Conformational Analysis of Humic Acids from Amazonian Dark Earth Soils observed by AFM

Conformat ional analysis of humic acid (HA), extracted from an Amazon anthropogenic soil and an adjacent soil which did not have anthropogenic A horizon, was done by atomic force microscopy (AFM), through the deposition of humic acid layers on muscovite mica strips using drop-casting deposition technique. This short communication presents, for the first time, AFM images of the sub-micron level structure of humic acid o f the Amazonian Dark Earths, compared to natural pedogenic Amazonian soil. The adsorbed anthropogenic HA form ring-shaped aggregates with diameters on the scale of several tens of nanometers, possibly showing evidences of a supramolecular formation. The formation of these structures was not verified for HA from pedogenic soil. In this case, it was observed particles with g lobular shape and a homogenous distribution of them on the mica surface. Structural characteristics of each sample were observed by nuclear resonance magnetic spectroscopy (NMR). The spectra analysis indicated that pedogenic HA are richer in aliphatic groups, as methoxylic and in polysaccharides structures, while the h igher concentrations of aromatic carbon, including also phenolic carbon, were observed in the anthropogenic HA, justifying its higher hydrophobic character.


Introduction
Soil Organic Matter (SOM ) play an important role on many aspects of the nature of soil and environmental processes. Hu mic Substances (HS) are the major o rganic constituents of soils and aquatic environments and are generated by the microbiological and chemical degradation, and transformation of organic matter (OM), resulting in chemical structures which are more stable than the starting material [1][2][3][4]. Most of the soils in the Amazon Basin are acidic, with low cation-exchange capacity, fert ility and production potential. In this environment, where soil fert ility is a limiting factor for sustainable agricultural develop ment, occurs the "Terra Preta de Índio" soil [5], also known as Amazonian Dark Earth, Anthropogenic Dark Earth, Indian Black Earths, or Archaeological Dark Earths. These soils, that have an archeo-anthropedogenic horizon, wh ich is a surface horizon with variable depth in soil profiles, exhibit elevated OM contents and either ceramic p ieces or lithic art ifacts [6]. The OM present in these soils shows high stability and reactivity characterist ics [7]. The orig in o f th is OM is thought to be mostly pyrogenic and is related to the domestic/agricultural activity of pre-colu mb ian populations that colonized the Amazon basin since 3000 B.C. [8]. Due to its high fertility, compared to the adjacent soils, it is has been frequently used in subsistence agriculture (beans, corn, etc.) and/or for commercial p roduction of a high diversity of products (papaya, coconut, cupuaçu etc.).
The chemical nature and reactivity of HS are still little understood. Several researchers [9][10][11] have suggested a new model for structure of HS, in wh ich small and heterogeneous humic molecu les are self-assembled in supramolecular conformat ions stabilized only by weak forces. Several techniques have been employed to characterize the size, shape, conformat ion, structure and composition of HS. Fluorescence spectroscopy, for examp le, has become widely recognized as a relatively simple, sensitive, and powerful tool for the evaluation of humification [12,13] and the molecular size of hu mic substances [14,15]. Nuclear Ressonance Magnetic also is co mmon ly used to characterize co mplex HS including hu mic and fu lvic acids [16][17][18][19]. In addit ion, other techniques such as capillary electrophoresis [20], ultravio let and infrared spectroscopy [21,22] and scanning electron microscopy (SEM ) [23] have also been frequently employed.
Atomic Force Microscopy (AFM) technique can image surfaces with atomic resolution by scanning a sharp tip across the surface at forces smaller than the forces between atoms [24]. AFM has been emp loyed to study the morphologies of humic and fulv ic acid [25][26][27][28] and it is a powerful tool to characterize small colloids, as well as collo id agglomerat ion, adsorption onto surfaces, or modificat ion in the morphologies, affected by changes in the physical-chemistry properties. Namjesnik-Dejanovic and Maurice [29] reported that high or low concentrations of fulvic acids, fro m river water, result in d ifferent structures, which can be differentiated by AFM. Chen and coworkers [30] combined AFM with SEM to investigated macro mo lecular structures of humic substancesThey reported that, depending on the pH and ionic strength of the initial solutions, humic substances deposited on mica and silica, adopted different spherical and large-area island conformations.
In a general way, a better understanding of HS structures will p rovide in formation o f origin and genesis, reactivity, and role in environ mental processes. The objective of this work was to study the anthropogenic soil hu mic acid in comparison to an adjacent soil which d id not have anthropogenic A horizon, using AFM and, comp lementally, NM R. The purpose of our investigation was to obtain new data of morphology, adsorption and orientation of HA structures on mica fro m anthropogenic HA and shows evidences from supramolecular formation for the first time in A mazonian Dark Ea rth.

