Sluggish and Ion-Resilient Behavior of Interfacial Aqueous Layer on Single-Layer Graphene Oxide: Insights from In Situ Atomic Force Microscopy.

Understanding interfacial interactions of graphene oxide (GO) is important to evaluate its colloidal behavior and environmental fate. Single-layer GO is the fundamental unit of GO colloids, and its interfacial aqueous layers critically dictate these interfacial interactions. However, conventional techniques like X-ray diffraction are limited to multilayer systems and are inapplicable to single-layer GO. Therefore, our study employed atomic force microscopy to precisely observe the in situ dynamic behaviors of interfacial aqueous layers on single-layer GO. The interfacial aqueous layer height was detected at the subnanometer level. In real-time monitoring, the single-layer height increased from 1.17 to 1.70 nm within 3 h immersion. This sluggish process is different from the rapid equilibration of multilayer GO in previous studies, underscoring a gradual transition in hydration kinetics. Ion strength exhibited negligible influence on the single-layer height, suggesting a resilient response of the interfacial aqueous layer to ion-related perturbations due to intricate ion interactions and electrical double-layer compression. Humic acid led to a substantial increase in the interfacial aqueous layers, improving the colloidal stability of GO and augmenting its potential for migration. These findings hold considerable significance regarding the environmental behaviors of the GO interfacial aqueous layer in ion- and organic-rich water and soil.

Direct observation of humic acid-promoted hydrolysis of phytate through stabilizing a conserved catalytic domain in phytase.

As a potential phosphorus (P) pool, the enzymatic hydrolysis of organic phosphorus (Po) is of fundamental importance due to the release of bioavailable inorganic phosphate (Pi) for agronomic P sustainability. However, little is known about the role of soil organic matter (SOM) in the hydrolysis process of phytate by phytase and the subsequent chemical behaviors involving the hydrolysis product (Pi) at different soil interfaces. Here, by using liquid-cell atomic force microscopy (AFM), we present a model system to in situ quantify the nucleation kinetics of phytase-released Pi when precipitating with representative soil multivalent cations (Ca2+/Fe3+) on typical soil mineral/organic interfaces in the presence/absence of humic acid (HA), which involves complex phytase-interface-HA interactions. We observed that a higher HA concentration resulted in a faster nucleation rate of amorphous calcium/iron phosphate (ACP/AIP) on bare and organically-coated (-OH/-COOH) mica surfaces compared with the HA-free control. Besides, the nucleation rate of ACP/AIP induced by organic interfaces was much more significant than that induced by clay mineral interfaces. By combining enzyme activity/stability experiments and AFM-based PeakForce quantitative nanomechanical mapping (PF-QNM) measurements, we directly quantified the contribution of noncovalent phytase-HA interaction to the increase in enzymatic activity from complex phytase-interface-HA interactions. Furthermore, the direct complexation of phytase-HA resulted in the stabilization of a conserved active catalytic domain (ACD) in phytase through the enhanced formation of both an ordered, stereochemically-favored catalytic domain and an unordered non-catalytic domain, which was revealed by Raman secondary structure determination. The results provide direct insights into how HA regulates the catalytic activity of phytase controlling Po fates and how soil interfaces determine the behaviors of released Pi to affect its availability, and thereby contribute to P sustainability in soils.

Molecular mechanisms for the humic acid-enhanced formation of the ordered secondary structure of a conserved catalytic domain in phytase.

Changes in the secondary structure of phytase, particularly the conserved active catalytic domain (ACD, SRHGVRAPHD) are extremely important for the varied catalytic activity during hydrolyzing phytate in the presence of humic acid (HA). However, little is known about the molecular-scale mechanisms of how HA influences the secondary structure of ACD found in phytase. First, in situ surface-enhanced Raman spectroscopy (SERS) results show the secondary structure transformation of ACD from the unordered random coil to the ordered ß-sheet structure after treatment with HA. Then, we use an atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) technique that can in situ directly probe the single-molecule interaction of ACD with HA and underlying changes in ACD secondary structure in the approach-retraction cycles in real time. Based on the SMFS results, we further detect the HA-enhanced formation of H-bonding between amide groups in the ACD backbone after noncovalently interacting with HA in the absence of phytate. Following the addition of phytate, the calculated contour length (Lc) and the free energies (ΔGb) of functional groups within ACD(-1/2) binding to mica/HA collectively demonstrate the formation of the organized intermediate structural state of ACD following its covalent binding to phytate. These spectroscopic and single-molecule determinations provide the molecular-scale understanding regarding the detailed mechanisms of HA-enhancement of the ordered ß-sheet secondary structure of ACD through chemical functionalities in ACD noncovalently interacting with HA. Therefore, we suggest that similar studies of the interactions of other soil enzymes and plant nutrients may reveal predominant roles of dissolved organic matter (DOM) in controlling elemental cycling and fate for sustainable agriculture development.

Molecular Understanding of Humic Acid-Limited Phosphate Precipitation and Transformation.

Phosphorus (P) availability is widely assumed to be limited by the formation of metal (Ca, Fe, or Al) phosphate precipitates that are modulated by soil organic matter (SOM), but the SOM-precipitate interactions remain uncertain because of their environmental complexities. Here, we present a model system by quantifying the in situ nanoscale nucleation kinetics of calcium phosphates (Ca-Ps) on mica in environmentally relevant aqueous solutions by liquid-cell atomic force microscopy. We find that Ca-P precipitate formation is slower when humic acid (HA) concentration is higher. High-resolution transmission electron microscopy observations demonstrate that HA strongly stabilizes amorphous calcium phosphate (ACP), delaying its subsequent transformation to thermodynamically more stable phases. Consistent with the formation of molecular organo-mineral bonding, dynamic force spectroscopy measurements display larger binding energies of organic ligands with certain chemical functionalities on HA to the initially formed ACP than to mica that are responsible for stabilization of ACP through stronger HA-ACP interactions. Our results provide direct evidence for the proposed importance of SOM in inhibiting Ca-P precipitation/transformation. We suggest that similar studies of binding strength in SOM-Fe/Al-P may reveal how both organic matter and metal ions control P availability and fate, and thus the eventual P management for agronomical and environmental sustainability.