Lignin has to be degraded before the molecular composi- tion of its phenolic components can be analyzed. The most common method for the degradation of lignin is alkaline ox- idation with cupric oxide (CuO), developed by Hedges and Parker in 1976. This method releases a number of pheno- lic acids, aldehydes, and ketones, which can be divided into four groups: the vanillyl group (V) consisting of vanillic acid, vanillin, and acetovanillone; the syringyl group (S) con- sisting of syringic acid, syringaldehyde, and acetosyringone; the cinnamyl group (C) consisting of trans-ferulic acid and p-coumaric acid; and the p-hydroxyl group (P) consisting of p-hydroxybenzoic acid, p-hydroxybenzaldehyde, and p- hydroxyacetophenone. Hedges and Mann (1979) analyzed fresh plant tissues and showed that the phenols of the sy- ringyl group are only obtained from angiosperm, but not from gymnosperm plant tissues. Likewise, the phenols of the cinnamyl group are only obtained from nonwoody and not from woody plant tissues, whereas the phenols of the vanil- lyl group are found in all kinds of vascular plant tissues (an- giosperm and gymnosperm, woody and nonwoody). These results led to the introduction of the lignin oxidation product (LOP) parameters C / V and S / V, for which C, for exam- ple, is defined as the sum of all lignin oxidation products of the C group (Hedges and Mann, 1979). The phenols of the p-hydroxyl group can originate from gymnosperm and non- woody angiosperm plant tissues, but are also oxidation prod- ucts of protein-rich organisms such as bacteria and plankton. Therefore, the P group is not used in the parameters to de- termine the source of lignin (Jex et al., 2014). The parameter 6 8 gives the sum of the eight analytes of the C, S, and V
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The second hypothesis (Figure 7B) is that the SpMnSOD enzymes are able to further reduce peroxide to hydroxyl radical, which is the reactive oxidant to attach lignin. It is reported that bovine erythrocyte Cu/Zn superoxide dismutase can generate hydroxyl radical, but that E. coli MnSOD, which in our hands shows very little lignin oxidation activity, does not generate hydroxyl radical. 35 The observation of oxidation products containing additional phenolic hydroxyl groups (e.g. products 3, 6, 7) is consistent with the known ability of hydroxyl radical to carry out phenolic hydroxylation, 36 hence this appears to be a possible mechanism for lignin oxidation via these enzymes. We note that Nature uses hydroxyl radical to attack lignin in a different context: brown rot fungi utilise Fenton chemistry to generate hydroxyl radical to attack lignin. 37,38 There are also literature reports of the production of hydroxyl radical in white rot fungus Phanerochaete chrysosporium, 39-41 though subsequent data implied that this is not a major contributing mechanism in white-rot fungal lignin degradation. 42
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The nitrated lignin assay data shows considerable variation in the rates of microbial lignin oxidation (see Fig 1), with rates of up to 5-fold higher than mean values in certain cases. There appears to be selectivity for certain bacterial supernatants for certain types of wheat lignin, implying some form of molecular recognition of structural features of the lignin. The different bacteria tested show different behaviour in the assays: Sphingobacterium sp. T2 showed approximately 10-fold higher activity than other strains, as observed previously (Taylor et al 2012), The high activity of this strain has been found to be due to the presence of two extracellular manganese superoxide dismutase enzymes with activity for lignin oxidation (Rashid et al 2015). Supernatants from Sphingobacterium sp. T2, S. viridosporus and M. phyllosphaerae generally showed higher activity in the
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Abstract. Lignin oxidation products, oxygen uptake rates, molar organic carbon to nitrogen (OC/N) ratio (from bulk elemental analysis) and Rp values (from loss on ignition ex- periments, the ratio of the refractory to total organic matter, OM) were determined for sediments along transects of Loch Creran and Loch Etive. Lignin data indicated the importance of riverine inputs contributing to land-derived carbon in the lochs as total lignin (3, mg/100 mg organic carbon, OC) de- creased from 0.69 to 0.45 and 0.70 to 0.29 from the head to outside of Loch Creran and Loch Etive, respectively. In addition, significant correlations of lignin content against to- tal OM and OC (p<0.05) also suggested a distinct contribu- tion of terrestrial OM to carbon pools in the lochs. The gen- eral trend of decreasing oxygen uptake rates from the head (20.8 mmole m −2 day −1 ) to mouth (9.4 mmole m −2 day −1 ) of Loch Creran indicates decomposition of OM. Biodegrad- ability of the sedimentary OM was also characterized by the increase of Rp values from the head to mouth of the lochs: 0.40 to 0.80 in Loch Etive and 0.43 to 0.63 in Loch Creran. Furthermore, the molar OC/N ratio decreased from 11.2 to 6.4 in Loch Creran, and from 17.5 to 8.2 in Loch Etive. De- rived rate constants for OM degradation were found to de- crease from LC0 to LC1, and increase from RE5 to RE6. This work demonstrates that oxygen uptake rates, Rp values and molar OC/N ratio are able to serve as useful proxies to indicate the biodegradability of sedimentary OM.
