Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance Research Articles

The Limitation of Unproductive Binding of Cellulases to Lignin by Ozone Pretreatment

The limitation of enzymatic saccharification of lignocellulose is attributed to the nonproductive adsorption between lignin and cellulase. This study aims to investigate the effects of ozone pretreatment on the physical structure and chemical properties of milled wood lignin (MWL). The objective is to reduce the non-productive adsorption of cellulase on lignin. The structure–activity relationship between the physical structure of MWL and the occurrence of nonproductive adsorption was analysed using two-dimensional heteronuclear single quantum coherence–nuclear magnetic resonance (HSQC-NMR) and phosphorus-31 nuclear magnetic resonance spectrum (31P-NMR), etc. The results indicate that ozone pretreatment resulted in a decrease in the phenolic hydroxyl content and S/G ratio, an increase in the carboxyl content, and a negative zeta potential of MWL. The maximum adsorption capacity decreased from 25.77 mg/g to 10.09 mg/g, the Langmuir constant decreased from 13.86 mL/mg to 10.11 mL/mg, and the binding strength decreased from 357.14 mL/g to 102.04 mL/g, as determined by Langmuir isothermal adsorption. This suggests that ozone pretreatment resulted in a reduction in the hydrophobicity of lignin and a weakening of the electrostatic attraction between lignin and cellulase, thereby effectively reducing the non-productive adsorption of cellulase on lignin. This study provides an environmentally friendly pretreatment technique and comprehensively analyses the structural changes of ozone-treated MWL. These findings contribute to a deeper understanding of the interaction between lignin and cellulase.

Application of 15N-Edited 1H-13C Correlation NMR Spectroscopy─Toward Fragment-Based Metabolite Identification and Screening via HCN Constructs.

Many key building blocks of life contain nitrogen moieties. Despite the prevalence of nitrogen-containing metabolites in nature, 15N nuclei are seldom used in NMR-based metabolite assignment due to their low natural abundance and lack of comprehensive chemical shift databases. However, with advancements in isotope labeling strategies, 13C and 15N enriched metabolites are becoming more common in metabolomic studies. Simple multidimensional nuclear magnetic resonance (NMR) experiments that correlate 1H and 15N via single bond 1JNH or multiple bond 2-3JNH couplings using heteronuclear single quantum coherence (HSQC) or heteronuclear multiple bond coherence are well established and routinely applied for structure elucidation. However, a 1H-15N correlation spectrum of a metabolite mixture can be difficult to deconvolute, due to the lack of a 15N specific database. In order to bridge this gap, we present here a broadband 15N-edited 1H-13C HSQC NMR experiment that targets metabolites containing 15N moieties. Through this approach, nitrogen-containing metabolites, such as amino acids, nucleotide bases, and nucleosides, are identified based on their 13C, 1H, and 15N chemical shift information. This approach was tested and validated using a [15N, 13C] enriched Daphnia magna (water flea) metabolite extract, where the number of clearly resolved 15N-containing peaks increased from only 11 in a standard HSQC to 51 in the 15N-edited HSQC, and the number of obscured peaks decreased from 59 to just 7. The approach complements the current repertoire of NMR techniques for mixture deconvolution and holds considerable potential for targeted metabolite NMR in 15N, 13C enriched systems.

1H-13C HSQC-NMR Analysis of Cranberry (Vaccinium Macrocarpon) Juice Defines the Chemical Composition of Juice Precipitate.

Shelf-stable cranberry juice precipitate has not been well characterized. Here, we describe using 1H-13C heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) spectroscopy for cranberry juice analysis, focusing on proanthocyanidins and the precipitate. HSQC-NMR cross-peaks from juices were categorized as aliphatic, olefinic, aromatic, carbohydrate backbone, or anomeric signals. An average cranberry juice precipitate had significantly more aromatic and significantly less carbohydrate backbone signals than an average supernatant. The precipitate was a collection of biomolecules held together by a mix of weak and strong intermolecular forces. Proanthocyanidin signals from precipitates of juices showed 22 ± 2 to 29.9 ± 0.7% A-type interflavan linkages and 34 ± 2 to 48 ± 3% of flavan-3-ol units with trans stereochemistry between the C2 and C3 positions. Based on this work, 1H-13C HSQC-NMR is useful to analyze cranberry juice and reveals the complex chemical nature of components in the soluble and insoluble phases.

