# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 2 Fungi, Bacteria and Their Microenvironment
### Bacterial-Fungal Interactions: A Harsh Existence  
At the cell-to-cell level, bacteria and fungi interact at many different levels of intimacy that can be considered from two perspectives: in terms of physical associations and in terms of molecular communication [32]. Physical associations can range from seemingly disordered polymicrobial communities (i.e., biofilms) to highly specific symbiotic associations of fungal hyphae and bacterial cells (i.e., ectosymbiotic or endosymbiotic). Molecular interactions, on the other hand, involve a complex and diverse set of chemicals and compounds, and the interaction can be contextualized as antibiosis, signaling, and chemotaxis, metabolite exchange, metabolic conversion, adhesion, protein secretion, genetic exchange, and physicochemical changes. More often than not, multiple mechanism of interaction can be employed by one microorganism. The multitude of interactions and their effect on the partners or surrounding environment is extensive and well reviewed in [1; 32; 33; 34]. Irrespective of the type of interaction, bacteria and fungi need to recognize each other prior to initiating any kind of target-oriented interaction. However, sensing and recognition mechanisms are often hindered by the particularity of soil and by the phenotypical characteristics of the organism (Figure 1). When considering bacteria, they have their highest diversity in soil micro-aggregates, where they are either \"swimming\" in the soil solution, or attached to soil particles. This is not surprising, as it is in accordance with the higher concentrations of organic matter found in the micro-pores, the more stable conditions in water content, and a lower predation pressure by protozoan or other predators [35; 36; 37; 38; 39]. One of the first studies focusing on the distribution of bacteria in soil revealed that specific bacterial populations are typically residing inside micro-aggregates, with 40-70% of these bacteria being localized in the 2-20 \(\upmu\)m and in <2 \(\upmu\)m size micro-pores [14]. In some cases, cells can even penetrate pores smaller than themselves [40]. In contrast, more recent studies reported that pores in the 30 to 150 \(\upmu\)m size range harbor a greater abundance of specific bacterial groups [41]. However, studies are scarce and only represent a snapshot in time, in a very dynamic environment. In fact, microbes explore a constantly changing environment in search of their _realized niche_, let it be by either active exploration of soil by fungal hyphae or by chemotactic movement, or passive transportation in bacteria. Any potential in bacterial mobility is limited by surface tension, capillary forces, and viscous drag that increase the energy requirement for their motility, particularly in partially saturated pore networks. Motility was found to cease, virtually completely, when the thickness of the water film was smaller than 1.5 \(\upmu\)m [42]. For these reasons, microbial cells are often found as individual cells when associated with highly localized dissolved organic matter, or as patches of dense populations when linked to more conspicuous substrates [43]. In bulk soil, the average distance between neighbouring bacterial cells was found to be around 12.46 \(\upmu\)m, with inter-cell distances shorter near the soil surface (10.38 \(\upmu\)m) than at depth (>18 \(\upmu\)m), due to changes in cell densities [44]. Simplified calculations suggests that, despite the very high number of cells and species in soil (10\({}^{8}\) cells per gram of soil), the number of neighbours that a single bacterial cell has within an interaction distance of ca. 20 \(\upmu\)m is relatively limited (120 cells on average) [44]. Similar crude estimates were also found when the surfaces of soil pores were used to calculate the exclusion zone of cells on soil aggregates (a radius of 178 \(\upmu\)m) [45]. A completely different story is depicted when filamentous fungi are considered.