## 2 Models of the Structure of Humic Substances
### The Supramolecular Aggregate Model  
Alongside the development of polymeric and micellar-like models, a flourishing line of research was developed on the use of the fractal theory in the study of the aggregation of HS [59]. The most salient scientific results concerning the application of the fractal model to HS will be illustrated in Section 3.3.1 dedicated to the scattering studies. After the advent of polymeric and micellar models, more credits are given to the hypothesis that HS are supramolecular aggregates of small organic compounds, held together by weak dispersive forces other than covalent bonding [60; 61; 62; 63; 64]. Hence, according to the concept of supramolecular structures, HS can be considered as relatively small and heterogeneous molecules of various origin, which self-organize in supramolecular conformations. Humic superstructures of relatively small molecules are now imagined as complex aggregates stabilized only by weak forces such as dispersive hydrophobic interactions (van der Waals, \(\pi\)-\(\pi\), and CH-\(\pi\) bonding) and H-bonds, the latter being progressively more important at low pH values. The representation of HS according to the model based on the spontaneous association of molecular components through weak bonds, provides several important implications: (1) accumulation of organic matter in soils is driven by hydrophobic interactions; (2) any HS supramolecular structure can undergo interchange processes with hydrophilic molecules generated through the degradation of biological tissues; (3) the hydrophobic bonds also lead to the consequent stabilization of the organo-mineral complexes and the general structure of soil [60]. Experimental evidences that HS have a hierarchical or \"structure within a structure\" architecture with at least two sequential levels of organization were provided by [PERSON] et al. [65; 66; 67]. In details, they developed a method in which a given HA\({}_{0}\) sample was firstly subjected to Soxhlet extraction using a C\({}_{6}\)H\({}_{6}\):CH\({}_{3}\)OH azeotrope for 72 h to collect two main fractions, respectively, a lipid-like L\({}_{0}\) and a non-amphiphilic HA\({}_{1}\), corresponding to about 67% of the total organic carbon (TOC). Then, the L\({}_{0}\) fraction was separated into further two sub-fractions: HA\({}_{2}\) accounting for almost one-third of TOC (possessing amphiphilic properties and soluble in an alkaline aqueous solution) and L\({}_{1}\) (lipid-like and soluble in nonpolar solvents). Differences in heat capacities between the pristine HA and the reconstituted physical mixtures (HA\({}_{1}\) + L\({}_{0}\) and HA\({}_{1}\) + HA\({}_{2}\) + L\({}_{1}\)) were compared. Thus, it was deduced a first level of organization correspondent to self-assembled nanoparticles formed by the amphiphilic HA\({}_{2}\) and the lipid L\({}_{1}\) fractions, which further interact with the components of HA\({}_{1}\) yielding the overall HA\({}_{0}\) composite (second level of organization). However, the interactions that trigger and stabilize the structure of these assembled molecular complexes are still unknown. The diffusion coefficients of carbohydrates, carboxyl-rich alicyclic molecules and aliphatic compounds in DOM strongly depend on their concentrations indicating their gradual association [68]. Based on this observation, [PERSON] et al. [69] hypothesized that free hydrophilic molecules at lower concentration can play a role as hydrotropic compounds and stabilize and solubilize the aromatic aggregates in DOM while at higher concentration slowly aggregate forming physically stabilized gel-like structures. Earlier results showing the different stability of HS aggregates at increasing temperatures [70], led to a new model in which HS are described as a hydrophobic inner core stabilized by outer layers of amphiphilic and polar molecules that impart a hydrogel-like structure [71]. Recent research developed by [PERSON] et al. [72], emphasized the role played by \"strong\" H-bonds among polar domains, in the formation of stable hydrogels-like structure.