Characterization of natural organic matter (NOM) derived from sewage sludge compost. Part 1: chemical and spectroscopic properties

In this study changes in the properties of natural organic matter (NOM) were studied during composting of sewage sludge in a laboratory experiment using the pile method. Typical physicochemical parameters were measured during 53 days of composting including humic fractions. The effects of humification on the molecular properties of humic acids (HA) were investigated by 13C CP/MAS NMR spectroscopy. On the basis of chemical analyses, 53 days of composting sewage sludge with structural material can be divided into three phases: (i) domination of rapid decomposition of non-humic, easily biodegradable organic matter (two to three weeks), (ii) domination of organic matter humification and formation of polycondensed, humic-like substances (the next two weeks), (iii) stabilization of transformed organic material and weak microbial activity. Spectroscopic characterization (13C NMR) of compost humic acids reveals changes in their structures during maturation. The changes are highly correlated with the processes taking place in bulk compost.     

Homogeneous modification of cellulose with succinic anhydride in ionic liquid using 4-dimethylaminopyridine as a catalyst

Cellulose, extracted from sugarcane bagasse, was successfully succinylated in ionic liquid 1-buty-3-methylimidazolium (BMIMCl) using 4-dimethylaminopyridine (DMAP) as a catalyst. Parameters investigated included the mass ratio of DMAP/succinic anhydride in a range from 0% to 15%, reaction time (from 30 to 120 min), reaction temperature (from 60 to 110 °C). The succinylated cellulosic derivatives had a degree of substitution (DS) ranging from 0.24 to 2.34. It was found that the DS of succinylated cellulosic derivatives using DMAP as a catalyst was higher than that without any catalyst under the same reaction conditions. The products were characterized by FT-IR, solid-state CP/MAS ¹³C NMR, and thermal analysis. FT-IR and solid-state CP/MAS ¹³C NMR spectra showed that succinoylation occurred at C-6, C-2 and C-3 positions. The thermal stability of the succinylated cellulose decreased upon chemical modification.     

A study of ordered structure in acid-modified tapioca starch by C CP/MAS solid-state NMR

Acid modification of tapioca starch earlier reported to increase the mechanical strength of tablets. The development of ordered structure (double helices) of these starches was monitored after equilibrating at 0.90 a_w (25 °C) using ¹³C CP/MAS NMR and X-ray diffraction. As the hydrolysis time increased, the intensity of the resonance for C1 and C4 amorphous fractions decreased while that for C1 and C4 double helix fractions increased. Relative crystallinity (%) obtained from ¹³C CP/MAS NMR and X-ray diffraction methods both increased sharply initially and then levelled off with hydrolysis time. The initial increase in relative double helix content and crystallinity was due to a hydrolytic destruction in the amorphous domain, retrogradation of the partially hydrolyzed amylose and crystallization of free amylopectin double helices. After 192 h, these two parameters were not significantly different (=0.05) indicating that the double helices that were not arranged into crystalline regions either had been hydrolyzed or crystallized.     

13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk-like fiber. The silk I structure has been recently resolved by (13)C solid-state NMR as a "repeated beta-turn type II structure." Here, we used (13)C solid-state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the (13)C CP/MAS NMR revealed a broad and asymmetric peak for the Ala Cbeta carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala-Gly-Ser-Gly-Ala-Gly sequences), 18% distorted beta-turn, 13% beta-sheet (parallel Ala residues), and 25% beta-sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr-rich region, 22% in both distorted beta-turn and distorted beta-sheet. Such a heterogeneous structure including distorted beta-turn can be observed for the peptides (AG)(n) (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala-Gly-Ser-Gly-Ala-Gly)(n) in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala Cbeta peak of (13)C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios.     

