# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 2 Fungi, Bacteria and Their Microenvironment
### The Hidden Properties of Soil
They can diffuse in and out of the smallest pores, including the very narrow 1.8-nm-wide spaces between clay particles [3]. These diffusion processes cause pH and element concentration gradients that are highly dependent on the abiotic properties of the soil (e.g., the mineral composition, morphology, and texture), the geochemistry of the surrounding fluids, as well as the activity of microorganisms secreting highly reactive organic acids (e.g., oxalic acids, citric acid) [24]. Air (gases) resides in between the fractions of the soil pore network that are filled with the soil solution. The soil atmosphere depends on the connectivity of the non-water-filled pores of soil with the atmosphere or with other open pores. It usually consists of varying amounts of gasses such as oxygen, carbon dioxide, nitrogen, and nitrogen dioxide, but also of volatile organic compounds (gasses with biotic origin). The composition of the soil atmosphere highly depends on the production or consumption of a specific gas by local organisms, the solubility and diffusion of the gases in the soil water and, consequently, also on the capacity for water retention of the soil itself, as described before [13; 25]. As an example, molecular oxygen has a very low solubility in water and its diffusion rate in water is 10,000-fold lower than in air [25]. Hence, soil micropores filled with water will rapidly turn anoxic upon consumption, limiting the growth and survival of many microbes. On the other hand, micropores can also offer protective microhabitats against \"toxic\" gases [26]. Independent of the soil component considered, the soil solution present in colloids can eventually diffuse out into wider pores, where it is readily available to microorganisms or is transported with the percolating water. It is also worth mentioning that filamentous fungi and plant roots stabilize the micro-aggregates of soil, and, in turn, together with other organisms living in the soil (e.g., nematodes, worms, larger animals), continuously increase soil solution conductivity through the soil. Nonetheless, the majority of substrates remain isolated and persist in the environment, possibly due to either physical protection or the separation from relevant enzymes [19]. In this regard, given their even smaller size, exoenzymes released by bacteria, archaea, and fungi into the soil solution can diffuse in and out of tiny pores and have significant roles both in the total fitness of the microbial population as well as in geo-chemical nutrient cycles of an ecosystem [27]. Respiration and decomposition rates differ considerably between sandy soils and soils rich in clay. This illustrates impressively how the dynamic interaction between the physical, air, and water components of soil directly influence the access, and thus the activity, of microbial populations to their substrate both in space and time. Sandy soilshave lower surface-to-volume ratios than clay soils, meaning less water retention, but also lower segregation between microenvironments. In this context, soil heterogeneity has to be considered. For example, organic matter placed in different regions of the pore network was shown to be decomposed at different rates [28] while a number of other studies reported increased respiration rates only after disruption of the soil structure in soils with high clay content (e.g., [29]). These results taken together suggest that both the local environmental conditions as well as the uneven distribution of microbial populations within the local environment strongly affect the mineralization rates [28; 30; 31] and the range over which organisms can disperse and interact.