Material and Methods
Hu mic acids (HA) fro m two d ifferent orig ins, pedogenic and anthropogenic soil, both obtained fro m the A mazon State (Brazil), were extracted and purified accord ing to the International Hu mic Substances Society (IHSS) method [31]. Suspensions of 100 mg L -1 of HA fro m anthropogenic Dark Earths and adjacent soil, were prepared suspending HA samples in deionized water. The suspension was shaken for at least three days. The pH was adjusted to 3.5 using HCl. Muscovite mica with a surface area of about 1 cm 2 was cleaved and kept into a Beaker containing 10 ml of a 100 mg L -1 HA suspension for 1 day, under constant stirring. The mica was then rinsed with deionized water, put in a Petri d ish and kept in a diseccator for 12 hours before obtaining the AFM images [32]. Unrinsed samples were also analyzed and used as comparison.
NMR experiments were performed with a Varian model Unity Inova 400 Spectro meter ( 13 C resonance frequency of 100.58 MHz), equipped with a solid Doty Supersonic probe. The HA samples (approximately 300 mg ) were conditioned in a zirconiu m cylindrical rotor with 5 mm of external diameter (Doty Supersonic). The variable amp litude cross polarization mag ic angle spinning technique (VA CP-MAS 13 C NMR) [33] was applied with a contact time of 1 ms, acquisition time of 12.8 ms, pulse delay of 500 ms, spinning speed of 6 kHz. The 13 C chemical shifts were referenced using hexametylbenzene − the resonance line in 17.2 ppm.
AFM imag ing were obtained at room temperature, using an Atomic Force Microscope TMX 2010 Topometrix, in-cluding special silicon nitride (Si 3 N 4 ) cantilevers with spring constant of 0,032 N m -1 and pyramidal tip with rad ius of 40 nm. To investigate the samples with AFM, it was used the contact mode tip and scan rate of 1.5 Hz. Surface scans of AFM were analyzed using a freeware scanning probe microscopy software based on MS-Windows named WSXM [34].

