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Natural Investment in Diversity:
The Role of Biological Communities in Soil

Frans A.A.M. De Leij
David B. Hay
James M. Lynch
School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK

Introduction

There is a persistent view that the commercial application of “environmental biotechnology” relies largely on the exploitation of single species, well-defined bio-chemical pathways, or the expression of novel gene sequences by genetically modified organisms. Consequently, the role of species assemblages and in particular the importance of interactions between the members of natural communities have been largely neglected in a commercial or industrial context. This oversight is important because most ecosystem processes appear to be governed by the activity of species at the guild and community level and not by species that function in isolation. Indeed, most well-studied bioremediation experiments and many examples of effective biological control show that natural, or “intrinsic,” processes are at least as important in achieving economic ends as specific and targeted bio-technological interventions.

This paper suggests that exploitation of microbial communities is a potentially rewarding alternative to the “classical” or “single-species” biotechnological approach. We emphasize the need for ecosystem study in the context of biotechnology development. The development of sampling and monitoring techniques has high priority for research. Monitoring is an important technology for tracking “intrinsic” beneficial processes, and sampling will provide data to improve our fundamental understanding of ecological processes in an applied context. In this paper, the commercial uses of microorganisms and microbial communities are examined in the context of bioremediation and biological control, but they could

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equally well be scrutinized in relation to such processes as soil formation and biodegradation.

Biological Control

Development of Single Isolates as Biological Control Agents

The commercial development of Bacillus thuringiensis is one of the most outstanding examples of successful technology based on a specific biological resource. B. thuringiensis has taken the dominant share of the biopesticide market: a little over 1% of total pesticide sales. That figure could rise to 10% during the next decade (Hokkanen and Lynch 1995). Although B. thuringiensis is often referred to as a biological control agent, its application and action have much more in common with chemical pesticides. The toxin crystal produced by the bacterium is the active ingredient of commercial products, and the ecology of the organism itself is largely irrelevant in pest control—in stark contrast with familiar examples of biological control in which the dynamics of host-parasite or host-predator interactions determine product efficacy.

Where biological solutions have been sought to combat pests in well-defined (and controlled) conditions, commercial success has sometimes been based on the exploitation of single species or isolates. For example, Peniophora gigantea is used to control Heterobasidion annosum in pine forests, and Agrobacterium radiobacter is commercially valuable for the control of A. tumifaciens (crown gall) in tree nurseries (Deacon 1991). Nevertheless, it is the specific match between the environmental requirements of the biocontrol agent and the conditions in which pest populations thrive that results in high product efficacy. Such matches appear to be the exception rather than the rule.

Involvement of Many Organisms in “Natural” Biological Control

Soil communities comprise a large variety of microbial species. It is estimated that a gram of natural soil might contain as many as 4,000 or 5,000 “species” with DNA-sequence similarities of less than 70% (Tørsvik 1990). This level of diversity is comparable in a variety of soil habitats, but microorganisms from different localities generally show markedly different patterns of species composition. The role of these microbial “species” and of species diversity in ecosystem function is largely unknown, but it is well established that several components of the normal soil flora serve to regulate the activities of pathogens. Plant-parasitic nematode populations in soil are regulated by a large variety of egg parasites, female parasites, nematode-trapping fungi, bacteria, and possibly viruses (Jatala 1986). Specificity is common, and in suppressive soils it is the combined activities of a consortia of antagonists that achieve control. Nematode-trapping fungi—which produce adhesive knobs, adhesive rings, or adhesive hyphal networks—are adapted to “catch” the free-living nematodes. Likewise, the nonmotile spores of Pasteuria penetrans (a bacterial parasite of root-knot and cyst nematodes) attach to juvenile nematodes. Fusarium oxysporum, Catenaria auxiliaris, and Nematophtora gynophila parasitize young females before egg-laying commences, and Peacilomyces

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lilacinus, Cylindrocarpon destructans, and Verticillium chlamydosporium are classified as specialized egg parasites. Many other organisms inhibit soil nematode populations in a nonspecific way through toxins, competition, and predation (Jatala 1986). The combined action of the whole community of specialized and nonspecialized organisms is responsible for keeping nematode populations below the economic threshold in nematode-suppressive soils.

There is also considerable species diversity within “functional groupings.” Kerry (1988) reported that as many as 150 species of fungi were isolated from eight cyst-nematode species, parasitizing 97% of adult female nematodes in suppressive soils; and Jatala (1986) reported that there are at least 100 species of nematode-trapping fungi. At any time, consortia of antagonists provide nematode suppression. The exact composition or activity of these consortia, however, is determined by spatial and temporal characteristics of the environment (Crump 1987). Different parts of the diverse community of organisms involved in nematode suppression are necessary to provide effective nematode control at any given time during the crop cycle.