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jostii RHA1 DypB was first bacterial enzyme to be identified to have activity towards polymeric lignin, a property thought to be held by fungal lignin peroxidases . This enzyme was found to show Mn(II) oxidation activity, which was required for oxidation of polymeric lignin . A multifunctional dye-decolorizing peroxidase Dyp2 from Amycolatopsis sp 75iv2 has also been reported to show activity for oxidation of lignin model compounds, and shows much higher Mn(II) oxidation activity than other bacterial DyPs . Amongst Gram-negative bacteria, strains of Pseudomonas have shown activity for lignin oxidation, and a peroxidase Dyp1B from Pseudomonas fluorescens Pf-5 has been identified, that shows activity for oxidation of phenolic substrates and, in the presence of Mn(II), polymeric lignin . Uniquely, this enzyme releases an oxidized lignin dimer product from treatment of wheat straw lignocellulose in the presence of Mn(II) . Bacterial DyP-type peroxidases therefore show great potential as biocatalysts for conversion of lignin from industrial processes such as pulp/paper manufacture and biofuel production into renewable chemicals [7,8].
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The mechanism of lignin oxidation was confi med to be elu- cidated by the amount of dioxygen consumption, which was measured by a dioxygen fl owmeter in this study. The obser- vation that the dioxygen consumption by 200 g of kraft lignin and by guaiacol or vanillyl alcohol was similar has led us to postulate that phenolic and nonphenolic moieties in kraft lignin are extensively oxidized. This postulatoin was sup- ported by the dioxygen consumption by 0.5 moles of GG (an equivalent of aromatic unit), which was larger than the diox- ygen consumption of 1 mole of guaiacol or vanillyl alcohol. The most plausible mechanism for the extensive oxidation of nonphenolic moieties in kraft lignin and GG is the oxidative cleavage of alkyl–aryl ethers by active oxygen species, a process that converts nonphenolics into phenolic moieties. Acknowledgments The authors gratefully acknowledge fi nancial support from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [Grant-in-Aid for Young Scientists (B), No.17780136].
The aromatic heteropolymer lignin constitutes 15-30% of the lignocellulose cell wall in plant biomass, and represents an abundant renewable raw material for conversion via either biocatalysis or chemocatalysis into aromatic chemicals. The microbial oxidation of lignin has been studied extensively in white-rot fungi, which produce extracellular lignin peroxidase and laccase enzymes for lignin oxidation [1, 2], but recently bacterial enzymes for lignin oxidation have also been discovered in some soil bacteria [3, 4]. Dye decolorizing peroxidases DypB in Rhodococcus jostii RHA1 , Dyp2 in Amycolatopsis sp. 75iv2  and Dyp1B in Pseudomonas fluorescens  have been shown to be active for oxidation of polymeric lignin and lignin model compounds, and Lin et al have reported that a B-type dyp gene in P. putida A514 is overexpressed 68-fold in the presence of alkali Kraft lignin . On the other hand, laccases are a group of enzymes that can oxidise a huge range of substrates without any additional cofactor, producing water as the only byproduct , making them a group of enzymes with a huge potential for scientific and industrial applications.
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Therefore, this study has been conducted to serve the purpose on improving and maximizing the yield of vanillin isolated from biomass sources. Besides of considering the raw material selection, the chemicals used as the oxidant and precipitating agent particularly for lignin oxidation and extraction processes were taken into account. Hence, high selective chemicals such as nitrobenzene oxidant for lignin oxidation process have been used to increase the yield of vanillin. Part of that, the crucial factors of lignin oxidation such as oxidation temperature, oxidation time and volume of oxidant that might affect the production yield of vanillin were also optimized and studied.
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Our study focuses predominantly on hardwood lignin oxidation using DDQ. Important new insights into the oxidation of the b – b and lignin-bound Hibbert's ketones (LBHK) units, as well as the reactivity of the b –O-4 linkage are presented. As is shown below, the material we refer to as lignin a OX can vary consid- erably in structure depending on the details of the oxidation protocol. It seems likely that the structure of lignin a OX will have a signicant knock-on eﬀect on the ability to produce aromatic monomers. We also show that DDQ-mediated oxida- tion of lignin is general across a range of hardwoods and can be tuned to alter the solubility of the residual lignin aer the removal of an initial set of aromatic monomers. This is of importance as one vision of the ideal use of lignin involves a series of carefully controlled depolymerisation steps that ultimately results in the stepwise production of a number of diﬀerent aromatic compounds in pure form. The overall aim of this work has therefore been to dene better what is meant by lignin a OX in the hope, ultimately, of facilitating the valor- isation of this important renewable resource.
