Dependence of Basal Spacing on the Amount of Aflatoxin and Water in the Complex

In the ranges of water and aflatoxin contents simulated, the basal spacing of the smectite varied from 9.8 to 27 Å. Increasing the water or AfB₁ contents generally led to an increase in basal d ₀₀₁ spacing (Fig. 1). No significant difference was noted in terms of interlayer expansion for structures with small water contents (0 and 3.7 mol/kg H₂O). This was due to the existence of empty void spaces between aflatoxin molecules in dry structures, which could be then filled with H₂O. For larger amounts of AfB₁ and H₂O, the basal spacing increased linearly with the contents of both molecules. For these smectite structures, water molecules filled the space between AfB₁ molecules, and the addition of new molecules led to expansion of interlayer distances.

Fig. 1 Dependence of d ₀₀₁ spacing of AfB₁-smectite complexes on AfB₁ and water content. The contour graphs were based on the 36 data points of molecular simulations

In the literature, the greatest d ₀₀₁ spacing of air-dried AfB₁-smectite complexes recorded at room temperature and ~60% humidity was ~15 Å (e.g. Deng et al., Reference Deng, Barrientos-Velazquez, Billes and Dixon2010). Basal spacings of >20 Å observed in high-water content conditions in these simulations offered more insight into to the AfB₁-smectite interactions in solution. As most X-ray diffraction analyses of AfB₁-smectite complexes were analyzed after drying the complexes, the d spacings of >20 Å in the presence of large amounts of water deserve experimental verification. Even at 0 mol/kg of water, the basal spacing of the AfB₁-smectite could be expanded to >18 Å when AfB₁ adsorption reached 0.9 mol/kg. Neither a large basal spacing like this nor this level of adsorption has been observed experimentally.

Influence of the Water Content on the Configurations of Adsorbed Aflatoxin in the Interlayer of Smectite

Water content affected to a great extent the orientations and configurations of the aflatoxin molecules adsorbed in the interlayer of the smectites. The individual aflatoxin molecules did not deform significantly from their nearly planar structure when the water content was varied, but the distribution of the AfB₁ molecules varied drastically when the interlayer water content was increased. For example, at the 0.37 mol/kg AfB₁ adsorption level, when the complexes were dry, the adsorbed AfB₁ molecules were oriented nearly parallel to the basal surfaces of the smectites with only one layer of the AfB₁ molecules scattered uniformly in the interlayer space (Fig. 2a). When the amount of water was increased to 7.5 mol/kg, AfB₁ molecules formed two layers (Fig. 2b). For a water content of 18.75 mol/kg, the AfB₁ molecules had more random orientations, and some of the aflatoxin molecules had inclination angles toward the basal surfaces of >60° (Fig. 2c). Introducing water into the AfB₁-smectite complexes made both the exchange ions and aflatoxin molecules more mobile and more dispersed in the interlayer. For larger AfB₁ loadings, some of AfB₁ molecules can be disoriented from parallel orientation, also for structures without water (Supplementary Materials, Fig. S1a). Addition of water molecules led to more random orientations of AfB₁ molecules for these structures (Fig. S1b, 1c), similarly as in the case of smaller AfB₁ loadings.

Fig. 2 Structure of Mnt04_Ba smectite with 0.37 mol/kg aflatoxin B₁ adsorbed: a dry; b with 7.5 mol/kg water; c with 18.75 mol/kg water adsorbed. Side view, top view, and distribution of atoms are shown in consecutive columns from left to right. The formation of AfB₁ π–π complexes for structures with water is visible

Aflatoxin molecules moved around in the interlayer and started stacking on each other at large water contents. They formed two-layer or even three-layer structures in some locations. A unique feature of self-association of aflatoxin molecules was observed on the structure corresponding to a larger amount of adsorbed water (Fig. 2b, c). In this self-associated AfB₁ structure, the aflatoxin molecules could form π–π stacking complexes, and the self-associated AfB₁ molecules excluded water to their surrounding spaces. With increasing numbers of water molecules, these π–π complexes could detach from the surface and disperse in the interlayer water (Fig. 2c). The self-association of AfB₁ molecules in the interlayer of smectite appeared to be consistent with the low water solubility of the toxin of 20–30 mg/L. Such a feature would be difficult to detect experimentally with spectroscopic methods.

