A micro-sampling approach to improve the
inventory of bacterial diversity in soil
L.G. Grundmann
1,*, F. GourbieÁre
Laboratoire d'Ecologie Microbienne du sol, UMR 5557, Universite Claude Bernard, 43 Bd du 11 November 1918, 69622 Villeurbanne Cedex, France
Received 15 May 1998; received in revised form 23 November 1998; accepted 25 February 1999
Abstract
A bias in the outcome of bacteria isolation from large soil samples has been observed in various diversity inventories. An improvement is proposed, based on a soil micro-sampling procedure. This procedure reduces errors due to soil heterogeneity and the micro-spatial bacterial distribution within the soil matrix.#1999 Elsevier Science B.V. All rights reserved.
Keywords:Soil sampling; Bacterial diversity; Bacteria isolation
1. Introduction
Studies designed to inventory diversity utilise very different types of approaches depending on the system studied. The study of bacterial diversity, especially in soils, presents speci®c problems which are probably not independent of the size of bacteria and of the complexity of the edaphic habitat. In fact, the present range of bacterial diversity obtained on bacteria iso-lated from current soil samplings underrepresents the range of microbial diversity: it is estimated that only 0.1±10% of the actual biodiversity is known based on isolated bacteria (Head et al., 1998). Unfortunately, the two approaches to biodiversity measurements, bacterial isolation and PCR-based sequence targeting in extracted soil DNA, suffer the same shortcomings involving sampling the heterogeneous matrix of soils,
a point which is not suf®ciently taken into account for bacterial and DNA isolation. Furthermore, although molecular techniques are powerful, these show some limitations in the case of diversity studies. These two types of shortcomings will be discussed in the following.
We propose a soil sampling procedure which takes into consideration the bacterial spatial distribution within the soil matrix and may widen the inventory of bacterial types.
2. Examples of sampling bias on bacterial diversity inventory
We carried out an experiment on 33 micro-samples of soil (pieces of soil inscribed in 50±250mm side cubes) (Table 1), selected from a clod of an agricul-tural soil. They were then cultivated in the appropriate mineral medium for culturing the genusNitrobacter, and serological tests were carried out on each culture. The smallest soil sample size had the largest percen-*Corresponding author.
Applied Soil Ecology 13 (1999) 123±126
1Laboratoire de Biologie Alpine, BP53x. 38 041 Grenoble
Cedex, France.
tage of samples simultaneously harbouring four ser-otypes (Table 1). However, this did not mean that the actual diversity was lower in larger samples. In the smallest samples, the competition was lowered due to the reduced number of cells, and the culturing prob-ability of each type of cell was larger (Gause, 1934). In larger samples, only the dominant serotypes were detected.
Warcup (1959) similarly succeeded in isolating fungal species from plant or fungal fragments col-lected from the soil whereas he failed to do so from suspensions of the bulk soil.
Another experiment, where a different soil frag-mentation technique was used, corroborated the importance of sampling strategy in bacterial diversity inventory. Instead of `dissecting' the soil into undis-turbed micro-samples, the soil was broken up into `global fractions'. A centrifugation method was used for splitting the soil (Hopkins et al., 1991; O'Donnell et al., 1995). These authors showed that different kinds of actinomycetes were isolated at different stages of the extraction procedure, and that certain organisms were only recovered on isolation media seeded from inocula derived from the application of the new extraction procedure. These results suggested that actinomycete-soil interactions may limit quantitative and representative sampling of actinomycetes from soil and that a soil-splitting technique was effective in breaking such interactions, thus improving the acti-nomycete inventory.
3. Limitation of molecular approach to bacterial diversity on extracted soil DNA
Recent advances in the inventory of bacterial diver-sity have been promoted by rRNA gene ampli®cation from extracted soil DNA without having to isolate the
organism. In addition, ampli®cation of DNA extracted from environmental samples with speci®c probes directed towards key metabolic genes could give an indication of the possible metabolic capabilities of soil bacteria (Seow et al., 1997). However, several studies indicate that molecular approaches tend to be biased towards the more dominant (abundant) organisms at the time of sampling (O'Donnell et al., 1995; Darrell et al., 1997). Integrated diversity studies based on molecular methods, physiological measurements and appropriate culture-based investigations are recommended (Darrell et al., 1997; Head et al., 1998). Diversity studies based on isolated DNA sequences are ef®cient and promising, but it is not always fruitful to deal with `virtual' organisms. In fact, the expression of many bacterial genes is involved in a multitude of fundamental soil processes (biochemical cycles, antibiotic biosynthesis, toxin production, etc.). To address functional issues, it is necessary also to work with the actual organism and its biochemical processes.
