition policy will be needed to avoid duplication of holdings between BRCs and unnecessary expenditure. This development will mean that BRCs will specialize in the preservation and maintenance of particular kinds of biomaterials, as it is unrealistic to expect individual funding bodies to support the maintenance and preservation of all types of microorganisms. Strategies will also be needed to ensure that members of the scientific community deposit biomaterials in BRCs: unfortunately, this is not common practice at present. Acquisition policies will also need to be sufficiently flexible to ensure that important specialist collections held in academia and industry are not lost due to the retirement of key individuals or to changes in direction of industrial and health care concerns. Customers will also need to have easy access to the holdings of BRCs so that they can contact the appropriate centers. The tightening of restrictions on the national and international transport of organisms strengthens the case for national/regional BRCs to provide a focus for help and advice on this issue but also provides the scientific community with centers that can approach the regulatory bodies to lobby on the need to avoid restrictive overregulation (66, 146). Additional issues subject to regulation include the handling and distribution of genetically modified microorganisms and transgenic animal and plant material (including cell lines), and the handling and distribution of biohazard and infectious agents.

Acquisition and distribution of data. There are increasing requirements for new linkages to be forged between biological resources and other databases replete with key information on nucleotide sequences, proteomics, and phenomics. BRCs will need to develop strategies to handle and interpret the vast amounts of data arising from developments in such areas (68, 442). This will ultimately lead to the development of resource centers as knowledge-based concerns, especially in the fields of systematics and functional genomics. It is well to remember that knowledge-based goods and services currently comprise around 60% of the wealth production in the 29 countries which belong to the Organization for Economic Cooperation and Development (66). It is vital to promote collaboration between BRCs and the proposed Global Biodiversity Information Facility, which is designed to coordinate the standardization, digitization, and dissemination of the world's biodiversity data in the interests of the scientific community.

Long-term funding and capacity building. BRCs which meet quality assurance guidelines are irreplaceable as the repositories of over a century of microbiology research and thereby provide a sensible basis for continued financial support. They are part of the infrastructure for science and technology by providing access to reliable authenticated cultures, conserving and replicating parts of these, and adding to associated catalogues and databases. The scientific community, including individual scientists and the biotechnology industry, needs to be assured that biological resources deposited in BRCs are held in perpetuity and that the appropriate caliber of staff can be given long-term support. Ideally, the deposits into and access to BRCs should be substantially underwritten by governments, particularly where costs are incurred from regulatory compliance associated with legal compliance (e.g., intellectual property rights), insurance indemnity for distribution, and the need to monitor the import, export, and distribution of holdings, notably dangerous pathogens. Cost is also highly relevant to the generation, gathering, and processing of information. The new strategy for microbial culture collections in the United Kingdom, as outlined in the Whittenbury Report (353), is indicative of governmental appreciation of the strategic value of collections of microorganisms as a resource for bioindustries and their contributions to the development and expansion of the overall national natural science and research base. There is also a need for such national policies to be directly embedded into an international perspective to ensure coordination and complementarity of the biotechnology infrastructure on a global scale.