The simulations also showed that, as expected from the experimental IR data of Deng et al. (Reference Deng, Barrientos-Velazquez, Billes and Dixon2010, Reference Deng, Liu, Barrientos-Velazquez and Dixon2012), mainly water bridges were formed between ions and aflatoxin carbonyl oxygen atoms as indicated by the radial distribution function (RDF) and running coordination number (RCN) plots between interlayer Ba²⁺ cations and carbonyl oxygens (Fig. 3). The first peak at ~2.8 Å on the RDF patterns was due to the direct Ba-carbonyl ion–dipole interactions and the second peak at radius of ~4.8–5.3 Å was due to the formation of water bridges between the exchange cation and the carbonyl groups. The peaks on the RDF patterns corresponded to the growth of the RCN function. The simulated ratio between the amount of water-bridged Ba to directly coordinated Ba was ~5.5:1 at the 7.5 mol/kg water content, and 40:1 for a water content of 18.75 mol/kg (calculated based on data from Fig. 3). These calculations suggested the importance of water bridging for the adsorption of AfB₁ by the wet smectites, but the simulation also suggested the direct ion–dipole interaction still made significant contributions to the bonding. The exchange cations can have the ion–dipole interactions with the oxygen atoms on the dihydrofuran rings (Fig. 2).

Fig. 3 Radial distribution function and running coordination number plots for Ba–O_carbonyl of AfB₁ for the Mnt04_Ba structure with 0.37 mol/kg of AfB₁ and variable amounts of water adsorbed

The calculated energies of aflatoxin molecule adsorption (Eq. 1) of the AfB₁-smectite complexes showed minima at water content levels of 7.5 mol/kg for all five montmorillonite structures that had layer charge which originated from the octahedral sheet (Fig. 4a, b, c, d, h). This amount corresponded to two layers of water in the interlayer of the smectites and gave a basal spacing of close to 15 Å. This basal spacing was in agreement with the experimental observation that the synthesized AfB₁-smectite had a basal spacing of ~15 Å at room humidity (Deng et al., Reference Deng, Barrientos-Velazquez, Billes and Dixon2010). With either more or less water, energies of AfB₁ molecule adsorption increased.

Fig. 4 Potential energies (kcal/mol) of adsorption of aflatoxin molecules on smectite structures. Smectite at a certain hydration level but without AfB₁ molecules was used as a reference state; note, therefore, that the amount of AfB₁ sorbed begins at 0.185 mol/kg

Although the grid for the energy plots was relatively sparse, it was still evident that the shapes of isoenergetic lines were not parallel to the abscissa axis but were inclined and corresponded partially to isolines of constant basal spacing (Fig. 1). The two water-layer structure of AfB₁-water-smectite complex may thus be the most energetically preferable and stable one. The initial AfB₁ molecules in the complex had the smallest (and preferable) energy of adsorption, but with increasing amounts of interlayer AfB₁, addition of AfB₁ molecules became less preferable (e.g. for Mnt04_Ba, 7.5 mol/kg H₂O, and 0.185 mol/kg AfB₁ it is –23 kcal/mol, but for 0.37 mol/kg AfB₁ adsorbed it is –14 kcal/mol – Fig. 4).

The potential energies of water molecule adsorption, ΔU ${\left(N\right)}_{H_{2}0}$ , on smectites with a constant number of AfB₁ molecules showed that structures with 0.185 mol/kg of AfB₁ had the smallest energies compared to all other AfB₁ contents and also dry structures (blue lines in Fig. 5). With the increase in AfB₁ contents, the addition of water molecules to the structure was energetically less favorable (green, orange, red, and brown lines in Fig. 5).

Fig. 5 Potential energies (kcal/mol) of water-molecule adsorption on smectite structures. Smectite with a certain number of AfB₁ molecules but without water was used as a reference state for each line

The initial decrease in energies for 0.185 mol/kg AfB₁ structures was related to expansion of interlayer space and the existence of vacuum easily accessible by H₂O molecules. This led to easier access by water molecules to the interlayer ions. The increase in potential energies of adsorption was caused by relative hydrophobicity of AfB₁ molecules. With increase in water content in the interlayers, the potential energy of H₂O adsorption also increased, which was related mainly to filling of coordinating spheres of cations, around which water molecules were sorbed preferentially (e.g. Fig. 2b).