Nanolignin-based high internal phase emulsions for efficient protection of curcumin against UV degradation

As an emulsifier, lignin exhibits excellent UV resistance on drug-loaded emulsion systems for drug delivery. However, due to the structural variation and complexity of lignins from various origins, their UV shielding performance varies with the techniques for lignin extraction, which impacts properties and the protection efficiency of lignin-based HIPEs (high internal phase emulsions). In this work, lignin nanoparticles, prepared from three lignin preparations of Eucalyptus, were used in HIPEs delivery systems to protect curcumin from degradation by UV radiation. Structures of the lignin preparations were characterized using 2D HSQC (heteronuclear single-quantum coherence) NMR (nuclear magnetic resonance), 31P NMR, and GPC (gel permeation chromatography). The residual curcumin level after 36 h UV exposure in the nanolignin-based HIPEs was over 72 %, much higher than that (< 10 % after 24 h UV exposure) in the oil phase without lignin, indicating that the nanolignin-based HIPEs with enhanced UV shielding ability protect curcumin better. Of the three lignin preparations, AL (alkali lignin), with the lowest molecular weight, highest contents of phenolic hydroxyl and carboxyl groups, and highest S/G ratio, displayed the best anti-UV radiation ability and the most uniform nanoparticle size.

Metabolomic approaches for the detection of Listeria monocytogenes and Staphylococcus aureus in culture media

This study aimed to determine the changes in metabolites in the culture media of foodborne pathogens using nuclear magnetic resonance (NMR) spectroscopy. Extracellular metabolites in tryptic soy broth inoculated with none, Listeria monocytogenes ATCC 19111, and Staphylococcus aureus ATCC 29213 and 27213 at 37 °C were collected and analyzed by 1D 1H NMR and 2D 1H–13C heteronuclear single quantum coherence (HSQC) NMR. Partial least squares discriminant analysis revealed an excellent variable influence on projection (VIP) score and predictive ability among the different culture media metabolites. Based on presence/absence, five extracellular metabolites (cadaverine, histidine, nicotinate, putrescine, and succinate) were only detected in culture media containing pathogens. According to the pathogen species, L. monocytogenes and S. aureus ATCC 29213 could be distinguished based on changes in 11 metabolites (tyrosine, betaine, glycine, pyroglutamate, fumarate, alanine, putrescine, arginine, inosine, threonine, and sucrose). Moreover, different strains of S. aureus (ATCC 29213 and 27213) were distinguished using hypoxanthine and putrescine. Overall, extracellular metabolites in culture media could be used to detect the presence/absence, species, and even different strains of foodborne pathogens.

Study the effect of zeolite pore size and acidity on the catalytic pyrolysis of Kraft lignin

In this study, three zeolites with different pore sizes and acidity, H-Beta, HY and HZSM-5, were used for the preparation of bio-oil by catalytic pyrolysis of Kraft lignin at 600 °C. The three-dimensional sizes and kinetic diameters of the bio-oil components were calculated using quantum chemical calculations, and the bio-oil was analyzed by heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR). The effects of zeolite acidity and pore size on the bio-oil upgrading process were studied for the pyrolysis products of Kraft lignin. The results showed that optimum pore size of the zeolites ranged from 6.500 Å–8.400 Å during the catalyzed pyrolysis of lignin. Among the zeolites, the H-Beta zeolite is the most effective in the lignin pyrolysis. The results also showed that the pore size and acidity of the zeolites had a significant effect on the product yields, coking and deoxygenation. The pore size affects the coking and dehydration reactions, while the acidity can promote the rearrangement of ether bonds or the breaking of lignin side chains and deoxygenation reactions. This study would be beneficial to improve the zeolite catalytic activity and minimize the oxygen content in bio-oil produced from the pyrolysis of Kraft lignin.

