Pseudomonas bohemica strain ins3 eliminates antibacterial hinokitiol from its culture broth.

A bacterial strain, Pseudomonas bohemica strain ins3 was newly isolated as a resistant strain against high concentrations of hinokitiol. This strain was revealed not only to show resistance but also completely remove this compound from its culture broth. In addition, its mechanism was revealed to be independent of conventional aromatic dioxygenases, ie catechol-1,2- or 2,3-dioxygenases.

Direct deuteration of hinokitiol and its mechanistic study.

Hinokitiol has a broad antibacterial activity against bacteria and fungi. While its biosynthetic pathway has been intensively studied, its dynamics in natural environments, such as biodegradation pathway, remain unclear. In this study, the authors report a direct deuterium labeling of hinokitiol as a traceable molecular probe to serve those studies. Hinokitiol was subjected to the H2-Pd/C-D2O conditions and deuterated hinokitiol was obtained with excellent deuteration efficiencies and in moderate yield. The 1H and 2H NMR spectra indicated that all ring- and aliphatic hydrogens except that on C-6 were substituted by deuterium. According to the substrate scope and computational chemistry, deuteration on tropolone ring was suggested to proceed via D+-mediated process, and which was supported by the results of the experiment with trifluoroacetic acid and Pd(TPP)4. On the other hand, the deuteration on aliphatic group was predicted to be catalyzed by Pd(II) species.

Ligninolytic Activity at 0 °C of Fungi on Oak Leaves Under Snow Cover in a Mixed Forest in Japan.

Despite the importance of litter decomposition under snow cover in boreal forests and tundra, very little is known regarding the characteristics and functions of litter-decomposing fungi adapted to the cold climate. We investigated the decomposition of oak leaves in a heavy snowfall forest region of Japan. The rate of litter weight loss reached 26.5% during the snow cover period for 7 months and accounted for 64.6% of the annual loss (41.1%). Although no statistically significant lignin loss was detected, decolourization portions of oak leaf litter, which was attributable to the activities of ligninolytic fungi, were observed during snow cover period. This suggests that fungi involved in litter decomposition can produce extracellular enzymes to degrade lignin that remain active at 0 °C. Fungi were isolated from oak leaves collected from the forest floor under the snow layer. One hundred and sixty-six strains were isolated and classified into 33 operational taxonomic units (OTUs) based on culture characteristics and nuclear rDNA internal transcribed spacer (ITS) region sequences. To test the ability to degrade lignin, the production of extracellular phenoloxidases by isolates was quantified at 0 °C. Ten OTUs (9 Ascomycota and 1 Basidiomycota) of fungi exhibited mycelial growth and ligninolytic activity. These results suggested that some litter-decomposing fungi that had the potential to degrade lignin at 0 °C significantly contribute to litter decomposition under snow cover.

Effects of hemicelluloses on dehydrogenative polymerization of monolignols with cationic cell wall-bound peroxidase.

To elucidate the influence of polysaccharides on hardwood lignification, dehydrogenative polymerization of monolignols, coniferyl alcohol (CA) and sinapyl alcohol (SA), was attempted with recombinant cationic cell wall-bound peroxidase (rCWPO-C) and horseradish peroxidase (HRP) in measurement cells of a quartz crystal microbalance with dissipation (QCM-D). Hardwood cellulose nanofibers were anchored; hemicelluloses, xylan, partially acetylated xylan (AcXY), galactoglucomannan, and xyloglucan, and the enzymes were subsequently adsorbed onto the QCM-D sensor surface, enabling fabrication of artificial polysaccharide matrices. The largest amount of rCWPO-C is found to be adsorbed onto AcXY among all the polysaccharides, which affords the largest amount and size of spherical dehydrogenation polymers (DHPs) from both CA and SA. In contrast, no DHP and a small amount of DHPs are formed from SA and CA, respectively, by HRP catalysis in all of the polysaccharide matrices. This study demonstrates important functions of a real tree-derived peroxidase, rCWPO-C, and AcXY for hardwood lignification.

Fabrication of Graphitized Carbon Fibers from Fusible Lignin and Their Application in Supercapacitors.

Lignin-based carbon fibers (LCFs) with graphitized structures decorated on their surfaces were successfully prepared using the simultaneous catalyst loading and chemical stabilization of melt-spun lignin fibers, followed by quick carbonization functionalized as catalytic graphitization. This technique not only enables surficial graphitized LCF preparation at a relatively low temperature of 1200 °C but also avoids additional treatments used in conventional carbon fiber production. The LCFs were then used as electrode materials in a supercapacitor assembly. Electrochemical measurements confirmed that LCF-0.4, a sample with a relatively low specific surface area of 89.9 m2 g-1, exhibited the best electrochemical properties. The supercapacitor with LCF-0.4 had a specific capacitance of 10.7 F g-1 at 0.5 A g-1, a power density of 869.5 W kg-1, an energy density of 15.7 Wh kg-1, and a capacitance retention of 100% after 1500 cycles, even without activation.

Thermal mobility of ß-O-4-type artificial lignin.