The argument for new efforts to achieve represen-tative bacterial isolation is illustrated in the following example. The recent changes made in the Proteobac-teria group illustrate the sometimes confusing inter-pretation of diversity from phenotypic and genotypic information. In some cases, phenotypic and genotypic characteristics converge, e.g. for Cyanobacteria (Olsen and Woese, 1993). Conversely, in the Proteo-bacteria group, large phenotypic diversity is found with only minor rRNA sequence divergence. Riboso-mal RNA sequences indicative of members of Pro-teobacteria are commonly found in marine environments (De Long et al., 1993; Fuhrman et al., 1993) and in soils (Borneman et al., 1996). The various phenotypic characteristics of these organisms have ecological and environmental consequences. It is not possible to draw phenotype inferences for these uncultured organisms without isolating them (Embley et al., 1995). It thus seems that we are bound to reconsider bacterial isolation from soil.
4. Soil traits to be considered for bacterial isolation
Assessment of biodiversity using bacterial isolation from soil as well as sequence targeting in soil-Table 1
Percentage of soil micro-samples of three different sizes simulta-neously harbouring fourNitrobacter serotypes
Volume of
extracted DNA are in¯uenced by the sampling pro-cedure. Two points seem to be of importance: (i) the size of the sample; and (ii) the bacterial micro-spatial distribution within the soil matrix.
The size of the soil sample is crucial, because it determines the number of bacteria which will be subjected to the same sample processing. Compari-sons of organism size with soil sample size can be pro®table. Bacterial cells are 1mm3on average, and are usually studied in 3±5 g of soil (3±5 cm3soil)
samples. To use the same scale ratio for the micro-arthropod group, Collembola (springtails), (109mm3 size) would necessitate 3.106cm3 soil samples, whereas experience has shown that 10 cm3 was an appropriate sample size (Peterson, 1982). It follows that there may be an inappropriate scale ratio in the case of bacteria, particularly when the goal is to inven-tory microscopic organisms which are highly abundant. If 1 cm3of soil harbours 107bacteria (which isa common density of bacterial ¯ora), then a piece of soil of 610ÿ5
cm3size (that is a piece of soil inscribed in a 0.5-mm side cube) contains on average 6102
bac-teria. Submillimetric particles can be handled with micro-manipulation techniques under a binocular microscope, as used for fungi (Warcup, 1959).
Soil is a somewhat discontinuous medium at the submillimetric scale (Hattori, 1973) and microbial niches in the soil are discrete. When preparing soil suspensions, cells originating from isolated micro-habitats are mixed together allowing negative inter-actions and competition during cultivation, leading to the elimination of some bacterial types. Antagonism zones are often seen on Petri dish cultures. In addition, sub-sampling soil suspensions favours the most abun-dant organisms. Ahmed and Oades (1984) noted: ``The spatial distribution of micro-organisms in soil and the need to overcome the wide range of micro-organisms±soil particle interactions are the main lim-itations in quantitatively and representatively sam-pling soil microorganisms.''
5. Use of minute soil samples to widen the inventory of bacteria diversity
These results suggest that the micro-fragmentation of soil (fractions of soil, sampling of speci®c habitats, dissection of minute pieces of soil) prior to culturing can help minimising cell interactions during sample processing for isolation. Soil fragmentation lowers the
competition (Tilman, 1994), giving a chance for minor populations to culture during enrichment cultures. It also minimizes the negative interactions in the soil suspension between bacteria of different types which were spatially separated in the soil matrix. Moreover, this sampling allows the targeting of spatially restricted habitats with diversity of interest (Klug and Tiedje, 1993). This micro-fragmentation can also be pro®table for soil DNA sequence identi®cation directly on soil extracted DNA (Felske and Akker-mans, 1998), or on enrichment cultures. Factors for improving sampling strategies are summarised in Fig. 1. The inconvenience of this approach is that a large number of samples has to be tested to be representative of the bulk soil sample. In addition, this sampling procedure should be combined with appropriate culturing techniques. It is not known whether nonculturability of bacteria is an intrinsic characteristic of cells, or that it arises due to the use of an inappropriate medium, or if it is a negative interaction related to the complexity of the inoculum and stress conditions (Bloom®eld et al., 1998).
6. Conclusions
The inventory of bacterial diversity in soil was greatly improved by molecular tools, but it is never-theless con®ned to `accessible' bacteria. Various results indicate that the cell accessibility to culturing or DNA ampli®cation depends upon the micro-spatial distribution of cells in the soil matrix and the inter-actions between bacteria during the sample proces-sing. This point is not suf®ciently taken into account in the usual bulk soil samplings and may conceal the actual diversity even if techniques of analysis are very ef®cient. The reduction of soil sample size was shown to improve the inventory of the bacterial diversity, because it takes into account micro-spatial distribution of soil bacterial populations.
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