Functional Lignin Building Blocks: Reactive Vinyl Esters with Acrylic Acid.

Introducing vinyl groups onto the backbone of technical lignin provides an opportunity to create highly reactive renewable polymers suitable for radical polymerization. In this work, the chemical modification of softwood kraft lignin was pursued with etherification, followed by direct esterification with acrylic acid (AA). In the first step, phenolic hydroxyl and carboxylic acid groups were derivatized into aliphatic hydroxyl groups using ethylene carbonate and an alkaline catalyst. The lignin was subsequently fractionated using a downward precipitation method to recover lignin of defined molar mass and solubility. After recovery, the resulting material was then esterified with AA, resulting in lignin with vinyl functional groups. The first step resulted in approximately 90% of phenolic hydroxyl groups being converted into aliphatic hydroxyls, while the downward fractionation resulted in three samples of lignin with defined molar masses. For the esterification reaction, the weight ratio of reagents, reaction temperature, and reaction time were evaluated as factors that would influence the modification efficacy. 13C NMR spectroscopy analysis of lignin samples before and after esterification showed that the optimized reaction conditions could reach approximately 40% substitution of aliphatic hydroxyl groups. Both steps only used lignin and the modifying reagent (no solvent), with the possibility of recovery and reuse of the reagent by dilution and distillation. An additional second esterification step of the resulting lignin sample with acetic acid or propionic acid converted 90% of remaining hydroxyl groups into short-chain carbon aliphatic esters, making a hydrophobic material suitable for further copolymerization with synthetic hydrophobic monomers.

Revisiting the Molar Mass and Conformation of Derivatized Fractionated Softwood Kraft Lignin.

The limited utilization of reliable tools and standards for determination of the softwood kraft lignin molar mass and the corresponding molecular conformation hampers elucidation of the structure-property relationships of lignin. At issue, conventional size exclusion chromatography (SEC) is unable to robustly measure the molar mass because of a lack of calibration standards with a similar structure to lignin. In the present work, the determination of the absolute molar mass of acetylated technical lignin was revisited utilizing SEC combined with multi-angle light scattering with a band pass filter to suppress the fluorescence. Fractionated lignin isolated using sequential techniques of solvent and membrane methods was used to enhance the clarity of light-scattering profiles by narrowing the molar mass distribution of lignin fractions. Further information on the molecular conformation of derivatized samples was studied utilizing a differential viscometer, and chemical structures were identified by NMR spectroscopy analysis. Through the help of fractionation, intrinsic viscosity values were determined for the different fractions as a function of molecular weight cut-off membranes. The derivatized acetone-soluble lignin was found to possess a lower molecular weight and an extremely compact structure relative to the derivatized acetone-insoluble fraction based on a significantly lower "α" value in the Mark-Houwink-Sakurada plot (0.15 acetone-soluble vs 0.33 acetone-insoluble). The differences in geometry were supported by the linkage analysis from NMR showing the acetone-soluble part containing fewer native linkages. In both of these examples, kraft lignin behaved like a solid sphere, limiting the ability to provide entanglements between molecular chains. From this standpoint, macroscopic properties of lignin are justified with this knowledge of a dense and extremely compact structure.

Screening, cloning, enzymatic properties of a novel thermostable cellulase enzyme, and its potential application on water hyacinth utilization.

Cellulose is the cheapest, natural, renewable organic substance that is used as a carbon source in various fields. Water hyacinth, an aquatic plant rich in cellulose, is often used as a raw material in fuel production. However, natural cellulase can be hardly used in industrial production on account of its low thermal stability and activity. In this study, a metagenomic library was constructed. Then, a new cellulase gene, cel1029, was screened by Congo red staining and expressed in the prokaryotic system. Enzymatic properties of Cel1029 were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions, and salt solutions. Finally, its ability of degrading water hyacinth was identified and evaluated. Cel1029 displayed high homology with endoglucanase in the glycoside hydrolase family 5 (GH5) and had high stability across a broad temperature range. More than 86% of its enzymatic activities were retained between 4 and 60 °C after 24 h of incubation. Single-factor analysis and orthogonal design were further conducted to determine the optimal conditions for the highest reducing sugar yield of water hyacinth. Interestingly, Cel1029 efficiently transformed water hyacinth with a reducing sugar yield of 430.39 mg/g in 22 h. These findings may open the door for significant industrial applications of a novel GH5 cellulase (NCBI Reference Sequence: MK051001, Cel1029) and help identify more efficient methods to degrade cellulose-rich plants.