Despite many studies that document the combined activities of several antagonists in the control of plant-parasitic nematodes, the commercial drive for biopesticide products has been in the development of solutions based on single microbial species or even single isolates. That approach is based on little understanding of host-parasite population dynamics, and the selection of agents for development is complicated by issues of ease of culture and product shelf-life. Biological control of cyst nematodes provides a good example; Nematophtora gynophila and Pasteuria penetrans are thought to be important agents of nematode control in natural soil (Davies and others 1992; Kerry and others 1982), but Verticillium chlamydosporium is the only organism chosen for commercial development, mainly because it is easy to mass-produce it in vitro.

Beneficial Use of Simple Consortia of Species if Vitro Culture is Possible.

Experimental tests have shown that the fungus Verticillium chlamydosporium can reduce plant-parasitic nematode populations in soil by as much as 90% (De Leij and others 1992b) but that this can be achieved only under specific environmental conditions: temperatures must be close to 20°C, appropriate host plants must be available for fungal colonization, and nematode population densities must be low (De Leij and others 1992a,b,c). Thus, V. chlamydosporium has a specific “window of opportunity”, and its utility as a commercial biopesticide in the field is not large. Furthermore, the high multiplication rate of cyst and root-knot nematodes means that parasitism rates as high as 90% (common in laboratory tests) are insufficient to prevent nematode population increases and economic damage to crops.

Higher levels of nematode control can be achieved with combined application of V. chlamydosporium and Pasteuria penetrans (an obligate bacterial parasite of nematodes). This approach provides control that is comparable with the use of nematicides and is much more efficacious than the use of either organism in isolation (De Leij and others 1992a). V. chlamydosporium is unable to penetrate the

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plant root and parasitizes only egg masses on the root surface (De Leij and others 1992a); P. penetrans also parasitizes nematodes that develop deep inside the root. Even though this simple consortium approach shows promise, it is commercially unattractive because of production constraints. Furthermore, the combination of nematode host specificity of P. penetrans and the relative small environmental window of opportunity of V. chlamydosporium is commercially unattractive.

Management of the System as a Means of Inducing Biological Control

Agricultural management provides an alternative to biological intervention. Simple practices, such as crop rotation, prevent the buildup of pests and diseases to economically damaging levels. In continuous cropping, initial accumulation of pests and diseases is tolerated to allow populations of natural antagonists to reach levels that provide long-term control. Augmentation of soil with organic manure is widely used to increase nonspecific biological activities that suppress pest and disease populations. Hams and Wilkin (1961), for example, reported that augmentation of soils with farmyard manure or green manure reduced plant damage attributed to plant-parasitic nematodes; the introduction of organic substrates probably promotes general microbial activity that is antagonistic to nematode populations. Similarly, amending soil with organic residues that have relatively high carbon-to-nitrogen ratios can control fusarium root rot; the free nitrogen required by the microbial biomass to degrade these organic amendments leads to insufficient nitrogen availability for pathogen growth (Snyder and others 1959). Others (for example, Park and others 1988) have suggested that induction of fusarium-suppressive soils is a more specific process whereby nonpathogenic Fusarium oxysporum isolates interact with siderophore-producing fluorescent pseudomonads to provide conditions that are nonconducive for the pathogen. Lemanceau and co-workers (1992, 1993) showed that pathogenic F. oxysporum isolates were more sensitive than nonpathogenic isolates to the iron deficiency induced by Pseudomonas putida, and this difference resulted in effective biological control.

In general, disease suppression in soils is attributed to biological processes. Experiments have shown that suppression can be transferred to nonsuppressive soils by adding small quantities of suppressive soil to soils that are conducive to disease (for example, Stirling and Kerry 1983). However, attempts to attribute disease suppression to specific components of the natural microbial community have largely met with failure. Processes and species interactions at the community level, rather than the specific ecosystem services or functions of individual species, are likely to be responsible for disease control and pest control in suppressive soils. Research on and economic exploitation of processes at the community level are therefore potentially rewarding. It is also an environmentally sound, sustainable, and in many situations realistic approach. Suppressive soils need not be only “hunting grounds” for potential biological control products; an understanding of suppressive-soil community ecology is likely to lead to augmentative and manipulative management practices that are of considerable economic benefit.

