# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 2 Fungi, Bacteria and Their Microenvironment
### The Hidden Properties of Soil  
Basic physical laws govern and dictate the properties of each of the microscopic components that compose soils; the surface-to-volume ratio increases with the decreasing dimensions of an object, leading to surface effects, such as surface tension, capillary forces, adhesion, and viscous drag. Soil is a highly heterogeneous medium, consisting of a mixture of solid material, and of water- or air-filled pores [4] (Figure 1). Thus, soil can be theoretically interpreted, either as the (organized) arrangement of aggregates/particles in the soil (i.e., soil structure) [5; 6], or as the connectivity, tortuosity, and heterogeneity of the pore space between these soil components (i.e., soil architecture) [7] (for a complete argumentation on soil structure versus soil architecture, see [PERSON] et al. [3]). Albeit the hierarchical organization of aggregates highly depends on the amount of energy that is applied to take the soil apart, soils can be classified based on the distribution of their different aggregate species (i.e., soil texture). The aggregates are generally divided depending on their size, i.e., from (very) stable micro-aggregates (<2 um), which are mainly composed of organic matter and clays, to less stable macro-aggregates, which are commonly composed of silt (2-63 um) or sand (63 um to 2 mm) [8]. Physical and chemical processes driven by both biotic and abiotic factors cause changes in aggregate size and continuous particle rearrangement. These constant changes in spatial organization of the solids and voids affect the architecture and connectivity of the pore space, which, in turn, affects the distribution of water and gases [9], as well as the diffusion of substrates (e.g., organic matter) [10; 11] and solutes (e.g., elements and ions) [12] in soil. Figure 1: The complexity of soil depends on the scale of analysis and is ultimately a mosaic of dynamic microenvironments defined at a specific point in time and space. Fungi and bacteria, together with other soil residents, live, grow and interact in this reality to the best of their fitness, perpetually looking for their _realized niche_. Water is one of the most important, but at the same time is most variable component of soil. Soil's water content depends on soil composition, rainfall, drainage, evaporation, temperature, and plant cover. Water is the medium connecting spatially separated areas in the soil matrix and becomes the solvent in which organic matter, microelements, and metabolites of different biological origins are dissolved or suspended (i.e., soil solution). The degree of water retention in soil microenvironments mainly depends on the soil pore neck size [13]. In macro-pores, localized in and between macro-aggregates, water is often well drained, whereas it is fully retained in micro-pores (localized between micro-aggregates) due to capillary action [13; 14]. As a consequence, when alternating saturation/desiccation cycles that occur due to changes in precipitation or temperature, water status is more conserved in the micro-pores, generating fine water pockets rich in moisture that remain spatially disconnected from one another [14; 15; 16; 17; 18]. Micro-pores are also important for the retention of important biological molecules, such as organic matter, proteins, and nucleic acids [19; 20; 21; 22; 23]. As a result of the clay-cation exchange capacity (CEC) [4], they can all be adsorbed and retained by the negative charges of clay in soil micro-aggregates. Indeed, [PERSON] and [PERSON] [14] found that organic matter is not homogenously distributed in soils, and higher concentrations (50-80%) were detected in micro-pores. Furthermore, at the micro-scale level, micro-elements are to be considered for the growth and survival of microorganisms. Micro-elements are often present in the soil solution, as they are originating from both metabolically catalysed redox reactions as well as from abiotic chemical weathering of rock surfaces.