These molecular simulation results confirmed the assumption that in the interlayer space of smectite, AfB₁ molecules filled the available surface area between those domains covered by the hydrated ions (Deng et al., Reference Deng, Liu, Barrientos-Velazquez and Dixon2012). Note, however, that the plots in Fig. 4 were received for a certain number of water molecules, which was a value that did not correspond directly to humidity. This was because, depending on the type of interlayer cation for the same humidity, the number of adsorbed water molecules was found to be different (e.g. Laird, Reference Laird2006).

Influence of Interlayer Cation on the Structure of Aflatoxin-clay Complex

The calculated energy plots of AfB₁, ΔU(N)_AfB1, and H₂O molecule adsorption, ΔU ${\left(N\right)}_{H_{2}0}$ , showed substantial differences between divalent (Ba²⁺ and Ca²⁺) and monovalent (Na⁺) cations (Figs. 4b, d, h and 5b, d, h). Both energies were much greater for monovalent ions for various AfB₁ and water contents. For Ca²⁺ and Ba²⁺ the plots were approximately the same.

The values of ΔU ${\left(N\right)}_{H_{2}0}$ for the Mnt04_Na structure (Fig. 5h) showed less dependence on the absolute water content than structures with divalent ions (Fig. 5b, d). All these observations could be attributed mainly to the differences in hydration enthalpy of the ions: Na⁺, –406 kJ/mol; Ca²⁺, –1579 kJ/mol; and Ba²⁺, –1309 kJ/mol (Smith, Reference Smith1977), as H₂O was mainly in the coordination spheres of interlayer cations (Fig. 2b, c).

The differences for ΔU(N)_AfB1 (Fig. 4b, d, h) were related rather to available space for AfB₁ adsorption on the surface. The divalent cations form only outer-sphere complexes in the center of Mnt04 interlayers (Fig. 6b, d), while monovalent Na⁺ ions tended to form both outer-sphere and inner-sphere complexes at the surface (Fig. 6h). Thereby, Na⁺ ions are steric hindrances for hydrophobic AfB₁ molecules (thick black lines in Fig. 6h – some Na⁺ ions are close to the surface). For the same number of AfB₁ and H₂O molecules, the maxima of carbon and oxygen atoms of AfB₁ at the surface were slightly greater for divalent ions (thin black lines in Fig. 6b, d, h). In parallel, the plateau in the center of the interlayer for these atoms had greater atomic density for structures with Na⁺ ions (for the z-position around zero in Fig. 6b, d, h). The results above are in good agreement with experimental data which shows that smectites with divalent cations are much better adsorbers of AfB₁. According to Deng et al. (Reference Deng, Liu, Barrientos-Velazquez and Dixon2012), this is related to a better size match between AfB₁ molecules and the adsorbing sites on smectite; in the case of divalent cations the hydrophobic smectite surface is more accessible to AfB₁ molecules and this is reflected in Fig. 6b, d, h.

Fig. 6 Distribution of atoms in the interlayer for the structures studied with 0.37 mol/kg of AfB₁ and 7.5 mol/kg of water contents. O18 and O22 are the carbonyl oxygens of AfB₁

Calculation of RDFs and RCNs for structures corresponding to 0.37 mol AfB₁ and 7.5 mol of water per kg of clay showed that differences in the hydration enthalpies of interlayer ions played an important role in the number of water bridges formed between ions and AfB₁ molecules of the same valency (Fig. 7); the more negative the hydration enthalpy, the more water bridges form between AfB₁ molecule and cations – in the case of Ba²⁺ ions there were clearly more direct ion–dipole interactions than for the Ca²⁺ form (as visible on RCN plots – Fig. 7a). For the Na⁺ form, the number of ion–dipole interactions was greater because of the larger number of ions in the interlayer, but the distances of water bridges were less localized (as visible on RDF plots – Fig. 7a). Considering the RDF and RCN for pairs: the ions (Ba, Ca or Na) and oxygens of water (Fig. 7b) showed 8-fold coordination of divalent cations and 4-fold coordination for the monovalent Na⁺ cation. This effect is clearly related to the water:ion ratio for the systems studied. Generally, the number of cations that coordinate carbonyl oxygens of AfB₁ via direct ion–dipole interactions (RCN plot: Fig. 7a) was inversely proportional to the number of water molecules surrounding the cations in the first coordination spheres (RCN plot: Fig. 7b).