A 13C CP/MAS NMR spectroscopy and AFM study of the structure of Glucagel™, a gelling β-glucan from barley

The structure of Glucagel™, a mixed-linked (1→3), (1→4)-β-d-glucan extracted from barley, was examined using ¹³C CP/MAS NMR spectroscopy and atomic force microscopy (AFM). Results from ¹³C CP/MAS NMR spectroscopy showed that Glucagel™ contained regions with two distinct conformations. In some of the regions the β-glucan chains associated to form a unique conformation, the A-conformation, while in the other regions the β-glucan chains were in an amorphous conformation. Dilute solutions of Glucagel™ were prepared for imaging by dissolving Glucagel™ in water at 90 °C. If the dilute solution was immediately deposited onto mica and the surface dried, then no fine detail was seen in the AFM image. However, when dilute solutions of Glucagel™ were left for several days before being deposited onto the mica surface, individual fibres could be clearly imaged. These results suggested that in gels formed from Glucagel™, junction zones occur because of the interaction of two β-glucan chains in the A-conformation.     

Modification of wood with isopropyl glycidyl ether and its effects on decay resistance and light stability

China fir (Cunninghamia lanceolata var. lanceolata) and maple (Acer sp.) wood were etherified with isopropyl glycidyl ether and the decay resistance and light stability of the modified wood were assessed. CP/MAS (13)C NMR and FT-IR analyses indicated that new ether bonds containing isopropyl groups formed after reacting wood with isopropyl glycidyl ether. Modified wood samples were very resistant to decay when exposed to brown-rot fungus Laetiporus sulphureus or white-rot fungus Lenzites betulina for 60 days in the soil-block test. The isopropyl glycidyl ether treatment of wood was effective in decreasing formation of phenoxyl radicals upon UV irradiation and thus protecting wood from photodiscoloration.     

Structural analysis of silk with 13C NMR chemical shift contour plots.

The polymorphic structures of silk fibroins in the solid state were examined on the basis of a quantitative relationship between the 13C chemical shift and local structure in proteins. To determine this relationship, 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues, and the C alpha chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13C NMR chemical shift data in aqueous solution reported for 40 proteins. The 13C CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk fibroin in silk I and silk II forms were used along with 13C CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk fibroin in beta-sheet and alpha-helix forms for the structure analyses of silk fibroins. The allowed regions in the 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and alpha-helix, were determined using their 13C isotropic NMR chemical shifts in the solid state. There are two area of the phi,psi map which satisfy the observed Silk I chemical shift data for both the C alpha and C beta carbons of Ala and Ser residues in the 13C chemical shift contour plots.     

Cellulose synthesized by Acetobacter xylinum in the presence of multi-walled carbon nanotubes

The structure of bacterial cellulose is affected by the bacterial strain used, culture media and cultivation conditions. In this study, acid-treated multi-walled carbon nanotubes (MWNTs) were added into a static culture medium and their effect on bacterial cellulose structure was studied by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), CP/MAS (13)C NMR and X-ray diffractometry. The bacterial cellulose ribbons and the MWNTs interwound and formed a three-dimensional network architecture. Band-like assemblies with sharp bends and rigidity were also produced in the presence of MWNTs. The intermolecular hydrogen bonds in bacterial cellulose produced in the presence of MWNTs were weakened. The crystal structure, cellulose I(alpha) content, crystallinity index (CrI) and crystallite size all changed. The results may suggest that the acid-treated MWNTs containing hydroxyl groups interact with the sub-elementary bacterial cellulose fibrils, subsequently interfering with the aggregation and crystallization.     

Structure of 2-C-(hydroxymethyl)-D-ribose (hamamelose) in the solid-state analyzed by CP MAS NMR and X-ray crystallography

D-Hamamelose, a branched-chain ribose (2-C-(hydroxymethyl)-D-ribose), has been synthesized and its solid-state structure analyzed by (13)C CP MAS NMR spectra and X-ray data. The presence of the complex pattern of resonances in the anomeric region, as well as in the ring carbon region, in (13)C CP MAS NMR spectrum indicated that the mixture of four cyclic forms, alpha- and beta-furanoses, as well as both alpha- and beta-pyranoses were present in the solid-state. X-ray analysis of crystals showed that D-hamamelose belongs to the monoclinic system with unit cell: a=4.790A, b=8.671A, c=8.880A and beta=98.89 degrees , space group P2(1). The furanose ring has the (2)E conformation.     