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CELPB15 was isolated from planetary ball-milled wood after 15 hours of milling. The extractable lignin yield was 86%, leaving the residual lignin (RELPB15) as insoluble residue after 96% dioxane extraction. Thus, RELPB15 represents only 14% of the original lignin in wood. The structure of RELPB15 is of particular interest since ozonation suggests unusually low β-O-4’ linkages (cf. Fig. 3.3 as indicated by very low E+T yield) as compared to CELPB15. Although insoluble in most solvents, RELPB15 became completely soluble in DMSO after acetylation. NMR analyses (spectrum not shown) clearly showed much lower methoxyl content and a much higher p-hydroxyphenyl-propane (H) unit content as compared to the corresponding CEL (Table 3.3). Likewise, clear differences are evident in the FTIR spectrum of RELPB15 as compared with that of CELPB15 as shown in Figure 3.6. The RELPB15 spectrum has lower absorption at 1467 cm -1 and a completely different absorption pattern at 800-900 cm -1 region. The latter indicates a shift from a typical guaiacyl-propane
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performed for all the samples in order to enhance the SEM images. Synthesized carbon nanoparticles were characterized using a transmission electron microscope (TEM), JEOL 2010F FEG TEM/STEM, employing a 200 kV operating voltage. Brunauer-Emmet-Teller (BET) surface area analysis of the freeze-dried lignin samples and the synthesized carbon nanoparticles were measured with a NOVA station-C, Quantachrome through nitrogen gas sorption at 77.3 K. First, the samples were flow de- gased at 55˚C for 8 - 16 hours to remove the volatiles. BET surface areas were taken from a multipoint plot over a P/Po range of 0.05 - 0.35.
The optimum pH for laccases are highly dependable on the substrate. In acidic medium the pH optima are found in the range 3.0-5.0 (26). Mostly, optimal pH that varies considerably has got a bell shaped profile, The substrate, oxygen or the enzyme itself are the main causes of these variations(30). The difference in redox potential between the phenolic substrate and the T1 copper could increase oxidation of the substrate at high pH values, but the hydroxide anion (OH - ) binding to the T2/T3 coppers results in an inhibition of the laccase activity due to a disruption of the internal electron transfer between the T1 and T2/T3 centres. These two opposing effects can play an important role in determining the optimal pH of the bi-phasic laccase enzymes (30). This could be understood with the example that laccase produced by Trametes modesta was fully active at pH 4.0 and very stable at pH 4.5 but its half-life decreased to 125 min at pH 3.0 (31).
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Banana is an important fruit in China. Banana production played important role in economic development in tropical region. Banana production in China was always cut because of lodging caused by typhoon. Getting new cultivars with high resistance to lodging is the basic resolution to resolve this problem. Screening and identifying the germ plasm resource is the first step to breed new cultivars. Banana plant height was high. A single banana plant needs large area. It is difficult to screen the germ plasm resource by identifying the physical strength of banana pseudostem. This research focused on studying the rela- tionship between pseudostem and plant height, pseudostem diameter, acid so- luble lignin, acid insoluble lignin, total lignin, pore numbers of pseudostem cross section, and the expression of 4-coumarate:CoA ligase (4CL). Results showed that the plant with high physical strength in seedling stage always has high physical strength in mature stage. The physical strength of banana seedling pseudostem was closely related to pseudostem diameter and total lignin. Pseu- dostem diameter and total lignin can be used to predict the physical strength of mature banana pseudostem. Work on identifying and screening the physical strength of banana germ plasm pseudostem can be reduced by measuring seedl- ing pseudostem diameter and total lignin in pseudostem of banana germplasm.
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Understanding how genetic modification affects the physico-chemical properties of wood is important for these technologies to be realized outside of lab-scale experimentation. Genetic modifications, especially in the case of lignin content and structure, are performed to promote more efficient biomass degradation and lignin removal (or separation/fractionation) in an industrial setting. However, there are limitations to alterations of the natural chemical structure of wood. Lignin plays an important role in providing the tree with strength and rigidity that are required to support the tree’s crown and foliage, and this can be affected by lignin content (Gindl and Teischinger 2002). Lignin is also important in water conduction as it provides hydrophobic properties allowing vessels to transport water, and it protects wood from microbial infection (Chen et al 2001). Lignin-deficient wood has been observed to result in collapsed or deformed cell walls resulting from hydraulic pressure in water conduction elements (Voelker et al 2011). Providing geneticists with information concerning the physical properties and biodegradability of transgenic tree species is necessary to guide and refine genetic research to provide biomass that is both enhanced in terms of industrial usage efficiency while still exhibiting phenotypical characteristics necessary for the species to endure environmental stresses.
