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Fig. 5.1. Representation of the logistic growth curve, with stages of bacterial growth on a single substrate.

stage 3

stage 4

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Fig. 5.1. Representation of the logistic growth curve, with stages of bacterial growth on a single substrate.

are also in competition with plant roots. For example, uptake of amino acids by soil bacteria is saturated at millimolar concentrations that occur naturally (Vinolas et al., 2001). Nutrient uptake is constrained by the diffusion rate of nutrients from microsites. The rate of diffusion varies with soil compaction, moisture, ped particle size and microorganism locomotion through the porous reticulum (Focht, 1992). Bacteria respond physiologically within minutes to changes in growth conditions or solution composition. Molecular biologists are very familiar with their sensitivity to slight changes in conditions, from culturing transformation-competent cells. When preparing cultures for transformation with DNA, slight variations (minutes) in the time to cool growing cells, or to sediment cells, or abundance of cells, greatly affect their competence. Cells that are not cooled in the active state quickly enough are already inactivated before they are prepared for transformation. Handling these cooled cultures too roughly also contributes to inactivating cells. Roughly a 5-10 min delay can reduce active (competent) cells by up to 100 times. If the same manipulations are carried out at room temperature, most cells become inactive within minutes of handling. Therefore, when soil is sampled, handled, mixed, diluted or partially desiccated at room temperature, inactivation of the bacteria is unavoidable. This is particularly significant if the cells are transferred from the soil solution to a diluted solution or water. It is difficult to cool intact soil rapidly enough to maintain active cells in the active state. Mostly, species that are described as active from handled samples are those that are active under the new conditions, and not necessarily all those that were active when the soil was sampled.

The growth of a bacterial culture on single substrate is useful to understand some of the underlying physiology that regulates growth (Fig. 5.1). Under suitable conditions, a stimulus such as the addition of nutrient resources will initiate cell activity. Cell activation from the inactive (non-growing) state requires a lag period for the molecules to be activated or synthesized (stage 1). The duration of the lag period depends on how much growth is required before cells can enter cell division. An increase in cell numbers is observed when cells have grown sufficiently to begin cycles of cell divisions (stage 2). Cells grow and divide until nutrients are limiting. At this point, a decrease in food resources and the accumulation of metabolic by-products decrease the rate of growth (stage 3). Very few species are able to accumulate storage material, especially under normal soil conditions. There are exceptions; for example, Arthrobacter crystallopoietes can accumulate a glycogen-like compound. Eventually, nutrients are insufficient to maintain the cell cycle and divisions end, although cells continue to remain active (stage 4). After some time of inadequate growth, cells become inactive (stage 5, late stationary phase). At this stage, some species overproduce or lose capsule material into the environment. This is easily observed in stationary phase cultures, and contributes to soil aggregate formation and biofilms. When soil conditions are inadequate to support metabolic activity, the cytoplasm becomes inactive and bacteria are said to be quiescent. Some species are able to form spores to survive unfavourable conditions. Once bacteria are stressed by lack of nutrients or unfavourable physical conditions, the cytoplasm becomes quiescent. To be reactivated and grow again, they need to be stimulated by suitable conditions. The conditions required to reactivate the cytoplasm are not necessarily the same as those favouring optimal growth. This may require a different nutrient solutions or changes in the physical environment (such as temperature). The longer bacteria are quiescent or encysted, the greater the loss of viability.

From a molecular perspective, growth of bacterial cells during the lag phase (stage 1) involves the activation of the genes necessary to transcribe the metabolic proteins and cell membrane transport proteins. These are regulated at the level of the operon (co-regulated genes), and at the level of the regulon (co-regulated operons). Entry into the growth and division cycle requires the accumulation of sufficient ribosomes, proteins and nucleic acid precursors (stage 1). There is a minimal size requirement to maintain cells in the cell cycle. It is generally observed as an increase in cell size from stationary phase cells (stage 5). During the period of rapid growth (stages 2-3), cells accumulate further cytoplas-mic molecules and ribosomes, and reach an optimal size. The duration of the period of DNA replication and cell separation (or filamentation) is independent of growth rate, and does not vary under fixed conditions. The period of growth to minimal size varies with the rate of nutrient intake and temperature. Therefore, not only the size of individuals but also the duration of the cell cycle (growth phase to division) varies as conditions change.

When concentrations of adequate nutrient substrates are too low, cells may continue to take in molecules, but at a rate too low to maintain growth and division. Thus, even though cells are not increasing in number, they can be metabolically active. The threshold growth rate and nutrient abundances vary for each species and its substrates. Depending on the soil physical environment and nutrient availability, bacterial growth rates can vary from 20 min under optimal laboratory conditions, to >3000 h (see references in Kjelleberg, 1993). Under conditions where the soil solution offers poor growth, or under nutrient stress, large cells or microcells develop over time. This involves a change in both volume and chemical composition of the cell. The aim is to maintain an optimal amount of active cytoplasm until the conditions for growth are better. Variations in size and morphology caused by starvation are studied mostly in marine and freshwater species, particularly in marine Vibrio (Kjelleberg, 1993; Lengeler et al., 1999). Many Gram-negative species reduce their energy needs by decreasing cell size. There are similarities in this process to spore formation in Gram-positive species. At first, there is a decrease in the copy number of plasmids, followed by changes in the protein composition and membrane lipid composition. Many enzyme pathways are inactivated and other enzymes synthesized. In the initial phase (30 min), proteolysis begins and starvation-induced proteins are synthesized. The accumulation of starvation-induced proteins imparts more resistance to heat and ultraviolet light. It is accompanied by switching to new nutrient substrates. In the following hours, membrane lipid composition changes, the flagellum is lost, and high-affinity substrate transport proteins are synthesized, as well as new starvation-induced proteins. In the third phase, metabolic activity decreases and cell size is gradually reduced. There is degradation of ribosomes and proteins, and cell wall material become a source of nutrients to maintain the cytoplasm. These become a source of phosphate and nitrogen for metabolism. This period continues for several days, and it is accompanied by synthesis of further high-affinity transporters and extracellular hydrolytic enzymes. The microcells can be about 30 times smaller than well-fed cells and can pass through 0.2 ^m membrane pores. The morphology of the cells with starvation varies, according to what the nutrient limitations are, and some nutrient limitations lead to larger cells (Fig. 5.2). If only certain nutrients are deficient, metabolic activity can continue but without cell division.

Attempts at studying single species of bacteria on single substrates, or mixed species on single substrates, are not very informative about the mechanisms of natural growth on complex substrates. Natural samples usually contain a variety of species which contribute to decomposition,

Fig. 5.2. Size variation in Vibrio with different nutrient limitations over 100 h, and effect on viability (CFU: colony-forming units) (data from Kjelleberg, 1993).

and cells grow on complex substrates that have a variety of molecular components. In the presence of multiple substrates, cells will switch from growing on a preferred substrate to another, as each is exhausted sequentially. Moreover, cultures are materially closed systems without exchange of nutrients with the environment. They are also shielded from the positive (syntrophy with other decomposers, symbiosis) and negative (predation, defensive molecules) effects of interactions with other co-occurring soil organisms.

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