Quantitation of aliphatic suberin in Quercus suber L. cork by FTIR spectroscopy and solid-state (13)C-NMR spectroscopy

This work determined that the percentage of suberin in cork may be found by solid-state (13)C cross polarization/magic angle spinning (CP/MAS) NMR spectroscopy and by FTIR with photoacoustic detection (FTIR-PAS) spectroscopy. A linear relationship is found between the suberin content measured through CP/MAS spectral areas and that measured gravimetrically. Furthermore, application of a partial least squares (PLS1) regression model to the NMR and gravimetric data sets clearly correlates the two sets, enabling suberin quantification with 90% precision. Suberin quantitation by FTIR-PAS spectroscopy is also achieved by a PLS1 regression model, giving 90% accurate estimates of the percentage of suberin in cork. Therefore, (13)C-CP/MAS NMR and FTIR-PAS proved to be useful and accurate noninvasive techniques to quantify suberin in cork, thus avoiding the traditional time consuming and destructive chemical methods.     

Isolation and characterization of chitin from bumblebee (Bombus terrestris)

Insect chitin possessing shell-like structure was prepared from the bumblebee corpses by a consequent treatment with 1M HCl and 1M NaOH. The bumblebee chitin was compared with crustacean (shrimp) chitin by using elemental analysis, Fourier-transform infrared (FT-IR) and solid-state (13)C cross-polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and confocal microscopy. Both chitins (bumblebee and shrimp) exhibited identical spectra, while the bumblebee chitin had a 5% lower degree of acetylation and was characterized by a fine membrane texture.     

Chemical and in vivo studies of interaction between cadmium and vitamin B6.

The interaction of vitamin B6 pyridoxine with cadmium acetate in ethanolic solution has been studied. The new compound Cd(PN-H)(OOCCH3) (PN-H = pyridoxinato anion) was isolated and its structure studied in the solid state by IR and 13C and 113Cd CP/MAS NMR spectroscopies. The effect of pyridoxine on survival rate among male Sprague rats injected intraperitoneally with 5 mg CdCl2.H2O/kg was also investigated. Vitamin treatment seems to increase (Protocol C) or does not affect the cadmium lethality. Although the analysis of the metal burden in some organs seems to suggest a light increase of the cadmium level in the liver, this change has no significance at a statistical level.     

Preparation of cellulose films from solution of bacterial cellulose in NMMO

Bacterial cellulose (BC) was dissolved in N-methylmorpholine N-oxide (NMMO) to prepare regenerated BC films (RBC) with phase inversion. The solubility of BC, supermolecule on structure, morphology, thermal and physical properties of the films were investigated by Fourier transform infrared spectroscopy (FT-IR), solid-state cross polarization/magic angle spinning ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR), wide-angle X-ray diffraction (WAXD), scanning electron microscope (SEM), and thermogravimetric analysis (TGA). The investigation suggested BC was dissolved completely in NMMO. From the C₆ signal shifts to the amorphous area, the crystallinity of materials decreased from 79.20% to 38.17%, and the transformation from cellulose I to II occurred. It was also found that the banded structure of the native materials was replaced by homogeneous and densified sections, so RBC films had better mechanical and barrier properties, and do thermal stability was similar to that of the native BC.     

Homogeneous modification of sugarcane bagasse cellulose with succinic anhydride using a ionic liquid as reaction medium

The homogeneous chemical modification of sugarcane bagasse cellulose with succinic anhydride using 1-allyl-3-methylimidazolium chloride (AmimCl) ionic liquid as a reaction medium was studied. Parameters investigated included the molar ratio of succinic anhydride/anhydroglucose units in cellulose in a range from 2:1 to 14:1, reaction time (from 30 to 160min), and reaction temperature (between 60 and 110 degrees C). The succinylated cellulosic derivatives were prepared with a low degree of substitution (DS) ranging from 0.071 to 0.22. The results showed that the increase of reaction temperature, molar ratio of SA/AGU in cellulose, and reaction time led to an increase in DS of cellulose samples. The products were characterized by FT-IR and solid-state CP/MAS (13)C NMR spectroscopy, and thermal analysis. It was found that the crystallinity of the cellulose was completely disrupted in the ionic liquid system under the conditions given. The data also demonstrated that homogeneous modification of cellulose with succinic anhydride in AmimCl resulted in the production of cellulosic monoester. The thermal stability of the succinylated cellulose decreased upon chemical modification.     