HSQC-NMR analysis of bamboo (Phyllostachys nigra)-cultured cell lignin produced under different phytohormone conditions

Abstract Bamboo-cultured cells (BCCs) were produced under three phytohormone conditions. BCC lignin was then isolated and characterized by heteronuclear single-quantum coherence-nuclear magnetic resonance (HSQC-NMR) analysis. HSQC-NMR analysis revealed that all three BCC lignin samples were composed of guaiacyl (G), syringyl (S), oxidized syringyl (S′), and p-hydroxyphenyl (H) units. p-Coumaric acid (pCA) and ferulic acid (FA) were identified as well. Main lignin substructures, including β-O-4, β-5, and β-β, were also detected. However, β-O-4/α-O-4, spirodienone, dibenzodioxocin, or tricin structures were absent in the BCC lignin. The BCC lignin contained higher proportions of H, FA, and β-5 structures, but less proportions of S, S′, and β-O-4 structures than the mature bamboo lignin. The removal of auxin 2,4-dichlorophenoxyacetic acid (2,4-D) from the subculture medium promoted G unit formations. Nevertheless, it suppressed H and pCA unit formations. Cytokinin 6-benzyladenine (BA) promoted H and β-β structure formations as well but suppressed β-O-4 formations than in the mature bamboo and BCC lignin produced under phytohormone free conditions.

1H-13C heteronuclear single quantum coherence NMR evidence for iodination of natural organic matter influencing organo-iodine mobility in the environment

The complex biogeochemical behavior of iodine (I) isotopes and their interaction with natural organic matter (NOM) pose a challenge for transport models. Here, we present results from iodination experiments with humic acid (HA) and fulvic acid (FA) using 1H-13C heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy. Even though not a quantitative approach, 1H-13C HSQC NMR corroborated that iodination of NOM occurs primarily through aromatic electrophilic substitution of proton by I, and also revealed how iodination chemically alters HA and FA in a manner that potentially affects the mobility of iodinated NOM in the environment. Three types of iodination experiments were conducted with HA and FA: a) non-enzymatic iodination by IO3− (pH 3) and I− (pH 4 and 7), b) addition of lactoperoxidase to promote I−-iodination in the presence of the co-substrate, H2O2 (pH 7), and c) addition of laccase for facilitating I−-iodination in the presence of O2, with or without a mediator (pH 4). When mediators or H2O2 were present, extracellular oxidases and peroxidases enhanced I− incorporation into NOM by between 54% and 3400%. Iodination of HA, which was less than that of FA, enhanced HA's stability (inferred from increases in aliphatic compounds, decreases in carbohydrate moieties, and thus increased molecular hydrophobicity) yet reduced HA's tendency to incorporate more iodine. As such, HA is expected to act more as a sink for iodine in the environment. In contrast, iodination of FA appeared to generate additional iodine binding sites, which resulted in greater iodine uptake capability and enhanced mobility (inferred from decreases in aliphatic compounds, increases in carbohydrates, and thus decreases in molecular hydrophobicity). These results indicate that certain NOM moieties may enhance while others may inhibit radioiodine mobility in the aqueous environment.

Chemical Structures of Adhesive and Interphase Parts in Sucrose/Citric Acid Type Adhesive Wood-Based Molding Derived from Japanese Cedar (Cryptomeria japonica).

In sucrose/citric acid based wood adhesive, the detailed bonding mechanism has still been unknown. Here, we investigated the detailed chemical structures of this adhesive wood (Japanese cedar)-based molding by using heteronuclear single quantum coherence–nuclear magnetic resonance (HSQC-NMR). NMR peaks associated with the furan-type structure appeared, suggesting that the furan compound was formed from sucrose and converted to a furan polymer during the adhesive process and that some of the furan structures in the polymers were ester-bonded with citric acid. The secondary forces between the furan polymers and wood components were thought to contribute to the adhesive effect. In our analysis of the interphase structure, primary hydroxyl groups of both polysaccharides and of lignin substructures were found to be esterified with citric acid. Additionally, some of the glycosidic bonds in polysaccharides were cleaved during the acidic condition produced by citric acid. The above results provided evidence of the polymerization of sucrose-derived 5-HMF, the esterification of wood components, and the degradation of polysaccharides during the molding process. Citric acid functioned as a clamp between the obtained furan polymer and the wood components. The sucrose/citric acid based wood adhesive can be defined as a hybrid-type wood adhesive, involving both secondary forces and chemical bonding interactions.

