Microbial Diversity and Ecosystem Functions - the Unmined Riches

*Corresponding author

Financial support: ATIPE grant of the French Science organization (CNRS).

Microorganisms represent an enormous and largely unexploited source for biotechnological applications. Microbial diversity can be regarded on one hand as a problem, for example due to the large variety of microorganisms causing (new emerging) diseases and on the other hand as a solution due to the rich biotechnological potential including disease control. Prokaryotes developed the basic types of metabolism and a wide range of activities, which allowed them to colonize all ecosystems and create the biosphere. As they are also the movers and shakers of global cycles of elements, microorganisms contribute significantly to the stability and functioning of ecosystems, which can be threatened by man-made disturbances including such originating from biotechnological applications. Thus, the sustenance of the environment requires a thoughtful management of microorganisms.

Microorganisms are somewhat fuzzily defined by their size and not by their position in the tree of life. The ancestral prokaryotes, i.e. cells with the hereditary information not bound in a nucleus (karyon), were likely the first forms of live on earth and they exist two times longer (4 Gyr) than eukaryotic organisms such as plants, fungi and animals (2 Gyr), which have the DNA bound in a nucleus. In marine and freshwater, the main microorganisms are viruses, prokaryotes, protists (mainly flagellates) and algae, whereas in soil the main microorganisms are viruses, prokaryotes, protists (mainly amoebae) and fungi (if fungi can be called microorganisms). Although the species composition and the various microbial processes may be different on sea and land, the functional groups and the quality of the processes are similar (Fenchel, 1994) . One specific characteristic of groups of microorganisms is their short generation time.

The abundance of prokaryotes in the open ocean, in soil, and oceanic and terrestrial subsurfaces is 1.2 x 10²⁹, 2.6 x 10²⁹, 3.5 x 10³⁰ and 0.25 - 2.5 x 10³⁰, respectively (Whitman et al. 1998). Prokaryotes represent likely the largest living surface on earth and this enormous interface between the living and the abiotic world is one of the reasons for their significance in transforming organic matter. It has been estimated that there are ca. 7.5 x 10²⁹ viruses in the world ocean (Weinbauer and Herndl, 2002) . No total virus numbers are known from soil, and the few data from sediments indicate that viral numbers are about as high as those of prokaryotes. Assuming a diameter of 50 nm for a typical virus, the string-of-pearl of oceanic viruses would be 400,000 light years long. In comparison, the diameter of our galaxy is only ca. 25,000 light years. Thus, prokaryotes have been described correctly as the unseen majority (Whitman et al. 1998) and this is even more correct for all microorganisms.

Microorganism are also associated with plants and animals, not only in the form of pathogens, but also in more beneficial ways such as gut bacteria in ruminants, which transform grass into digestible biomass, or as symbionts in the rhizosphere, which fix molecular nitrogen. Such associations are now under scrutiny to assess their significance for the survival and performance of plants and animals. This kind of interactions between microorganisms and hosts represents a classical ecological relationship. The majority of such studies focused on interactions between the host and the pathogen, however, ecological interactions of both the host and the pathogen with the biotic and abiotic components of their environments are crucial for the initiation and outcome of infections, their transmission and their epidemiology. Mankind (and its domestic animals and plants) is continuously exposed to infectious agents, and indeed carries a number of potentially infectious agents as part of its normal microbial community, however, only a small fraction of these contacts leads to infections and, of these infections, only a small fraction results in overt disease. It is likely that products have evolved in these associations, which can be used in biotechnology. For example, from prokaryotic endosymbionts one might better understand, how defense mechanisms of plants and animals are circumvented, which could also help understand how pathogens infect a host.

The associations of microorganisms with hosts and other habitats are critical determinants for many topics relevant for mankind such as the quality of the environment, global change, sewage plants, or the decline of food. Using black box approaches for the main groups of microorganisms has revolutionized our knowledge on the carbon and energy flow through food webs. For example, soil humus and dissolved organic matter in aquatic systems are the main biologically available sources of organic carbon on earth, which is predominantly transformed by the microbial food web. Thus, everything that affects the microbial food web on a global or local scale also affects the flow of carbon and energy and ultimately biogeochemical cycles. This, in turn may affect for example fisheries, agriculture and climate.

Diversity and gene transfer

The term biodiversity stands for the diversity of life at all organizational levels. The older term species diversity is described by species richness, species eveness and species difference. These parameters can be combined in diversity indices such as the Shannon - Weaver index to obtain a single value for an ecosystem. Species richness is the number of species in a system, species eveness is the significance of a species in a system e.g. in terms of numbers, biomass or activity and species difference is the taxonomic relatedness of species. Aspects of functional diversity can be estimated as the catabolic or metabolic versatility of isolates or habitats, which gives a measure of the distribution of catabolic or metabolic capabilities (Weinbauer and Höfle, 1998;Wenderoth and Reber, 1999) . It has been suggested to use the term ecodiversity for species diversity and to consider biodiversity and ecodiversity as two sides of the same coin (Margalef, 1997) .