Anticorrosive epoxy coatings from direct epoxidation of bioethanol fractionated lignin.

The development of lignin-based anticorrosive epoxy coatings for steel protection is beneficial for both alleviating the fossil resource depletion and value-added utilization of lignin but remains a challenge due to the inherent heterogeneous structure of lignin. Here, we selectively extract the low molecular weight (MW) fraction of a crop residue-derived enzymatic hydrolysis lignin (EHL) through a bioethanol fractionation process and prepare epoxy resin by direct epoxidation of the bioethanol fractionated lignin (BFL). The coatings are then fabricated using 20-100 wt% of BFL-based epoxy resin (LEp) as the commercial epoxy resin substitute. The low MW and high p-hydroxyphenyl content of the BFL offer high solubility and good workability for BFL and LEp during epoxidation and coating production, respectively. Lignin-based coatings with 20-40 wt% LEp exhibit good adhesion property (5B) and superior corrosion resistance, compared to the commercial epoxy coating. Although coating with high LEp concentrations (i.e., 60-100 wt%) resulted in decreased adhesion strength, the coating with 100 wt% LEp still displayed corrosion protection performance comparable to that of the commercial epoxy coating. Overall, this study provides a simple and effective approach to converting lignin to epoxy resins for a wide variety of surface coating applications.

Discovery of lignin-transforming bacteria and enzymes in thermophilic environments using stable isotope probing.

Characterizing microorganisms and enzymes involved in lignin biodegradation in thermal ecosystems can identify thermostable biocatalysts. We integrated stable isotope probing (SIP), genome-resolved metagenomics, and enzyme characterization to investigate the degradation of high-molecular weight, 13C-ring-labeled synthetic lignin by microbial communities from moderately thermophilic hot spring sediment (52 °C) and a woody "hog fuel" pile (53 and 62 °C zones). 13C-Lignin degradation was monitored using IR-GCMS of 13CO2, and isotopic enrichment of DNA was measured with UHLPC-MS/MS. Assembly of 42 metagenomic libraries (72 Gb) yielded 344 contig bins, from which 125 draft genomes were produced. Fourteen genomes were significantly enriched with 13C from lignin, including genomes of Actinomycetes (Thermoleophilaceae, Solirubrobacteraceae, Rubrobacter sp.), Firmicutes (Kyrpidia sp., Alicyclobacillus sp.) and Gammaproteobacteria (Steroidobacteraceae). We employed multiple approaches to screen genomes for genes encoding putative ligninases and pathways for aromatic compound degradation. Our analysis identified several novel laccase-like multi-copper oxidase (LMCO) genes in 13C-enriched genomes. One of these LMCOs was heterologously expressed and shown to oxidize lignin model compounds and minimally transformed lignin. This study elucidated bacterial lignin depolymerization and mineralization in thermal ecosystems, establishing new possibilities for the efficient valorization of lignin at elevated temperature.

Genomics and metatranscriptomics of biogeochemical cycling and degradation of lignin-derived aromatic compounds in thermal swamp sediment.

Thermal swamps are unique ecosystems where geothermally warmed waters mix with decomposing woody biomass, hosting novel biogeochemical-cycling and lignin-degrading microbial consortia. Assembly of shotgun metagenome libraries resolved 351 distinct genomes from hot-spring (30-45 °C) and mesophilic (17 °C) sediments. Annotation of 39 refined draft genomes revealed metabolism consistent with oligotrophy, including pathways for degradation of aromatic compounds, such as syringate, vanillate, p-hydroxybenzoate, and phenol. Thermotolerant Burkholderiales, including Rubrivivax ssp., were implicated in diverse biogeochemical and aromatic transformations, highlighting their broad metabolic capacity. Lignin catabolism was further investigated using metatranscriptomics of sediment incubated with milled or Kraft lignin at 45 °C. Aromatic compounds were depleted from lignin-amended sediment over 148 h. The metatranscriptomic data revealed upregulation of des/lig genes predicted to specify the catabolism of syringate, vanillate, and phenolic oligomers in the sphingomonads Altererythrobacter ssp. and Novosphingobium ssp., as well as in the Burkholderiales genus, Rubrivivax. This study demonstrates how temperature structures biogeochemical cycling populations in a unique ecosystem, and combines community-level metagenomics with targeted metatranscriptomics to identify pathways with potential for bio-refinement of lignin-derived aromatic compounds. In addition, the diverse aromatic catabolic pathways of Altererythrobacter ssp. may serve as a source of thermotolerant enzymes for lignin valorization.

n-p Heterojunction of TiO2-NiO core-shell structure for efficient hydrogen generation and lignin photoreforming.