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Bioremediation

Intrinsic and Augmentative Approaches

The degradative enzymatic capabilities of microorganisms and microbial species assemblages play an important role in the remediation of polluted environments (see Crawford and Crawford 1996 for a review of principles and applications of microorganism exploitations and Lynch and Wiseman 1998 for details of microbial and microbial-product use in ecotoxicant monitoring). Natural communities show considerable potential to recover from small- and even medium-scale pollution effects, and with time biotic and abiotic factors interact to reduce contaminants to nondetectable levels. This is “intrinsic bioremediation”, remediation that relies solely on natural processes with little or no intervention (see Ellis and Gorder 1997 for review).

As a commercial technology, the “do nothing” (but monitor) approach does not require investment in physical removal or discharge technology and is easily integrated with other pollution-control and remediation technologies. In many situations, this is a realistic and economical approach to pollution abatement. As a consequence, biodegradation by naturally occurring populations of microorganisms is a major mechanism, for example, in the removal of petroleum from coastal waters (well documented in the Prince William Sound, Alaska, after the Exxon Valdez oil spill). Rapid acclimation of the resident microbial population is common after hydrocarbon contamination (for example, Braddock and others 1995) and is evidence of the capacity of the community to respond to pollution. Detailed research has also shown that a 10-fold increase in population size of hydrocarbon degraders can follow substantial petroleum contamination of coastal waters (Atlas 1995a). Furthermore, petroleum degradation rates can be increased (by a factor of 3–5) by enrichment with inorganic fertilizers (Atlas, 1995b: Coffin and others 1997; Pritchard and others 1995). Similarly, in terrestrial environments, there is considerable evidence that natural microbial species assemblages respond to pollution in ways that ameliorate or remove contaminants and that this activity can be enhanced by manipulation of the physiochemical conditions to augment remediation (Liu and Suflita 1993).

Intrinsic cleanup does not require extensive knowledge of the abiotic and biotic processes and interactions by which remediation is achieved. Nevertheless, understanding of “the system” is beneficial where it leads to the ability to enhance decontamination rates by manipulating and controlling environmental conditions or augmentation of biological processes for human benefit.

Complex Interactions, Biological Diversity, and the Exploitation of Intrinsic Bioremediation Processes

Molecular studies have shown that diverse microbial species assemblages (and genes) are involved in the complete catabolism of complex substrates (for example, Vallaeys and others 1995). Metabolic capabilities are often widely dispersed among distinct taxonomic groups and environments (Mueller and others 1994), and the metabolic capabilities of microbial communities as a whole are

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characterized by considerable functional overlap (Pritchard and others 1995). This could be indicative of “functional redundancy,” but it is more likely that patterns of susceptibility and resistance to pollutants interact with metabolic and cometabolic activities to create a mosaic of functions underpinning microbial community integrity in the context of environmental heterogeneity. Furthermore, studies of mixed cultures (enrichments of xenobiotic-degrading microorganisms in liquid culture, for example) have demonstrated the importance of secondary use of substrates in microbial populations. Complex interactions—including the provision of specific cofactors, removal of toxic products, modification of growth rates, cometabolism, and gene transfer—have now been implicated in microbial communities and are important in degradation (Weightman and Slater 1979). Much can be learned from degradation studies of pesticides: the herbicide dalapon, for example, is not readily degraded by any organism, but enzyme products in small and well-structured microbial communities can bring about complete substrate metabolism (Senior and others 1976).

Diversity of metabolic function is undoubtedly important in the ability of microbial communities to achieve bioremediation, but it is also important because of indirect and “cascade” interactions that enable complete degradation. Genetic diversity also underpins community-level responses to environmental change, species compensation, and complementarity (see Frost and others 1995); and genetic diversity is likely to be particularly important in the degradation of substrates in changeable, fluctuating, and perturbed environmental conditions (which are often characteristic of polluted environments).