Effects of backbone and side chain on the molecular environments of chiral cavities in polysaccharide-based biopolymers

The effects of the backbone and side chain on the molecular environments in the chiral cavities of three commercially important polysaccharide-based chiral sorbents--cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC), amylose tris(3,5-dimethylphenylcarbamate) (ADMPC), and amylose tris[(S)-alpha-methylbenzylcarbamate] (ASMBC)--are studied by attenuated total reflection infrared spectroscopy (ATR-IR), X-ray diffraction (XRD), 13C cross-polarization/magic-angle spinning (CP/MAS) and MAS solid-state NMR, and density functional theory (DFT) modeling. These sorbents are used widely in preparative-scale chiral separations. ATR-IR is used to determine how the H-bonding states of the C=O and NH groups of the polymer depend on the backbone and side chain. The changes in the polymer crystallinity are characterized with XRD. The changes in the polymer helicity and molecular mobility for polymer-coated silica beads (commercially called Chiralcel OD, Chirapak AD, and Chiralpak AS) are probed with 13C CP/MAS and MAS solid-state NMR. The IR wavenumbers and the NMR chemical shifts for the polymer backbone monomers and dimers and the side chains are predicted at the DFT/B3LYP/6-311+g(d,p) level of theory. It is concluded that the molecular environments of the C=O, NH, and phenyl groups show significant differences in intramolecular and intermolecular interactions and in the nanostructures of the chiral cavities of these biopolymers. These results have implications for understanding how the molecular environments of chiral cavities of these polymers affect their molecular recognition mechanisms.     

Thermal, dynamic and structural properties of drug AT1 antagonist olmesartan in lipid bilayers

It is proposed that AT1 antagonists (ARBs) exert their biological action by inserting into the lipid membrane and then diffuse to the active site of AT1 receptor. Thus, lipid bilayers are expected to be actively involved and play a critical role in drug action. For this reason, the thermal, dynamic and structural effects of olmesartan alone and together with cholesterol were studied using differential scanning calorimetry (DSC), 13C magic-angle spinning (MAS) nuclear magnetic resonance (NMR), cross-polarization (CP) MAS NMR, and Raman spectroscopy as well as small- and wide angle X-ray scattering (SAXS and WAXS) on dipalmitoyl-phosphatidylcholine (DPPC) multilamellar vesicles. 13C CP/MAS spectra provided direct evidence for the incorporation of olmesartan and cholesterol in lipid bilayers. Raman and X-ray data revealed how both molecules modify the bilayer's properties. Olmesartan locates itself at the head-group region and upper segment of the lipid bilayers as 13C CP/MAS spectra show that its presence causes significant chemical shift changes mainly in the A ring of the steroidal part of cholesterol. The influence of olmesartan on DPPC/cholesterol bilayers is less pronounced. Although, olmesartan and cholesterol are residing at the same region of the lipid bilayers, due to their different sizes, display distinct impacts on the bilayer's properties. Cholesterol broadens significantly the main transition, abolishes the pre-transition, and decreases the membrane fluidity above the main transition. Olmesartan is the only so far studied ARB that increases the gauche:trans ratio in the liquid crystalline phase. These significant differences of olmesartan may in part explain its distinct pharmacological profile.     