Demethylation, phenolation, and depolymerization of lignin for the synthesis of lignin-based epoxy resin via a one-pot strategy

A one-pot method to synthesize demethylated-phenolated lignin (DPL) and demethylated-phenolated lignin-based epoxy resin (DPLINP) using phenol as a phenolic reagent and polymerization inhibitor was proposed in this study. This method avoids the occurrence of polycondensation caused by demethylation and simplifies the synthesis of lignin-based epoxy resins. Phenolated lignin (PL)-based epoxy resin (PLINP) and demethylated lignin (DL)-based epoxy resin (DLINP) were prepared to investigate the effects of different modification methods on the resulting lignin and epoxy resin. The results of 1H nuclear magnetic resonance (NMR), Fourier transform infrared, and gel permeation chromatography analyses indicated that the introduction of phenol could significantly reduce the occurrence of polycondensation while also decreasing the molecular weight and increasing the content of phenolic hydroxyl in DPL compared with DL and PL. 2D heteronuclear single quantum coherence NMR analysis was used to further investigate the structural changes of lignin during the modification process. The results obtained via dynamic mechanical analysis, flexural test, and impact test demonstrated that demethylated-phenolated lignin-based epoxy network (DPLIEN) possessed an improved cross-linking density, glass transition temperature, and mechanical performance compared with all other epoxy resins. Moreover, the polycondensation of DL caused by demethylation deteriorated the mechanical performance of demethylated lignin-based epoxy network (DLIEN). The thermal stability of DPLIEN was decreased compared with phenolated lignin-based epoxy network (PLIEN), as the removal of methoxy groups may have reduced the protective char layer formation in DPLIEN. This study provides a novel method for the modification of lignin and synthesis of lignin-based epoxy resins.

Synthesis and characterization of an isocyanate-terminated hyperbranched polymer and its waterborne study

An isocyanate-terminated hyperbranched polymer (HBI) was successfully synthesized by reacting hyperbranched polyester (BoltornTM HB-20) with isophorone diisocyanate (IPDI). Gel permeation chromatography (GPC) was used to monitor the molecular weight and its distribution changes during the synthesis. The structure and composition of IPDI were characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The structure of HBI was confirmed by one-dimensional 1H, 13C NMR, and distortionless enhancement by polarization transfer (DEPT) NMR spectroscopy as well as two-dimensional heteronuclear multiple bond coherence (2D-HMBC) and heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Furthermore, HBI was reacted with methoxypolyethylene glycols (MPEG) to obtain a water-dispersible polyisocyanate with hyperbranched structure.

Bond Structures between Wood Components and Citric Acid in Wood-Based Molding.

Citric acid-based wood adhesive is considered a chemical-bonding wood adhesive. However, the detailed structures of the bonds between wood components and citric acid remain unknown. Here, we examine the chemical bonding structures between citric acid and wood by heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) analysis of wood-based molding using Japanese cedar (Cryptomeria japonica) and citric acid. In the HSQC-NMR spectrum of the wood molding, some esterified C/H correlation peaks appeared. The primary hydroxyl groups of polysaccharides, such as cellulose and galactoglucomannan, and the primary hydroxyl groups of the β-O-4 and β-5 substructures in lignin were found to be esterified with citric acid. In contrast, the secondary hydroxyl groups, except for xylan, barely reacted because of the steric hindrance. Additionally, the C/H correlation peak volumes of the reducing ends of mannan and xylan in the anomeric region increased after molding. It was clarified that the glycosidic bonds in the hemicelluloses were cleaved under the acidic molding condition with citric acid. The HSQC-NMR analysis revealed that the esterification of hemicellulose and lignin, and degradation of hemicellulose, proceeded under the molding condition. These results will promote understanding of the adhesive mechanism of citric acid-based wood adhesive and of the properties of the molding.

Extraction of High-Purity Lignins via Catalyst-free Organosolv Pulping from Low-Input Crops.

A catalyst-free organosolv pulping process was applied to cup plant (Silphium perfoliatum, S), Miscanthus grass (Miscanthus x giganteus, M), and the Paulownia tree (Paulownia tomentosa, P), resulting in high-purity lignins with no signals for cellulose, hemicellulose, or other impurities in two-dimensional heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra. Different biomass particle sizes used for the organosolv pulping (1.6-2.0 mm (1); 0.5-1.0 mm (2); <0.25 mm (3)) influenced the molecular weight and chemical structure of the isolated lignins. Principal component analysis (PCA) of 1H NMR data revealed a high intergroup variance of Miscanthus and Paulownia lignins, separating the small particle fraction from the larger ones. Furthermore, monolignol ratios identified via HSQC NMR differ significantly: Miscanthus lignins were composed of all three monolignols (guaiacyl (G), p-hydroxyphenyl (H), syringyl (S)), while for Paulownia and Silphium lignins only G and S units were observed (except for P3).

