Growth of bacterial cells in colonies on adequate substrate, under adequate abiotic conditions, occurs on the surface of soil particles and in the soil solution. Bacterial growth on a substrate forms colonies that are more or less compact depending on the genera (see Chapter 1). Growth of colonies is arrested by substrate limitation, spatial congestion, excessive metabolic by-products or growth inhibition by other organisms. Grazing by bacterivores reduces the colony population and often disaggregates the colony. It contributes to dispersal as well as to reducing the population growing on the substrate at a microsite or in a pore. Elimination of all individuals of a colony is unlikely, especially if the colony is disaggregated by the disturbance. The soil porous reticulum provides ample refuge for at least some of the bacterial cells to escape grazing (Vargas and Hattori, 1986; and see Hattori, 1994). These remaining cells continue to grow and divide on the substrate as long as the conditions are suitable. Dispersed cells settle on new substrate that may or may not be suitable for growth. Regular reduction of colony size reduces substrate depletion rate at first, and reduces secondary metabolite accumulation locally. As a result, the period of activity on the substrate is extended, as the colony continues with a growth phase. Displaced cells on adequate substrate can grow into new colonies, which contribute to the increase in biomineralization observed in the presence of grazers. These are mimicked in vitro by mixing soil microcosms.
The soil particles provide a variety of substrates and microclimates over very short distances, at the submillimetre scale. In fact, the microscale heterogeneity of the soil is such that in grasslands, which are less heterogeneous than forest soils, only 1 g of soil can represent the more abundant bacteria at the site (Felske and Akkermans, 1998). Since 1 g of soil will tend to have 107-1010 bacterial cells, it suggests that most colonies are probably very small. The distribution of bacterial colonies is best described as consisting of many small colonies on various substrate surfaces, representing an array of species and functional groups. A statistical study from soil thin sections predicted that 80% of the microcolonies existed in pores of <1 ^m, and >90% were in pores of <5 ^m in diameter (Kilbertus, 1980). Different species that are active at any one time are growing on the various substrates available. Within a ped, aeration is limited and the centre can be anaerobic. Therefore, over short distances (10-100 ^m), there are gradients of microclimates, with aerobic and anaerobic activity in close proximity (Focht, 1992). Many species represented in each sample are inactive because the abiotic conditions are not suitable, or their substrate is absent. Many individuals are inactive simply because they are not on a suitable substrate, even though other individuals of the species could be growing in the vicinity. A study of bacterial diversity at the submillimetre scale revealed that at the microhabitat scale (<50 ^m), isolates were very different between samples (Grundmann and Normand, 2000). The authors identified the chemolithotrophic Nitrobacter species using specific primers. They identified 20 isolates from 5 g of soil, and 17 isolates from several microsam-ples. The genetic distance between isolates, compared with reference strains, showed there was as much diversity between isolates in microhabitats from the same field as we know for the whole genus.
There are attempts at investigating the role of the bacterial assemblage in the soil at this scale of resolution. One approach is to use tagged cells which can be identified from samples. For example, in an elegant, study Jaeger et al. (1999) designed Erwinia herbicola with a reporter gene to determine the presence, activity and location of this strain in experiments. The reporter gene was the ice nucleation gene inaZ from Pseudomonas syringae. The assay for the presence and abundance of transformed cells is by a simple droplet-freezing protocol, followed by counting the number of ice nucleation sites/mm according to established procedures. The study showed that this approach was adequate to map the abundance and location of the cells and their substrate along root tips. In this study, both tryptophan and sucrose abundance in root exudates could be mapped along the root by monitoring the active tagged cells.
Other innovative techniques are being exploited to visualize soil microheterogeneity. These methods include computed tomography and magnetic resonance imaging techniques (Gregory and Hinsiger, 1999). These techniques can already provide resolution of soil physical structure at 100 ^m. Minirhizotron video-microscopy techniques are also improving in magnification and are increasingly useful in monitoring decomposition. Infrared spectroscopy has also been applied to identify strains and species in very small growing colonies, without disturbing the colony. Fourier transform infrared absorption spectra (FT-IR) can discriminate between bacterial species and strains on agar plates within hours of culture establishment (Choo-Smith et al., 2001). This assay relies on comparison of the spectra against reference spectra, and requires a small number of cells.
Another method is to use microcosms which are sampled by obtaining thin slices of soil, with relatively low disruption to the matrix structure. This approach was exploited using labelled litter that could be traced into the microcosm as it decomposed (Gaillard et al., 1999). The authors note that since regular mixing or disruption of soil-bacteria suspensions or of soil microcosms increases the decomposition rate (organic matter mineralization), this may be caused by redistribution of cells on to suitable substrate regularly. In the natural soil, this mixing and redistribution of peds is carried out by the invertebrates and particularly by Oligochaeta species. Fractionation studies of microdetritus and macrode-
tritus into various aggregate size categories showed that soluble substrates as well as particulate microaggregates (or peds) of 2-50 ^m were the site of this metabolic activity (Chotte et al, 1998). These studies cannot determine the zone of influence of a microdetritus point source across peds, since the soil matrix is disturbed. In order to do this, Gaillard et al. (1999) devised replicated compact soil core microcosms. Each soil core contained labelled (13C and 15N) wheat straw (1 cm clippings) as the source of litter, and was incubated at 15°C for up to 100 days. The soil cores were fractionated at regular intervals to remove the straw, the adhering soil, and the top and bottom half of each core. The bottom half was sliced into 800 ^m sections. The data from these soil cores were compared with controls without added straw. The results of the time course incubation indicate that almost all the activity due to straw occurs within 4 mm of the straw (Fig. 5.6). The straw 13C accumulation in the soil was higher at the end of the incubation. In contrast, the 15N accumulation was higher at the beginning of the incubation. Lastly, the straw and adhering soil were observed under low-temperature scanning electron microscopy (SEM) for visual observations of the spatial arrangement of bacterial cells and hyphae. There was a clear gradient of decreasing abundance and size of colonies away from the straw, with fungal hyphae dominating adjacent to the straw. The 3-4 mm zone of influence, or detritusphere, identified in this experiment probably varies with soil bulk density and porosity, as well as the composition of the litter from which the microdetritus or macrodetritus is derived. The detritusphere is probably greater in fresh leaf litter which leaches more nutrients.
In the environment, more complete decomposition occurs in the presence of a complex community of primary saprotrophs that contribute a variety of enzymes (or various functional groups) (Filip et al., 1998). This decomposition is sensitive to the composition of the soil solution and soil organic matter (SOM). For example, humic substances from the organic matter added to nutrient broth stimulated growth of mixed cultures up to six times the control levels, without humic substances (Filip et al., 1999). It is more difficult to obtain bacterial growth from soil isolates in single species cultures on natural substrates. In part, this is due to limitation of the enzymatic panoply that would otherwise be available to decompose complex substrates. For example, when bacteria are diluted then cultured, much less growth occurs below a threshold number of species, whereupon it is assumed the enzymatic or metabolic panoply has been reduced too much (see Salonius, 1981; Beare et al., 1995; Martin et al., 1999). Another limitation is whether corresponding bacterial physiotypes are active. This point is illustrated in a study of methanogenesis in anoxic rice fields (Chidthaisong and Conrad, 2000). The study considers the metabolic pathways of the bacteria that would be active under the anaerobic conditions of submerged rice fields. Bacteria that use ferric ions, nitrate,
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