The species concept poses some problems when being applied to prokaryotes and viruses. The problem of prokaryotic taxonomy is that prokaryotes have only few morphological traits useful for identification. They are better described by their metabolic capabilities, which can only be adequately measured when species are isolated on culture plates. However, only a small number of prokaryotes (typically less than a percent) grows on culture plates. This is sometimes called the 'Great Plate Count Anomaly'. This GPCA is also the reason for the low number of isolated prokaryotic viruses, since hosts have to be isolated before an attempt can be made to isolate a specific virus. Prokaryotes (and viruses) are truly the black hole of taxonomy (Wilson, 1992) . There is also evidence that the ecodiversity of small eukaryotes, which have more morphological and behavioral features than prokaryotes, is higher than previously assumed (Lopez-Garcia et al. 2001; Moon-van der Staay et al. 2001) .

In soil, it was estimated that there might be up to10,000 bacterial species per 100 g soil (Torsvik et al. 1990) . Recent evidence suggests that the domains Archaea and Bacteria, which together are also known as prokaryotes, are equally abundant in the deep sea and that ca. one third of the prokaryotes in the ocean are Archaea (Karner et al. 2001) . We have still no clue, what the Archaea are doing, since there is no isolated Archaea known from the ocean. However, it is now clear that Archaea are not only inhabiting extreme habitats. Overall, the more effort is applied to study the diversity of a sample, the more species are detected. This indicates that we have no clue of the actual microbial diversity in a sample or a system. Thus, the species diversity of oceanic and soil prokaryotes remains unknown. Based on the finding that every well investigated cellular species has at least one specific virus (e.g. ca. 500 viral species are known which infect Homo sapiens), we can estimate that the ecodiversity of viruses is at least as high as that of cellular organisms. Thus, microorganisms are also the unseen majority in terms of ecodiversity. Novel techniques are now available to circumvent the GPCA and are being currently refined to obtain genetic fingerprints of natural prokaryotic communities and to assess parameters such as species richness and taxonomic relatedness.

An important mechanism by which microbes can interact in the environment is gene transfer (Lorenz and Wackernagel, 1994) . Studies on the process of conjugation, transduction and transformation provided direct evidence for the exchange of genetic information between prokaryotes. The ability of microorganisms to exchange DNA or release DNA, which transforms or transduces other bacterial cells enable populations to adapt or evolve. Prokaryotes have several possibilities to transfer genes including a mechanism called transduction, where genes are transferred by the activity of viruses. This results in a horizontal spread of genes (as opposed to a 'vertical' spread by cell division) within a community and may contribute to diversity. Although there is considerable interest in horizontal gene transfer, it has not been often investigated in the environment due to methodological problems. One of the interests (or concerns) to study gene transfer is the potential of unwanted gene transfer such as the spread of genes such as e.g. for antibiotics or frost resistance. The huge number of prokaryotes (and viruses) on earth certainly provides for an enormous potential of gene transfer. An evaluation of whole genome sequencing also suggests that lateral gene transfer occurs frequently (Doolittle, 1999). Recent evidence suggests that antibiotic resistance of bacteria due to fish farming can be spread worldwide within years (Sørum, 2002) . It is not known how this spread occurs, but transport in ship ballast or atmospheric transmission is possible. Although it is not clear, how this resistance was transmitted, it is clear that such events can occur in natural communities on a global scale with a previously unimagined speed. This also points to the need of a better risk assessment of biotechnological applications. 

Links between diversity and ecosystem functioning

 Ecosystem functions provided by microbes are the transformation of inorganic carbon into biomass by primary producers, nutrient regeneration and cycling, conversion of organic matter including humus that would otherwise be lost from the food web into living biomass, regulation of biogeochemical cycles and consequently climate. Functions such as degradation of organic matter (including oil or pesticides) require complex metabolic pathways. Microorganisms account for the main portion of the global metabolism (and biomass). Thus, also for functioning, the microorganisms might be the unseen majority. A combination of fluorescent in situ hybridization (FISH) with microautoradiography (MICRO-FISH, STAR-FISH) is a means to link diversity and function for in situ studies (Ouverney and Fuhrman, 1999; Cottrell and Kirchman, 2000) . This technique uses radiolabeled substrates to determine the uptake of specific substrates by cells and specific taxonomic probes to assess, which species are taking up such substrates. Genus- to species-specific FISH probes, which are available for some taxa, can be used in the future. With such and other methods, we might learn more about the role of as-of-yet uncultured prokaryotic species, and this may allow us to get a handle on their culturability. Moreover, by using molecular tools it has been recently shown that unicellular cyanobacteria in the ocean might fix molecular nitrogen (Zehr et al. 2001) and that aerobic anoxygenic phototrophs are abundant and diverse in the ocean (Beja et al. 2000) . Together these unexpected and major findings indicate that our knowledge on species and functional diversity in ecosystems is rudimentary.