Study on the chemical modification of alkali lignin towards for cellulase adsorbent application

The research of cost-efficient lignin-based adsorbents is a practical strategy for the recovery of cellulase. In this study, alkali lignin was modified to increase the phenolic hydroxyl (Ph-OH) content for cellulase adsorption applications. After phenolation, compared with the lignin reference, the maximum adsorption cellulase capacity of lignoresorcinol (LigR) and lignopyrogallol (LigP) was improved from 76.5 mg/g to 842.1 mg/g and 911.4 mg/g, respectively. The enzyme activity of the adsorbed cellulase on LigR was higher than that on LigP, which could migrate to the fresh substrates during enzymatic hydrolysis. The adsorbed cellulase could be easily recovered from two lignin-based adsorbents by adjusting pH. The distinct cellulase adsorption behavior of two lignin-based adsorbents was closely related to the high Ph-OH contents and low S/G ratio in phenolated lignin samples characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Heteronuclear Single Quantum Coherence-Nuclear Magnetic Resonance (HSQC-NMR).

Enhanced Softwood Cellulose Accessibility by H3PO4 Pretreatment: High Sugar Yield without Compromising Lignin Integrity

Softwood lignocellulose is a potential feedstock for the production of biofuels and bioproducts. However, the highly cross-linked nature of softwood lignocellulose restricts enzyme access to its sugars. Thus, harsh pretreatment conditions (180-280 °C) and/or high enzyme loading are required to unlock sugars. These requirements negatively affect the economic viability of softwoods in biorefineries. Here we show that HPO pretreatment of pine and Douglas fir under a mild reaction temperature (50 °C) and atmospheric pressure enabled a high (∼80%) glucan digestibility with low enzyme loading (5 filter paper units (FPU)/g glucan). The dissolution and regeneration of softwoods disrupted the hydrogen bonding between cellulose chains, thereby increasing the cellulose accessibility to cellulase (CAC) values by ∼38-fold (from ∼0.4 to 15 m/g biomass). Examination of HPO-pretreated softwoods by cross-polarization/magic angle spin (CP/MAS), C- nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR) revealed that breaking of the orderly hydrogen bonding of crystalline cellulose caused the increase in CAC (higher than 11 m/g biomass), which, in turn, was responsible for the high glucan digestibility of pretreated softwoods. The HPO pretreatment process was feedstock independent. Lastly, 2D C-H heteronuclear single quantum coherence (HSQC) NMR showed that the lignin was depolymerized but not condensed; thus, the lignin can be available for producing high-value products.

Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR.

The Focal Adhesion Targeting (FAT) domain of Focal Adhesion Kinase (FAK) is a promising drug target since FAK is overexpressed in many malignancies and promotes cancer cell metastasis. The FAT domain serves as a scaffolding protein, and its interaction with the protein paxillin localizes FAK to focal adhesions. Various studies have highlighted the importance of FAT-paxillin binding in tumor growth, cell invasion, and metastasis. Targeting this interaction through high-throughput screening (HTS) provides a challenge due to the large and complex binding interface. In this report, we describe a novel approach to targeting FAT through fragment-based drug discovery (FBDD). We developed two fragment-based screening assays—a primary SPR assay and a secondary heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) assay. For SPR, we designed an AviTag construct, optimized SPR buffer conditions, and created mutant controls. For NMR, resonance backbone assignments of the human FAT domain were obtained for the HSQC assay. A 189-compound fragment library from Enamine was screened through our primary SPR assay to demonstrate the feasibility of a FAT-FBDD pipeline, with 19 initial hit compounds. A final total of 11 validated hits were identified after secondary screening on NMR. This screening pipeline is the first FBDD screen of the FAT domain reported and represents a valid method for further drug discovery efforts on this difficult target.