Fig. 7 Radial distribution functions and running coordination numbers for: a carbonyl oxygens–interlayer cations; b cations–oxygen of water molecules, for montmorillonite with a charge of 0.4 per O₁₀(OH)₂ substituted with different ions – 0.37 mol/kg AfB₁ and 7.5 mol/kg of water sorbed

The potential energy of the addition of AfB₁ to pure water was 9.5 kcal/mol. With an increasing number of water molecules in the interlayers, the potential energy of addition of AfB₁ molecules to the structure had values less than addition of AfB₁ to pure water (~0 kcal/mol; Fig. 4). For a hydration level of 7.5 mol/kg, the energy of addition of AfB₁ molecules to a clay complex could even be <–20 kcal/mol for the first AfB₁ molecules adsorbed (Fig. 4a, b, d).

To explain differences in energy of aflatoxin adsorption, the contributions of different energy terms to the total energy of AfB₁ addition were calculated (Eqs. 2 and 3; Table 1). Clearly, the largest differences between smectites were in the electrostatic terms. A less negative term in Na-montmorillonite indicated weaker interactions between the ions, the atoms of the surface and the AfB₁ molecules. These observations are in agreement with experimental results that showed poor AfB₁ adsorption on Na-montmorillonites (Deng et al., Reference Deng, Liu, Barrientos-Velazquez and Dixon2012; Barrientos-Velazquez et al., Reference Barrientos-Velazquez, Marroquin Cardona, Liu, Phillips and Deng2016b).

Table 1 Energy contribution (kcal/mol) after addition of AfB₁ molecules to montmorillonites with various cations along with the basal spacing of the complexes

*smectite structure assumes addition of 0.37 mol/kg of AfB1 and having 7.5 mol/kg of water

In the case of addition of AfB₁ to smectite intercalates, a small deformation of molecular structure (the molecular energy term was 79.4–80.0 kcal/mol, similar for each cation) was observed compared to the addition of AfB₁ to water (molecular energy term: 68.5 kcal/mol; Table 1). The introduction of AfB₁ into smectites led to some deformation of the molecules, therefore, but AfB₁ was retained in the interlayer through electrostatic + van der Waals interactions either with ions or with the surface.

Influence of Layer Charge Density of Montmorillonite on Aflatoxin B₁ Adsorption

Aflatoxin adsorption energy, ΔU(N)_AfB1, calculated from Eq. 1 for Ba-montmorillonites having layer charges of 0.3, 0.4, and 0.5 cphuc (Fig. 4a, b, c), showed that layer charge had a large influence on the thermodynamics of the addition of AfB₁ to the structure. For less moist conditions, the energy of adsorption was substantially less for low-charge montmorillonite (Fig. 4a). The smallest energy values were observed when the water content was close to 7.5 mol/kg. With increasing numbers of water molecules in the interlayers, the potential energy of the addition of AfB₁ molecules reached greater values; among Mnt_Ba structures, the highest energies were observed for Mnt05_Ba.

The calculated energy plots for adsorption of H₂O molecules, ΔU ${\left(N\right)}_{H_{2}0}$ , (Fig. 5a, b, c) showed that with an increase in layer charge, the energy of hydration became more negative. This was due to the first water molecules hydrating around ions being bonded more exothermally (e.g. Rudbeck, Reference Rudbeck2006). If, in the structure, there were more interlayer ions, then, statistically, more water would bond within the first shells of ions. On the other hand, with increasing numbers of water molecules, clay layers moved further apart. For higher charge-density smectites, clay layers tended to be closer to each other than for low-charge smectites, which could eventually lead to reduction of the amount of intercalated water molecules in experimental studies.

Visualizations of distributions of atoms (Fig. 6a, b, c) showed lower intensity maxima of carbon and oxygen atoms of AfB₁ at the surface for Mnt05_Ba, compared to Mnt03_Ba and Mnt04_Ba. The plateau in the center of the interlayer was, therefore, greatest for high-charge montmorillonite, indicating that AfB₁ molecules were more disordered and farther from the surface for this smectite. The basal spacing was smaller for the high-charge montmorillonite, which indicated squeezing of interlayer molecules (Table 2). Another distinct difference in these complexes was the location of the interlayer cations; they stayed in the middle of the interlayer space in the lower charge-density montmorillonite, and they stayed both in the middle and closer to basal oxygens of montmorillonite with charge density of 0.5 per half unit cell.