Hydrogen evolution from biomass photoreforming has been widely recognized as a promising strategy for relieving the pressure from energy crisis and environmental pollution, as it could generate sustainable H2 and value-added bioproducts simultaneously. Combining p-type semiconductors with n-type semiconductors to form n-p heterojunction is an effective strategy to improve the photocatalytic quantum efficiency by enhancing the separation of photogenerated electrons and holes, which could greatly facilitate the realization of such biomass photorefinery concept. However, the incompact contact between the n-type and p-type semiconductors often induces the aggregation of photogenerated electrons and holes. In this work, we design and synthesize an ultrafine n-p heterojunction TiO2-NiO core-shell structure to overcome the incompact contact in the n-p interface. When the n-p heterojunction photocatalysts are evaluated for photocatalytic water splitting and biomass lignin photoreforming respectively, the as-fabricated TiO2-NiO nanocomposite with 3.25% NiO demonstrates the highest hydrogen generation of 23.5 mmol h-1 g-1 from water splitting and H2 (0.45 mmol h-1 g-1) and CH4 (0.03 mmol h-1 g-1) cogeneration with reasonable amount of fatty acids (palmitic acid and stearic acid) production from lignin photoreforming. The excellent photocatalytic activity is ascribed to the synergistic effects of high crystallinity of TiO2 ultrafine nanoparticles, core-shell structure and n-p heterojunction with NiO nanoclusters. This present work demonstrates a simple and efficient method to fabricate ultrafine n-p heterojunction core-shell structure for noble-metal free catalyst for both water splitting and biomass photoreforming.

Alkaline sulfonation and thermomechanical pulping pretreatment of softwood chips and pellets to enhance enzymatic hydrolysis.

To assess the impact of alkalinity on sulfonation and the enzyme-mediated hydrolysis of softwood cellulose, Lodgepole pine chips were impregnated with 8% sodium sulfite and increasing loadings of sodium carbonate before thermomechanical pulping. It was apparent that alkali addition enhanced lignin sulfonation with an additional 4% loading of sodium carbonate proving optimal. TEM indicated that sulfonation predominantly occurred within the secondary-cell-wall lignin, increasing cellulose accessibility to the cellulase enzymes. Although increasing alkalinity did not significantly enhance lignin sulfonation, likely due to the lower acetyl content of the softwood chips, it increases mannan solubilization. Despite their smaller particle size, softwood pellets were more poorly sulfonated, probably due to their higher lignin content and lower amount of acid groups. This more condensed lignin structure was confirmed by 2D-NMR and GPC analyses which indicated that the EMAL derived from softwood pellets contained less native ß-O-4 linkages and had a higher molecular weight.

[Comparison of mechanical properties of three machinable resin ceramic composite materials].