Results and Discussions
A typical image of pedogenic HA adsorbed on mica surface is shown in Figure 1a. The globular and cone-shaped features were assigned to islands of HA adsorbed on mica surface, and were identified t wo general types of structures, which can be summarized as: part icles or spheroids and network of particles. AFM images show that large aggregates and small particles coexisted. The pH of HA suspension (3.5) favors the aggregate conformation due to a shielding of charges of functional groups and, under these conditions, intermo lecular interactions between the particles are also favored [35]. Statistical bearing analysis was performed on different samples, over the entire scan area, to evaluate the effect of the rinsing procedure on the configurat ion of adsorbed pedogenic HA structures and the format ion of adsorbed HA layers. On the rinsed surface it was also observed spheroids or isolated cone-shaped islands with smaller grain ( Figure  1b). This image shows individual HA structures and reveal that the molecules have a globular shape and are fairly homogenous distributed on the surface. Freshly cleaved mica surface was also scanned under similar conditions and a smooth and flat surface of mica was observed. The cone shape was partially caused by the pyramidal shape of the AFM tip [36]. For each samp le, five random positions were imaged, so me of them with different magnifications, so that the selected image is very representative.
An image of anthropogenic HA adsorbed on mica surface is shown in Figure 2. The adsorbed HA structures in both sample-treat ment methods (see Figure 2a and 2b) fo rmed islands with sub-micron size. In the unrinsed sample ( Figure  2a) continuous HA layers, in ring format, were observed. The islands of the unrinsed sample were significantly larger than those found in the rinsed ones. These homogeneous ring structures (see Figure 2c), formed at lo w salt concentration and low pH values, indicate evidences of supramolecular structures of HA in the natural environ ment and are strongly depending of the washing method. The quasi-continuous arrangements of the HA adsorbed on mica, before rinsing procedure, is a characteristic of supramolecu lar structures, which s mall part icles or mo lecules interact forming a large structure. If the structure was composed of isolated molecules, the rinsing procedure make either total removing of the HA from mica surface or keep the initial structure, with small changes. Figure 2b, obtained after the rinsing process, shows some spherical and aggregates of HA structures, with apparent colloidal diameters < 500 n m. The image of a mica surface is very smooth and the adsorption of HA on the mica is quite clear. The size and shape of the spherical colloids are in agreement with those observed in the Figure 1 and with reports from other research groups for similar HS [30,35]. However, neither ring structure nor hole in the midd le of the structure was found in these works. It should be noted that these shaped-ring structures were never observed in rinsed sample. This behavior can be due to two facts: (1) the air-drying process at room temperature was slow, possibly allowing lateral movement and stretching of the HA structures along the mica surface and (2) hydrophobicity effect. In the case 1, the adsorption of HA molecules tends to follow the orientation of the plates between the atomic layers of the mica surface. In these plates, the lowest interfacial energy could be maintained, and the interaction between the HA molecules and hydrophilic mica surface results in orientations that maximize the contact area at the solid/liquid interface [36].
However, the hydrophobic character (case 2) of humic material, represented by hydrophobic compounds (long alky l-chain alkanes, alkenes, fatty acids, sterols, terpenoids, and phenyl-alky l residues of lignin degradation), allows their self-association into supramo lecular structures separated fro m the water med iu m.
This behavior describes a modern structural description regards humic material as a supramolecular structure [37], constituted of relatively small b io-organic mo lecules self-assembled main ly by weak dispersive forces such as van der Waals force, π-π, CH-π and hydrogen bonds, building apparently large molecu lar chains. VA CP-MAS 13 C NMR spectra of pedogenic and anthropogenic HA, p resented in Figure 3, shows a similar features of soils HS [17,38], which can be observed in five regions of d istinct chemical shifts: C-alkyl   Figure 4b, the images illustrate a cut along the solid line shows the vertical profile. The sizes and shapes of the particles are not uniform for all the aggregates, which suggest that the aggregation of HA structures is optional and spontaneous. These homogeneous ring structures, formed in lo w pH values, again indicate evidences of supramolecu lar arrangements. In view to this, the results are apparently more consistent with the model of HA association of small mo lecules [37].
It is worth reminded that aggregation process of HS is still poorly understood [39]. These properties of HS still a topic of debate between HS scientists. Divergent descriptions of HS defended by its proponent have been reviewed by Clapp and Hayes [40,41]. The challenge of consolidating a fully ac-cepted structural model to HS remains. The macro mo lecular model [42], until recently well accepted, has been extensively questioned and a supramolecular model was proposed [9] and diverged yet [41,43]. Addit ional efforts must be created to have a definitive structural model fo r HS and spectroscopic and microscopic methods combined with other analytical tools to secure an important role in this endeavor.

Conclusions
AFM was successfully used to measure HA adsorption on a mica surface. The results gave evidence that structure formation in thin HA films can be caused by dewetting during film deposition, and solution physical-chemistry. Under the experimental conditions used in this study, the adsorbed molecules form ring-shaped aggregates with diameters on the scale of several tens of nanometers; smaller nanometer-scale rings present along the circu mference could potentially represent hydrophobic domains. The different shapes of HA under different conditions suggest a supramo lecular structure. The AFM data of the vertical dimension suggest smaller co llo ids; this might be due to a size selectivity of the adhesive forces and the attachment process to the mica, favoring smaller part icles. The data of the lateral dimension shows a larger spread (up to several 100 n m), which cannot be exp lained by the tip art ifact (∼40 n m). Additional studies to determine the changes in the HA structures as a function of solution conditions, is necessary. Other techniques such as small-angle X-ray scattering (SAXS), scanning electron microscopy (SEM ) and molecular dynamic may p rovide information important on HA of the Amazonian Dark Earths and already are being perfo rmed.