Difficulty of High-Technology Bioremediation Solutions in Natural Environments

Recombinant-gene technology appears to offer appealing prospects for the design of microorganisms for use in bioremediation. Genetic manipulation has been used to expand the array of substrates that can be used by wild-type microorganisms and to restructure existing metabolic pathways (thereby avoiding the production of deleterious metabolites; see Lui and Suflita 1993). Nevertheless, the “inundative approach”in which single species, strains, or isolates of bacteria (recombinant or wild-type) are cultured in vitro and released into the environment—has proved difficult for achieving viable populations of pollution degraders in situ. Experimental tests often show that recombinants are likely to be outcompeted by wild-type parental strains (Fleming 1994; Recorbet and others 1992; Vahjen 1997), and even indigenous species often fail to establish in field trials, because of abiotic or biotic factors (de Leij and others 1992c; Kerry and others 1993). Thus, the commercial use of recombinants and wild-type “superstrains” is not likely to be great in the context of bioremediation. As Hamer (1993) has stated, the utility of genetically engineered microorganisms in bio-remediation processes is likely to be restricted to specific in situ and ex situ applications because recombinants fail to match the degradative abilities of natural microbial species assemblages (despite the addition of metabolic capabilities) and fastidiousness is likely to preclude their use in all but the most highly protected and controlled environments.

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Wilson and Lindow (1993) refer to some 27 experimental releases of recombinant microorganisms in field trials. Much of the work has been done to assess the potential risks posed by releasing genetically modified organisms into the environment, but it is now clear that it remains a major technological challenge to achieve the establishment and persistence of recombinants in natural environments. The genetic and physiological “programming” of microorganisms to achieve viable and controlled phenotypic expression under variable (and largely unpredictable) physiochemical and biotic conditions is no small task (Delorenzo 1994).

High Priority of Ecological Research for Commercial Exploitation of Microorganisms in Bioremediation

Exploiting intrinsic bioremediative processes does not require a full understanding of the biochemical, physiological, and ecological interactions by which pollutant removal (or transformation) is achieved. But it is clear that the better these processes are understood, the easier it will be to improve intrinsic remediation efficiencies. A considerable body of evidence suggests that bioremediation rates in situ are determined by soil, sediment, and substrate chemistry. However, recent studies of pollutant mineralization (of hexadecane, phenanthrene, and naphthalene, for example) show that environmental factors (especially temperature, disturbance, and mixing) are at least as important as purely chemical interactions in determining rates of biodegradation by microorganisms (Sugai and others 1997), emphasizing the need for ecosystem-level study of system interactions in bioremediation processes.

Similarly, the study of interactions between microorganisms and other fauna has high priority. In terrestrial habitats, competition and grazing by microbial predators are thought to be important determinants of soil biodegradation rates (Travis and Rosenberg 1997), but models of microorganism-substrate interactions have yet to include a robust analysis of distribution and dispersal of such interactions in field situations (Dighton and others 1997). As a whole, there is a need for fundamental research to improve understanding of the complex interactions that determine the removal of pollutants by natural communities. Such research might lack the glamour of manipulative genetic technologies (to produce recombinants with “designer” functions), and the approach does not have the same value as “intellectual property,” but it is likely to be much more profitable. As Price (1997) concludes in a recent review of bioremediation of marine oil spills, “understanding fundamental microbial ecology is the priority for commercial clean-up technology.”

Discussion

Diverse communities are likely to comprise commercially valuable individual species and strains. This is well documented and often cited to support “species conservation” (Wilson 1992). However, diverse microbial species assemblages can act in concert (via complex ecosystem-level interactions) to achieve ecologically and economically valuable processes. Furthermore, it is the innate capacity of

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microbial assemblages to respond to change that makes the community as a whole valuable as a resource in combating pollutants, plant pests, and diseases. The gene frequency of degradative biochemical pathways appears to be characterized by high levels of “functional overlap”. Similarly, shared preferences for specific hosts are well documented among potential biological control agents. This could suggest “redundancy” in the normal community state but have considerable economic value in “variable” natural systems. Natural microbial communities comprise large numbers of unidentified or unculturable genotypes (Ward and others 1990) that can belie ecosystem integrity and contribute “function” after community disturbance. In fact, the nature and scale of microbial ecosystem processes have led some authors to conclude that such concepts as redundancy and species value have little meaning in the context of microbial community ecology (for example, Finlay and others 1997).

We conclude that intrinsic processes and ecosystem interactions can have important value for human society. Often, it is the community function as a whole (not a particular or “valuable” species) that is important. The maintenance of biodiversity itself is important for benefits derived from microbial communities. Microbial diversity needs to be conserved not only for the benefit of individual species and genotypes that function within the community as a whole, but also because microbial “ecosystem services” are often carried out at the community level. Besides development of appropriate environmental-management strategies aimed at preserving and stimulating the activity of naturally occurring communities, monitoring techniques have high priority for research and technology development. Intrinsic cleanup and biocontrol processes must be tracked so that further steps can be taken when natural abatement fails, and sampling data are also likely to improve our fundamental understanding of ecosystem processes.

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