Reduced complexity of multidimensional and diffusion NMR spectra of soil humic fractions as simplified by Humeomics

Background: Humeomics is a sequential step-wise chemical fractionation that simplifies the complex matrix of a humic acid (HA) and weakens its supramolecular interactions, thereby allowing a detailed characterization of the involved molecules. A recalcitrant residual end product of Humeomics, namely RES4, was successfully solubilized here in alkaline conditions and subjected to a semi-preparative high-performance size exclusion chromatography (HPSEC). Results: The resulting six size fractions separated by HPSEC were analyzed by different NMR techniques. 1D 1H-NMR spectra did not reveal significant molecular differences among size fractions, although all of them differed from the spectrum of the bulk RES4 especially in signal intensity for aliphatic materials, which were assigned by 2D NMR to lipidic structures. Diffusion-ordered spectroscopy (DOSY)-NMR spectra showed that the homogeneity of RES4 was significantly changed by the HPSEC separation. In fact, nominally large size fractions, rich in lipidic signals, had significantly lower and almost constant diffusivity, due to stable supramolecular associations promoted by hydrophobic interactions among alkyl chains. Conversely, diffusivity is gradually increased with the content of aromatic and hydroxyaliphatic signals, which accompanied the reduction of fractions sizes and was related to smaller superstructures. Conclusions: This study not only confirmed the occurrence of supramolecular structures in the recalcitrant humic residue of Humeomics, but also highlighted that more homogeneous size fractions were more easily characterized by NMR spectroscopy

Optimized procedure for the determination of P species in soil by liquid-state 31 P-NMR spectroscopy

Liquid-state 31P-NMR spectroscopy becomes progressively an important role for studying phosphorus (P) dynamics in soil. Soils of different origin and organic matter content were used to optimize sample preparation and re-dissolution procedures to improve characterization of P species in soil by 31P-NMR spectroscopy. The efficiency of P extraction from an untreated fresh soil was compared to that from freeze-dried and air-dried soil samples. A freeze-drying pretreatment not only provided the greatest extraction yields of total and organic P from both farmland and forest soils but also enhanced the intensity of signals for inorganic and organic P species in 31P-NMR spectra, except for polyphosphates. Re-dissolution of freeze-dried soil extracts in relatively dilute alkaline solution and addition of a small aliquot of concentrated HCl to the NMR tube prior to analysis improved the quality of NMR spectra. Finally, the visibility of relatively weak P signals, such as for phosphorus diesters, phosphonates, polyphosphate, phospholipids, and DNA were reproducibly enhanced when 31P-NMR spectra were generated after at least 15 h of acquisition time. The optimized procedure presented here ensured the greatest detectability of inorganic and organic P species by liquid-state P-NMR spectroscopy in soil extracts

The Wine: typicality or mere diversity? The effect of spontaneous fermentations and biotic factors on the characteristics of wine

Wine is probably one of the main fermented beverages for which the recognition of the \u201cterritoriality\u201d is fundamental for its appreciation. The sensory profile of wine is significantly affected by microbial activities, and indigenous microorganisms may significantly contribute to the expression of wine typicality. The microbial ecology of wines is complex and includes several species and strains of yeasts, bacteria and molds. Several works showed the positive effects of spontaneous fermentations on the quality of wine as a consequence of the growth of different species and/or strains together at high levels. Furthermore, a new style of \u201cnatural\u201d winemaking is gaining importance, since the resulting wines are obtained thanks to the action of spontaneous autochthonous agents and the use of chemical addition is not allowed. In this contest, natural winemaking could provide enhanced opportunities for products with unique characters and popularly recognized as typical. The present work reports on microbial ecology and molecular profile characterizing natural large-scale vinifications, and an innovative procedure, named \u201cfortified pied de cuve\u201d, to accelerate the alcoholic fermentation performed spontaneously is also reported. Furthermore, this work reports on how the biotic factors, such as migratory birds, contribute in disseminating of winerelated yeasts over long distances, opening up new fields of research that will allow to unravel connection between wine and environmental factors

Effects of post cure treatment in the glass transformation range on the structure and fire behavior of in situ generated siilica/epoxy hybrids

A new "in situ” sol–gel synthesis procedure was exploited to produce silica/epoxy nanocomposites with 6 wt.% maximum silica content. 3-Aminopropyltriethoxysilane (APTS) was used as a coupling agent. The experimental results (fourier- transform infrared spectroscopy, FTIR, small-angle X-ray scattering, SAXS, transmission electron microscopy, TEM, nuclear magnetic resonance, NMR, and dynamic mechanical analysis, DMA) support that the structure consists of nanosized silica particles (maximum 1.25 nm in size) embedded in a hybrid co-continuous network. A post cure non-isothermal heating from 15 to 100 °C (beyond the Tg of the neat epoxy) caused Tg and storage modulus to increase. The fire behavior, that, owing to severe regulations (i.e., in aerospace engineering), often prevents composites applications, was also studied. The formed silica domains prevented melt dripping phenomena during vertical flame spread tests. Cone calorimetry tests showed a remarkable decrease of the heat release rate (HRR) for all the hybrid systems with respect to the neat cured resin, even at very low silica loadings (i.e., 2 wt.%). This decrease was much more pronounced for the hybrid structures that were not subjected to the post cure thermal treatment. The use of multiple structural investigation techniques allowed to choose among multiple hypothesis and conclude that nanoparticles clustering is the main reason of the effects of the post curing treatments

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters

NMR evaluation of the interactions between humic substances and three molecules of agrochemical interest: beta-D-glucosidase and alkaline phosphatase enzymes and glyphosate.

This doctoral thesis presents results concerning the investigation, by liquid-state Nuclear Magnetic Resonance (NMR) technique, on interactions occurring among humic substances and three molecules largely diffused in soil: the herbicide glyphosate, the enzymes beta-D-Glucosidase and Alkaline Phosphatase. These agrochemical molecules were examined as a function of the increasing addition of humic substances. In detail, glyphosate was treated with both humic and fulvic acids (pH 5 and 7.2); the alkaline phosphatase with two different humic acids (pH 10.4) and beta-D-Glucosidase with fulvic acids (pH 5 and 7.2). All studied molecules interacted with employed humic substances and the complex formations were mainly driven by weak forces. These results were initially supported by the observation of resonance broadening and/ or chemical shift drift spectroscopic phenomena, as a function of the humic concentration. Then, by analyzing both the molecular spin relaxation and correlation times as well as the self-diffusion constants, significant and diagnostic variations emerged, thus, corroborating the hypothesis of the occurred interactions. Moreover, only in the case of glyphosate, it was possible to apply the saturation transfer difference (STD) NMR experiment and, by means of diffusion results, the fraction of herbicide bond to humic matter was calculated. Instead, concerning the soil enzymes, the attention was focused on a further purpose that consisted in researching possible modifications, due to humic interactions, affecting the enzymatic catalytic activity. Therefore, a progressive inhibition of the activity was detected in liquid-phase, as a function of both the molecular concentration and composition of humic substances. Finally, aiming to better reproduce a more realistic soil condition, the enzymatic inhibition, due to the interactions with natural organic matter, was further studied in liquid-solid phase. In fact, by preliminary experiments, beta-D-Glucosidase was immobilized on OH-Al-humate-montmorillonite complexes and a significant inhibition was again observed as well as its extents were more enhanced than the one detected for pure liquid catalysis