Direct dissolution of cellulose in NaOH/thiourea/urea aqueous solution

Untreated cellulose was directly and quickly dissolved in NaOH/thiourea/urea aqueous solution. The mechanism of dissolution was investigated by SEM, WXRD and (13)C NMR. The components of this solvent cannot dissolve cellulose on their own, and the interactions between NaOH and urea, as well as between NaOH and thiourea, play an important role in improving the dissolution of cellulose. Moreover, (13)C NMR spectra proved that NaOH, thiourea, and urea were bound to cellulose molecules, which brings cellulose molecules into aqueous solution to a certain extent and prevents cellulose macromolecules from associating. (13)C NMR spectra of the cellulose solution show that this novel mixture is a direct solvent. Optical microscopy and CP MAS (13)C NMR were used to study the process of dissolution. The results reveal that cellulose is dissolved completely and that cellulose I (cotton linter) first changes to amorphous cellulose chains in solution, and then to cellulose II during regeneration. Moreover, a new, more effective dissolution method is proposed, as confirmed by dynamic rheology measurements.     

High-resolution solid-state 13C NMR of the LH1 from Rhodospirillum rubrum

High-resolution solid-state 13C NMR spectra of the light-harvesting antenna complex (LH1) from Rhodospirillum rubrum were observed for the first time by cross-polarization (CP), magic angle spinning (MAS) methods with a total elimination of spinning side band technique (TOSS). Chemical shift analysis of the CP/MAS/TOSS 13C NMR spectra confirmed that the LH1 consists mainly of -helices in the solid state. Time constants of cross polarization (TCH) and relaxation time T1 in a rotating frame (T1H) were determined from the experiments at various contact times. Smaller values of TCH were obtained for the carbons attached directly with protons in a rigid state. Relaxation times T1H revealed the dynamic structure of the complex and showed that bacteriochlorophyll a in the LH1 has high internal mobility even in the solid state. The proton spin-lattice relaxation time in a laboratory frame (T1H) determined by the 13C NMR signal amplitude changes suggested that protons in the LH1 proteins have such strong interaction among them that the spins of all protons in the protein can diffuse through spin-lattice-relaxation.     

Celery (Apium graveolens) parenchyma cell walls: cell walls with minimal xyloglucan

The primary walls of celery ( Apium graveolens L.) parenchyma cells were isolated and their polysaccharide components characterized by glycosyl linkage analysis, cross-polarization magic-angle spinning solid-state ¹³C nuclear magnetic resonance (CP/MAS ¹³C NMR) and X-ray diffraction. Glycosyl linkage analysis showed that the cell walls consisted of mainly cellulose (43 mol%) and pectic polysaccharides (51 mol%), comprising rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). The amounts of xyloglucan (2 mol%) and xylan (2 mol%) detected in the cell walls were strikingly low. The small amount of xyloglucan present means that it cannot coat the cellulose microfibrils. Solid-state ¹³C NMR signals were consistent with the constituents identified by glycosyl linkage analysis and allowed the walls to be divided into three domains, based on the rigidity of the polymers. Cellulose (rigid) and rhamnogalacturonan (semi-mobile) polymers responded to the CP/MAS ¹³C NMR pulse sequence and were distinguished by differences in proton spin relaxation time constants. The arabinans, the most mobile polymers, responded to single-pulse excitation (SPE), but not CP/MAS ¹³C NMR. From solid-state ¹³C NMR of the cell walls the diameter of the crystalline cellulose microfibrils was determined to be approximately 3 nm while X-ray diffraction of the cell walls gave a value for the diameter of approximately 2 nm.     

Use of 13C conformation-dependent chemical shifts to elucidate the local structure of a large protein with homologous domains in solution and solid state

In order to clarify the difference between solution NMR and X-ray diffraction analyses concerning the presence of alpha-helical structure in protein A, the 13C conformation-dependent chemical shifts of the 13C-labeled carbonyl carbons for selectively labeled protein A were used. In the 13C CP/MAS NMR spectra, the higher-field shifts of the carbonyl carbons of 13C-labeled Thr and Val residues compared with the random coil chemical shifts both in solution and solid state imply the presence of the third helix in the polypeptide chain, in contrast to the crystal structure of Fc-bound B-domain. Thus, a combination of selective isotope labeling and conformation-dependent chemical shifts will be a good Indicator to monitor the local structure of homologous protein in solution and solid state.