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The benzene ring allows phenoxyl radicals to acquire a qui- noid structure with the arrangement of an unpaired electron on any of the carbon atoms. Thus, the bonds of almost any side substituent can occur in the β-position relative to the ring to increase the probability of their thermally stimulated cleavage. Thus, degradation with the elimination of aromatic fragments is one of the most important radiolytic transformations of lignin at high temperatures. Monohydric and dihydric phenols are the main liquid products of the degradation.
that more crosslinks were formed during curing process. Due to the increased amount of formaldehyde, more hydroxyl methyl phenols were created, thus, more condensation reactions could happen during the curing process. Those exothermic reactions will release energy, which was characterized by DSC. The exception of 20%PLPF with F:P ratio of 1.9:1 could be probably due to the high viscosity. Higher viscosity comes from higher molecular weight of the structure, which indicates that more condensation reactions have taken place during resol preparation, rather than curing process. Another observation is that as the lignin incorporated in the foams increases, the onset temperature of curing increased while less energy was released. This can be justified by 1) the lower reactivity of lignin towards formaldehyde compared with phenol, leading to lower amount of potential crosslinks; 2) attributed to the presence of lignin, LPF resins have a higher activation energy compared to PF resins (Alonso, Oliet, Pérez, Rodrıguez, & Echeverrıa, 2004).
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bars represent one standard deviation. ref4 are less pigmented than wild type, although they are not as yellow as the chalcone synthase-deficient tt4 mutant (Burbulis et al. 1996). Similarly, stems of ref3 and ref4 plants display virtually none of the purple an- impact of the ref and brt mutations on total lignin con-
This spectrum contain clear and new deference peaks at several position where are not found in IR spectrum of stander PVC; (Figure 4) one of these peaks that appear at 3650 cm –1 which attributed to the free hydroxyl groups stretching vibration which can formed as a result to thermal-oxidation process, while in the state of PVC contain deferent percent from Ca-lignin chelating com- plex; Figure 6, Figure 7, and Figure 8, where in this samples the peak became much less intense or not found.
with veratraldehyde (Nacalai Tesque) as the internal standard. In order to quantify the nitrobenzene oxidation products, vanillin acetate, syringaldehyde acetate, and p-hydroxybenzaldehyde acetate were prepared with the respective reagent grade chemicals (Nacalai Tesque). Quantifi cation of nitrobenzene oxidation products was carried out by gas chromatography (Shimadzu GC14B) with FID under the following conditions: column, Shimadzu CBP-5 column (25 m × 0.25 mm ϕ ); column temperature: 100°C (1 min), 100°C → 270°C (5°C/min increment), 270°C (10 min); injection port temperature, 250°C; detector tem- perature, 250°C; carrier gas, helium; fl ow rate, 1.5 ml/min.
compound liberated from the B-ring of the dimeric model compound must be stable and quantitatively detected under the conditions employed in this study, when the b-O-4 bond of the dimeric model compound is cleaved. A phenolic compound with a-carbonyl group, such as 4-hydroxy-3-methoxybenzaldehdye (vanillin), satisfies this prerequisite and is suitable for the B-ring of a b-O-4 type dimeric lignin model compound applied to this study. Because it was reported that this type of dimeric lignin model compound is surprisingly labile under alkaline conditions [11, 12], however, a phenolic compound with a-carbonyl group cannot be utilized as the B-ring of dimeric model compound in this study. In this context, b-O-4 type dimeric model compounds III and IV, carrying two fluo- rines on their aromatic B-ring, may be utilized to evaluate the contribution. Compound V is liberated accompanying the b-O-4 bond cleavages of compounds III and IV, and hence, should be stable under the conditions employed in this study where AOS are operative. To examine the sta- bility of compound V, it was subjected to oxygen-alkali treatment together with the generator of AOS, compound VI (Nos. 5 and 6 in Table 1). The recoveries of compound V were 95 and 91% at a reaction time of 360 min in the treatment Nos. 5 and 6, respectively. These results confirm that compound V is stable enough to be quantitatively detected when compound V is liberated from compounds III and IV in their oxygen-alkali treatments.