# Soil Is Still an Unknown Biological System
## 2 Properties of Soil as a Biological System
### The Main Research Approaches  
Soil biologists and soil biochemists have struggled and still struggle with knowing what is in soil and what it is doing, with the aim not only to better know the system but also to properly quantify soil functions [9]. They have carried out different research approaches. Studies using simplified systems, such as those only using two soil components, have brought a better understanding of interactions between soil components, such as the interactions of a single clay mineral with DNA, proteins, or microbial species [23; 24; 25; 38]. Of course, caution is required to extrapolate the results of these studies on simplified systems to soil, which contains a huge variety of microbial species and, for example, not pure clay minerals, being covered by other inorganic constituents and organic material. Even DNA and proteins are released in soil with other cellular components, which may interfere with their adsorption by surface-reactive particles. This brief discussion about the drawbacks of soil omics methods and the problems with interpreting relative data does not want to discourage their use but to help with the proper discussion of the obtained data. Nowadays, omics techniques are among the most powerful ones to determine microbial diversity and microbial functions in soil. Knowledge of the microbial taxa inhabiting soil is improved using molecular techniques, in particular amplicon sequencing and metagenome techniques. However, these studies are often based on the assumption that the detected microbial species carry out in soil the same functions observed in vitro [12]. The presence of functional genes is often taken as a sign that the relative function potentially occurs in the soil where these genes are detected without determining the gene expression or relating this presence to the measurement of the target activity [12]. Most of the microbial species inhabiting soil are generally in a dormant state, as already mentioned [13]. In addition, rare species can be active and responsible for the target activity when the expression of genes is related to the measured activity, as reported for rare species encoding alkaline phosphomonoesterase activity [14]. It is well established that metagenome studies should be combined with proteomic analyses to gain insight into soil functions because proteins are the final expression of each genome. Unfortunately, soil proteomics still has challenges and pitfalls, such as the fact that it is not technically possible to determine all expressed proteins in soil due to their huge number, and that the complete protein extraction from soil is not possible with a single extraction procedure because proteins have different properties (low and high molecular weights, hydrophilic and hydrophobic moieties, different isoelectric points) and thus different interactions with the surface-reactive particles of soil [39]. The holistic consideration of the soil system represents the best approach to quantify nutrient transformations in the soil-plant system, especially if it is combined with the use of labelled (for example, with \({}^{14}\)C or enriched with \({}^{15}\)N) compounds or the determination of changes in the abundances of stable isotopes (for example, \({}^{13}\)C) [8]. According to the holistic approach, the system is portioned into pools (for example, microbial biomass C, microbial biomass N) with a functional meaning, and fluxes between these pools can represent abiotic processes (for example, nitrate leaching or ammonia volatilization) or biotic transformations (for example, nitrification, mineralization, etc.). In this way, the distribution of the labelled compound can be monitored through the different pools and be discriminated from the respective nutrient already present in the soil (for example, the \({}^{15}\)N enriched fertilizer is distinguished by the native soil N) [8].