Assessing the potential of quantitative 2D HSQC NMR in 13C enriched living organisms.

In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D 1H-13C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo. This study represents a simple pilot study that compares two of the most popular quantitative 2D HSQC approaches to determine if quantitative results can be directly obtained in vivo in isotopically enriched Daphnia magna (water flea). The results show the perfect-HSQC experiment performs very well in vivo, but the decoupling scheme used is critical for accurate quantitation. An improved decoupling approach derived using optimal control theory is presented here that improves the accuracy of metabolite concentrations that can be extracted in vivo down to micromolar concentrations. When combined with 2D Electronic Reference To access In vivo Concentrations (ERETIC) protocols, the protocol allows for the direct extraction of in vivo metabolite concentrations without the use of internal standards that can be detrimental to living organisms. Extracting absolute metabolic concentrations in vivo is an important first step and should, for example, be important for the parameterization as well as the validation of metabolic flux models in the future.

Establishing lignin structure-upgradeability relationships using quantitative 1H-13C heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) spectroscopy.

Lignin depolymerization could provide an attractive renewable aromatic feedstock for the chemical industry. Past studies have suggested that lignin structural features such as ether content are correlated to lignin's upgradeability. An obstacle to the development of a conclusive causal relationship between lignin structure and upgradeability has been the difficulty to quantitatively measure lignin structural features. Here, we demonstrated that a modified HSQC-NMR method known as HSQC0 can accurately quantify lignin functionalities in extracted lignin using several synthetic polymer models. We then prepared a range of isolated lignin samples with a wide range of ether contents (6-46%). By using a simple ether cleavage model, we were able to predict final depolymerization yields very accurately (<4% error), conclusively demonstrating the direct causal relationship between ether content and lignin activity. The accuracy of this model suggests that, unlike in native lignin, ether linkages no longer appear to be randomly distributed in isolated lignin.

Separation of Benzoic and Unconjugated Acidic Components of Leonardite Humic Material Using Sequential Solid-Phase Extraction at Different pH Values as Revealed by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Correlation Nuclear Magnetic Resonance Spectroscopy.

Here, we report on sequential solid-phase extraction of leonardite hymatomelanic acid (CHM) on a non-ionic sorbent at four steadily lowered pH values: 7, 5, 3, and 2, yielding fractions with different acidic properties. Using nuclear magnetic resonance (NMR) spectroscopy and ultrahigh-resolution mass spectrometry, we revealed a gradual shift of dominating scaffolds in the fractions of CHM from reduced saturated to oxidized aromatic compounds. An increase on the average aromaticity of the CHM fractions was accompanied by a red shift in fluorescence spectra. These results were supported by heteronuclear single quantum coherence and heteronuclear multiple bond correlation NMR experiments. We have demonstrated that the CHM fraction isolated at pH 5 was dominated by aliphatic carboxyl carriers, while the pH 3 fraction was dominated by aromatic carboxyl acids. The developed fractionation technique will enable deeper insight on structure-property relationships and the design of the humic-based materials with tailored reactive properties.

Modified Hydrotropic Pretreatment of Eucalyptus under Alkali and Acid Conditions for Lignin Removal and Enhancing Enzymatic Hydrolysis

Hydrotropic pretreatment is an environmentally friendly technology that can be applied for lignin isolation from lignocellulosic biomass to enhance enzymatic hydrolysis. In this study, conventional hydrotropic pretreatment was modified with additional alkali and acid to investigate the lignin removal mechanism. Lignin recovered from the hydrotropic solution was analyzed by Heteronuclear Single Quantum Coherence-Nuclear Magnetic Resonance (HSQC-NMR). It was found that the amounts of β-O-4 linkages in the different types of hydrotropic lignin were more than 50%. Alkali-hydrotropic lignin particularly contained 74.9% of β-O-4 linkages. Also, the cleavage of α-O-4 formed more phenolic groups in alkali-hydrotropic lignin. The addition of acid in the hydrotropic treatment could reinforce the broken β-O-4 bond. Modified hydrotropic pretreatments more easily displaced the lignin from the fiber surface and improved the enzymatic accessibility of the pretreated substrates. Considering other impact factors including the fiber structure and total lignin content, enzymatic hydrolysis was heavily influenced and facilitated by the acid-hydrotropic method.