PURPOSE: This in vitro study was to compare the flexural properties, fracture toughness and hardness of three machinable composite materials. METHODS: Three kinds of resin composite ceramic Upcera Hyramic, 3M Lava Ultimate, Vita Enamic and a glass ceramic Vitablocs Mark II were chosen for the study. Bar-shaped specimens (16 mm×4 mm×1 mm, 2 mm) were prepared for flexural strength experiment; specimens (17 mm×4 mm×3 mm) were prepared for fracture toughness experiment and specimens of 4 mm thickness were prepared for hardness test. Flexural test and fracture toughness experiment were performed with an universal testing machine at a cross-head speed of 0.5 mm/min. Hardness test was performed with an micro hardness tester.Scanning electron microscope was used to observe the roughness of fracture surface. One-way variance analysis was used to determine the statistical differences with SPSS 17.0 software package. RESULTS: The mean flexural strength of the tested blocks at 1 mm thickness was Hyramic（207.7515±13.12）MPa＞Vita Enamic（182.0286±15.18）MPa＞Lava Ultimate（145.8469±8.98）MPa＞Vitablocs MarkⅡ（103.0542±18.19）MPa. The mean flexural modulus were Vitablocs MarkⅡ（49.49±5.50）GPa＞Vita Enamic（40.65±3.80）GPa＞Hyramic（14.89±2.38）GPa＞Lava Ultimate（7.09±1.24）GPa. The mean flexural strength of the tested blocks at 2 mm thickness was Hyramic（208.1986±25.07）MPa＞Lava Ultimate（172.9297±12.73）MPa＞Vitablocs MarkⅡ（158.6587±15.37） MPa＞Vita Enamic（155.3670±13.77）MPa. The mean flexural modulus were Vitablocs MarkⅡ（24.07±1.86）GPa＞Vita Enamic（19.64±0.98）GPa＞Hyramic（10.35±0.87）GPa＞Lava Ultimate（8.68±0.86）GPa. The mean fracture toughness was Vita Enamic（1.6357±0.16）MPa·m1/2＞Lava Ultimate（1.4286±0.11）MPa·m1/2>Vitablocs MarkII（1.3233±0.10）MPa·m1/2>Hyramic（1.0614±0.09）MPa·m1/2. The hardness of the experimental group was significantly lower than that of the control group. CONCLUSIONS: According to ISO 6872/2008, three kinds of machinable resin ceramic composites meet the needs of clinical strength.Hyramic showed higher flexural strength at different thickness, it is an ideal material for dental restoration. Vita Enamic has not only higher flexural strength at the thickness of 1 mm, but also good toughness, it is suitable for repair of patients that have limited occlusal space and great bite force, named occlusal veneer.

[Effect of the thickness of reinforced glass ceramics on the degree of conversion of dual-cure resin cements].

PURPOSE: To determine the effect of the thickness of reinforced glass ceramics on the degree of conversion (DC) of three dual-cure resin cements (Multilink N, RelyX Ultimate and NX3-Nexus). METHODS: Upcera reinforced glass ceramics and IPS e.max CAD test specimen were processed, with different thickness, transmittance was tested by ultraviolet and visible spectrometer. The DC was evaluated using Fourier transform infrared (FT-IR) spectrometer, then the degree of conversion of polymerization before and after curing was calculated. SPSS 21.0 software package was used for data analysis. RESULTS: Transmittance decreased along with the thickness of reinforced glass ceramics increased. At 2 mm -thickness transmittance of upcera lithium disilicate glass ceramics was superior to IPS e.max CAD. The DC of dual-cured resin cement was decreased with the increase of thickness. At the same 2 mm group, the DC of Multilink N and RelyX Ultimate under upcera lithium disilicate glass ceramics were superior to that under IPS e.max CAD groups. CONCLUSIONS: At 2 mm-thickness transmittance of upcera lithium disilicate glass ceramics was superior to IPS e.max CAD. At the same 2 mm group, the DC of Multilink N and RelyX Ultimate under upcera lithium disilicate glass ceramics were superior to that under IPS e.max CAD groups.

Exome Sequencing and Gene Prioritization Correct Misdiagnosis in a Chinese Kindred with Familial Amyloid Polyneuropathy.

Inherited neuropathies show considerable heterogeneity in clinical manifestations and genetic etiologies, and are therefore often difficult to diagnose. Whole-exome sequencing (WES) has been widely adopted to make definite diagnosis of unclear conditions, with proven efficacy in optimizing patients' management. In this study, a large Chinese kindred segregating autosomal dominant polyneuropathy with incomplete penetrance was ascertained through a patient who was initially diagnosed as Charcot-Marie-Tooth disease. To investigate the genetic cause, forty-six living family members were genotyped by SNP microarrays, and one confirmed patient was subject to WES. Through systematic computational prioritization, we identified a missense mutation c.G148T in TTR gene which results in a p.V50L substitution known to cause transthyretin-related familial amyloid polyneuropathy. Co-segregation analysis and clinical follow-up confirmed the new diagnosis, which suggested new therapeutic options to the patients and informed high risk family members. This study confirms WES as a powerful tool in translational medicine, and further demostrates the practical utility of gene prioritization in narrowing the scope of causative mutation.