Table 2 Energy contribution (kcal/mol) after addition of AfB₁ molecules to smectites with various layer charges along with basal spacing of the complexes

*smectite structure assumes addition of 0.37 mol/kg of AfB1 and having 7.5 mol/kg of water

Analysis of the potential energy contribution showed differences in the electrostatic term (Table 2). With increase in smectite charge, the attraction between interlayer cations and the clay layer became much stronger and aflatoxin molecules were more squeezed within interlayer spaces, especially for montmorillonite. This resulted in an increase of the electrostatic term and a decrease in the van der Waals term, with increasing montmorillonite layer charge (Table 2). The effect of layer charge on AfB₁ molecules suggested that the interlayer of high-charge smectites was not favored.

The layer charge on a montmorillonite was varied by Barrientos-Velazquez et al. (Reference Barrientos-Velazquez, Marroquin Cardona, Liu, Phillips and Deng2016b), who observed that an optimal layer charge was critical for maximum AfB₁ adsorption. Increasing the exchange capacity to >85 cmol/kg decreased significantly the aflatoxin adsorption capacity of montmorillonite. All the results achieved with the modeling, therefore, agreed quite well with those from experiment.

Influence of Location of the Layer Charge on Aflatoxin B₁ Adsorption

The influence of the location of the layer charge on aflatoxin B₁ adsorption was investigated by comparing AfB₁-beidellite with the AfB₁-montmorillonite complexes. Plots of the energy of aflatoxin adsorption on beidellite (Fig. 4e, f, g) showed that adsorption of AfB₁ on smectites with layer charge located in the tetrahedral sheet was much less favorable thermodynamically than if the charge was located in the octahedral sheet. This was because the interlayer ions tended to be located close to the basal surface in beidellites (thick black lines in Fig. 6e, f, g), while they tended to stay in the middle of the interlayers in montmorillonites (thick black lines in Fig. 6a, b, c). If the ions were located in the middle of the interlayer space, they had more freedom to adapt to optimal positions between AfB₁ molecules, while if the cations were directly on the clay surface they could expel AfB₁ molecules from the surface and formation of π–π stacking-complexes was more restricted. The distribution of the cations in the interlayer provides an explanation for the experimental AfB₁ adsorption in montmorillonite and beidellite observed by Barrientos-Velazquez et al. (Reference Barrientos-Velazquez, Marroquin Cardona, Liu, Phillips and Deng2016b). Greater aflatoxin adsorption by octahedral charged smectites (hectorite and montmorillonite) was observed when compared to tetrahedral charged smectites (beidellite and nontronite).

Ba-montmorillonite with a different location of layer charge in comparison to Ba-beidellite showed generally less negative values of ΔU ${\left(N\right)}_{H_{2}0}$ and, thus, a smaller gradual increase of ΔU ${\left(N\right)}_{H_{2}0}$ with increase in the amount of adsorbed water (Figs. 5a, b, c vs. 5e, f, g). This is related to partial hydrophilicity of the beidellite surface in comparison to montmorillonite (Szczerba et al., Reference Szczerba, Kalinichev and Kowalik2020), which results in smaller values of ΔU ${\left(N\right)}_{H_{2}0}$  for smaller amounts of water in the structures.

Comparison of energy contributions after addition of AfB₁ molecules to beidellites (Table 2) showed large variations in the electrostatic contribution compared to montmorillonites. In the case of beidellites, the ions were located in the positions close to substitutions in the tetrahedral sheets, and closer to the smectite surface (Fig. 6e, f, g). Ions were less mobile, only partially hydrated in closed shell positions, and, therefore, the electrostatic term was much greater for high-charge beidellites (Table 2). Generally, van der Waals terms decreased with the increase in layer charge, but electrostatic terms increased much more, resulting in a net increase of potential energy for high tetrahedrally charged smectites. These results were confirmed by an experimental test in which the aflatoxin affinity was significantly lower in nontronite, which is a tetrahedrally charged smectite, compared to montmorillonite (Barrientos-Velazquez et al., Reference Barrientos-Velazquez, Marroquin Cardona, Liu, Phillips and Deng2016b).