The 'insurance hypothesis' assumes that there is many species in an ecosystem, which can perform the same or very similar functions (Yachi and Loreau, 1999) . These redundant species can take over ecosystem functions once a dominant species is lost or low in performance. This insurance due to redundancy of species may result in a resilience of ecosystem functions, although ecodiversity may vary. Resilience can also mean the resistance of a system in terms of diversity and function against disturbances. Periodic variations such as due to seasons can by described as dynamic equilibrium. The relationship between diversity and ecosystem functioning and its contribution to the resistance of ecosystems are a hotly discussed issue in ecology. It is often assumed that diversity stabilizes ecosystems, however, this concept has also been criticized. Due to their fast generation times (usually days to weeks), microorganisms are well suited to test ecological theory. It seems that stress causes two effects in microbial communities, which might be tightly linked. One is a loss in structural diversity although re-diversification may occur in the surviving tolerant communities. The other is an increase in biomass respiration, apparently due to the fact that stressed microorganisms are forced to divert a relatively larger part of the available energy into the maintenance of the various biochemical functions, for example when stress is exerted by a high proton concentration or by metal contamination.

Implications for biotechnology in developing countries

A broad definition of biotechnology is the (industrial) use of living organisms to produce food, drugs or other products. Two types of implications emerge from our knowledge on microorganisms in the environment. One type of implications is that the diversity and functions of the unseen majority are also the unmined riches (Wilson, 1992), the source of new biotechnological applications. The other type of implication is that biotechnological applications such as all types of agriculture and fisheries or the release of genetically engineered microorganisms (GEMs) might threaten microbial biodiversity and the environment.

Species represent libraries of genetically encoded information (dealt with by genomics) and the translation of this information into function (dealt with by functional genomics and proteomics). Loosing species due to extinction deprives us from obtaining benefits from these species. This is also true for microorganisms, however, we know only little about extinction of microorganisms. With every species, but particularly with larger animal or plant species, that gets extinct, we also loose a variety of symbiotic or parasitic viruses, prokaryotes and protists, which exclusively harbor this species. Thus, we loose associations of species and all the information bound in these associations. To understand, how biotechnology applications such as e.g. the release of GEMs or genetically designed crops affect microbial diversity and the environment, we must first understand diversity, ecosystem functions and the link between the two. Although methodological progress has been made in the past decades, there are still no methods available to study all three aspects of ecodiversity (richness, eveness and difference) and the function of single species.

Phage therapy was shortly invented after the discovery of bacteriophages. The idea behind is to use phages to control pathogenic bacteria (Levin and Bull, 1996) . The advent of antibiotics and some methodological problems put an end to phage therapy in Western Europe and North America, whereas phage therapy was continued in several countries from Eastern Europe and the former Soviet Union and recently a review was published in English on this topic (Sulakvelidze et al. 2001) . The threat of resistance to antibiotics has revived the interest in phage therapy (Merril et al. 1996) . Isolating and designing phages would be a relatively inexpensive way of biotechnological application. Phage therapy might not only be used for fighting pathogens but also to control environmentally problematic prokaryotes such as GEMs.

That our knowledge on the diversity of microorganisms is just the 'tip of the iceberg' is now a commonplace. More refined isolation techniques are required, since this is necessary for many biotechnological applications. Cyanobacteria, i.e. photosynthesizing prokaryotes, have never been isolated, until they have been seen by their autofluorescence in epifluorescence microscopes. Once they were detected, it did not take long to isolate them and isolation is indeed easy. This example fuels the hope that we can isolate more prokaryotes once we know what we have to look for. Knowledge from in situ approaches may thus help here to develop new isolation techniques. Understanding the link between diversity and function not only provides information on the resilience of ecosystems to (man-made) disturbance, but will also help in developing isolation strategies of indigenous prokaryotes useful for biotechnological applications such as waste management.

Research on genomics of microbial communities has started already and it is just a matter of time until functional genomics and proteomics enter the field. Strategies have been proposed to assess the information of the whole genome of a habitat, the so called 'metagenome', without the need of cultivation (Rondon et al. 2000) . This involves comparison of the genome of bacteria with different lifecycles and cloning the metagenomic DNA from an environment into a bacterial artificial chromosome (BAC) vector. Clones can be screened for biological activity expressed in Escherichia coli or the libraries can be probed for sequences of interest. Another type of technology, which can use the information of BAC libraries are the DNA/RNA chips representing a high-through put technology. In combination with the nucleic acid based metagenome approach the proteome approach is useful. Proteins from bacteria, which are grown under varying conditions such as e.g. the absence or presence of heavy metals, can be sequenced to identify the respective gene. This allows for an identification of a gene and its function and the gene (or relatives of it) can be searched for in the BAC library. Cloning of unknown or even artificial genes into vectors will reveal new products such as e.g. proteins for heavy metal tolerance.

Some of these applications, such as genomics and proteomics, require rather expensive technologies (including database handling) and thus likely increase the gap between countries, which can afford that and countries, which cannot. Other applications, such as a rough assessment of ecodiversity of microbes and ecosystem functions, isolation of microorganisms, or phage therapy should be affordable by developing countries. It is a well known fact that on land and sea there is a longitudinal diversity gradient with higher diversity found towards the equator (Wilson, 1992) . Since this is also the area of a considerable concentration of developing countries, they probably possess the lion's share of the unmined riches in terms of biodiversity including microbial diversity. There is the danger that they will not fully benefit from their riches.

We thank Conny Maier for critically reading the manuscript.

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