Several lignin model polymers and their derivatives comprised exclusively of ß-O-4 or 8-O-4' interunitary linkages were synthesized to better understand the relation between the thermal mobility of lignin, in particular, thermal fusibility and its chemical structure; an area of critical importance with respect to the biorefining of woody biomass and the future forest products industry. The phenylethane (C6-C2)-type lignin model (polymer 1) exhibited thermal fusibility, transforming into the rubbery/liquid phase upon exposure to increasing temperature, whereas the phenylpropane (C6-C3)-type model (polymer 2) did not, forming a char at higher temperature. However, modifying the Cγ or 9-carbon in polymer 2 to the corresponding ethyl ester or acetate derivative imparted thermal fusibility into this previously infusible polymer. FT-IR analyses confirmed differences in hydrogen bonding between the two model lignins. Both polymers had weak intramolecular hydrogen bonds, but polymer 2 exhibited stronger intermolecular hydrogen bonding involving the Cγ-hydroxyl group. This intermolecular interaction is responsible for suppressing the thermal mobility of the C6-C3-type model, resulting in the observed infusibility and charring at high temperatures. In fact, the Cγ-hydroxyl group and the corresponding intermolecular hydrogen bonding interactions likely play a dominant role in the infusibility of most native lignins.

Synthesis of beta-O-4-type artificial lignin polymers and their analysis by NMR spectroscopy.

We describe the synthesis and NMR spectroscopic analysis of three artificial lignin polymers containing only the beta-O-4 substructure: syringyl-type homopolymer, p-hydroxyphenyl-type homopolymer and guaiacyl/syringyl-type heteropolymer. Using gel permeation chromatography, the weight-average degree of polymerization (DP(w)) of the three polymers was determined as 19.2, 38.6, and 13.9, respectively. The polymers were prepared based on the synthetic methodology of guaiacyl-type homopolymer, and were fully characterized using (1)H-, (13)C-, and (1)H-(13)C NMR spectroscopy of the acetylated and non-acetylated forms. The spectra of guaiacyl/syringyl-type heteropolymers were in good agreement with those of the beta-O-4 substructure of milled wood lignin obtained from the hardwood of Japanese white birch.

Low molecular weight lignin suppresses activation of NF-kappaB and HIV-1 promoter.

Human immunodeficiency virus type 1 (HIV-1) is a cytopathic retrovirus and the primary etiological agent of acquired immunodeficiency syndrome (AIDS) and related disorders. In cells chronically infected with HIV-1, nuclear factor-kappaB (NF-kappaB) activation by external stimuli such as tumor necrosis factor alpha (TNFalpha) augments replication of HIV-1. NF-kappaB involves in many diseases such as inflammation, cancer, and Crohn's disease. In this paper, we exhibit that (i) HIV-1gene expression was inhibited by lignin, (ii) fraction of small molecular mass in HBS lignin (less than 0.5kDa) had stronger inhibitory effects than large molecular mass (more than 1.3kDa), (iii) lignin also inhibited activation of NF-kappaB induced by TNFalpha, (iv) among six lignin dimer-like compounds, compound 6 containing beta-5 bond has more potent inhibitory activity than compounds 1, 2, 3, 4 and 5, which contain beta-1, beta-O-4, 5-5, or beta-beta structural units. These results suggested that small molecules of lignin inhibit HIV-1 replication through suppression of HIV-1 transcription from LTR including activation via NF-kappaB. Low molecular lignin may be a beneficial material or drug leads as a new class for AIDS and NF-kappaB-related diseases.

Optimization of simultaneous saccharification and fermentation conditions with amphipathic lignin derivatives for concentrated bioethanol production.

Amphipathic lignin derivatives (A-LDs) prepared from the black liquor of soda pulping of Japanese cedar are strong accelerators for bioethanol production under a fed-batch simultaneous enzymatic saccharification and fermentation (SSF) process. To improve the bioethanol production concentration, conditions such as reaction temperature, stirring program, and A-LDs loadings were optimized in both small scale and large scale fed-batch SSF. The fed-batch SSF in the presence of 3.0g/L A-LDs at 38°C gave the maximum ethanol production and a high enzyme recovery rate. Furthermore, a jar-fermenter equipped with a powerful mechanical stirrer was designed for 1.5L-scale fed-batch SSF to achieve rigorous mixing during high substrate loading. Finally, the 1.5L fed-batch SSF with a substrate loading of 30% (w/v) produced a high ethanol concentration of 87.9g/L in the presence of A-LDs under optimized conditions.

Production of mesoscopically patterned cellulose film.

Honeycomb and stripe patterned films were prepared from cellulose triacetate (CTA)/chloroform solution, as a result of the self-organization of the polymer during evaporation of the solvent. The honeycomb patterned CTA films were prepared by two methods, a direct pattern formation method and a transcription method. The latter method gave a well-organized microporous honeycomb pattern. Both types of patterned CTA films were saponified to yield the corresponding patterned cellulose films.

Dehydrogenative polymerization of coniferyl alcohol in artificial polysaccharides matrices: effects of xylan on the polymerization.

To elucidate the influence of wood polysaccharide components on lignin formation in vitro, models for polysaccharide matrix in wood secondary cell wall were fabricated from two types of bacterial cellulosic films, flat film (FBC) and honeycomb-patterned film (HPBC), as basic frameworks by depositing xylan onto the films. An endwise type of dehydrogenative polymerization, "Zutropfverfahren", of coniferyl alcohol was attempted in the films with/without xylan. The resultant dehydrogenation polymer (DHP) was generated inside and outside xylan-deposited films, whereas DHP was deposited only outside the films without xylan. The amount of the generated DHP in the xylan-deposited films was larger than that in the films without xylan. The frequency of 8-O-4' interunitary linkage in DHP was also increased by the xylan deposition. These results suggest that xylan acts as a scaffold for DHP deposition in polysaccharides matrix and as a structure regulator for the formation of the 8-O-4' linkage. In addition, mechanical properties, i.e., tensile strength and modulus of elasticity (MOE), of both cellulosic films were found to be augmented by the deposition of xylan and DHP. Especially, DHP deposition remarkably enhanced MOE. Such effects of xylan on DHP formation and augmentation of mechanical strength were clearly observed for HPBC, revealing that HPBC is a promising framework model to investigate wood cell wall formation in vitro.

Lignin gel with unique swelling property.

Lignin gels were prepared from acetic acid lignin by use of polyethylene glycol diglycidyl ether as cross-linker. The gels were found to swell in aqueous ethanol solution, in particular 50% (v/v) solution. In addition, they also swelled in alkaline solution and shrank upon heating. A literature search showed that investigation on gel swelling in aqueous ethanol has not been reported so far. Gels prepared from the cross-linker alone and its analogues did not show such swelling characteristics in aqueous ethanol. Therefore, the unique swelling property must be attributable to an intrinsic property of lignin.

Amphipathic lignin derivatives to accelerate simultaneous saccharification and fermentation of unbleached softwood pulp for bioethanol production.

Amphipathic lignin derivatives (A-LDs) were already demonstrated to improve enzymatic saccharification of lignocellulose. Based on this knowledge, two kinds of A-LDs prepared from black liquor of soda pulping of Japanese cedar were applied to a fed-batch simultaneous saccharification and fermentation (SSF) process for unbleached soda pulp of Japanese cedar to produce bioethanol. Both lignin derivatives slightly accelerated yeast fermentation of glucose but not inhibited it. In addition, ethanol yields based on the theoretical maximum ethanol production in the fed-batch SSF process was increased from 49% without A-LDs to 64% in the presence of A-LDs.

Influence of syringyl to guaiacyl ratio on the structure of natural and synthetic lignins.

Several kinds of natural woods and isolated lignins with various syringyl to guaiacyl (S/G) ratios were subjected to thioacidolysis followed by Raney nickel desulfuration to elucidate the relationships between the S/G ratio and the interunit linkage types of lignin. Furthermore, enzymatic dehydrogenation polymers (DHP) were produced by the Zutropf (gradual monolignol addition) method from mixtures of various ratios of coniferyl alcohol and sinapyl alcohol. The analysis of DHPs and natural wood lignins exhibited basically a similar tendency. The existence of both syringyl and guaiacyl units is effective for producing higher amounts of beta-O-4 and 4-O-5 structures, but it lowers the total amount of cinnamyl alcohol and aldehyde end groups. The relative frequency of the beta-beta structure increased, whereas that of beta-5 and 5-5 structures decreased with increasing syringyl units.

Chemical synthesis of beta-O-4 type artificial lignin.

An artificial lignin polymer containing only the beta-O-4 substructure was synthesized. The procedure consists of two key steps: 1) polycondensation of a brominated monomer by aromatic Williamson reaction; and 2) subsequent reduction of the carbonyl polymer. 13C-NMR and HMQC spectra of the polymer were consistent with beta-O-4 substructures in milled wood lignin isolated from Japanese fir wood. The weight average degree of polymerization (DP(w)) ranged from 19.5 to 30.6, which is comparable to enzymatically synthesized artificial lignin from p-hydroxycinnamyl alcohols (dehydrogenation polymer, DHP) and some isolated lignins. Using this new lignin model polymer, it will now be possible to reinvestigate the properties and reactivity of the main lignin structure in terms of its polymeric character.

Easy synthesis of beta-O-4 type lignin related polymers.

The beta-O-4 structure is the most abundant substructure in lignin. Lignin related polymers composed of only the beta-O-4 structure were prepared using simple aromatic compounds as starting materials. Acetophenone derivatives were brominated, polymerized in the presence of K2CO3 and reduced with NaBH4 to give the lignin related polymers. These are linear polymers which resemble natural lignins in their structures, although they do not have a gamma-hydroxymethyl group. The number average degree of polymerization (DPn) was determined with peracetate of the polymers by gel permeation chromatography. The DPn of guaiacyl type polymers ranged from 15.2-21.4, where the value for the syringyl type was 11.3 and for the p-hydroxyphenyl type 16.9. The Guaiacyl type polymer was very soluble in usual lignin solvents such as 1, 4-dioxane-water (96 : 4, v/v) and DMSO, but only slightly soluble in acetone-water (9 : 1, v/v).