LIGNIFICATION DURING SECONDARY WALL FORMATION IN COLEUS: AN ELECTRON MICROSCOPIC STUDY

The fine structure of lignin deposition was examined in developing secondary walls of wound vessel members in Coleus. KMnO₄, which was used as the fixative, selectively reacts with the lignin component of the cell wall and thus can be used as a highly sensitive electron stain to follow the course of lignification during secondary wall deposition. Lignin was first detected as conspicuuos electron-opaque granules in the primary wall in the region where the secondary wall thickening arises and as fine granular striations extending into the very young secondary wall. As the secondary wall develops lignification becomes progressively more extensive. In cross sections the lignified secondary wall appears as concentric, fine granular striations; in tangent al or oblique sections it is seen as delicate, beaded fibrils paralleling the long axis of the thickening. High magnification of tangential or oblique sections shows that the fibrillar appearance is due to the presence of alternating light and dark layers each approximately 25-35 A wide. It is assumed that the light layers are the cellulose microfibrils and the dark regions contain lignin which fills the space between the microfibrils. KMnO₄, by selectively reacting with lignin, thus negatively stains the cellulose microfibrils revealing their orientation and dimensions.     

Biodegradation of high molecular weight lignin under sulfate reducing conditions: lignin degradability and degradation by-products

This study is designed to investigate the biodegradation of high molecular weight (HMW) lignin under sulfate reducing conditions. With a continuously mesophilic operated reactor in the presence of co-substrates of cellulose, the changes in HMW lignin concentration and chemical structure were analyzed. The acid precipitable polymeric lignin (APPL) and lignin monomers, which are known as degradation by-products, were isolated and detected. The results showed that HMW lignin decreased and showed a maximum degradation capacity of 3.49 mg/l/day. APPL was confirmed as a polymeric degradation by-product and was accumulated in accordance with HMW lignin reduction. We also observed non-linear accumulation of aromatic lignin monomers such as hydrocinnamic acid. Through our experimental results, it was determined that HMW lignin, when provided with a co-substrate of cellulose, is biodegraded through production of APPL and aromatic monomers under anaerobic sulfate reducing conditions with a co-substrate of cellulose.     

UGPase和反义4CL基因对转基因烟草纤维素和木质素合成的调控

用根癌农杆菌介导法将源于紫穗槐的尿苷二磷酸葡萄糖焦磷酸化酶（UGPase）基因、反义4-香豆酸辅酶A连接酶（4CL）基因以及两者的双价基因分别转移至烟草中。PCR和Southern杂交检测证实外源基因已整合到转基因烟草基因组中。测定全纤维素和Klason木质素含量的结果显示,增强UGPase基因的表达可提高转基因植株的纤维素含量,但对木质素含量没有影响;抑制4CL基因的表达可显著降低转基因植株的木质素含量,但对纤维素含量没有影响;转移双价基因的转基因植株中纤维素含量增加而木质素含量降低。     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

木质纤维素的定量测定及降解规律的初步研究

为了准确地测定稻草及其发酵物中纤维素、半纤维素、木质素的含量,通过差重法进行定量测定,并以此评价白腐菌株Pleurotus sapidus对稻草秸秆的降解状况,结果表明：利用差重法测定稻草发酵物中纤维素、半纤维索、木质素的百分含量是可行的,并能很好地评价白腐菌对稻草的降解规律,即降解过程中纤维素、半纤维素、本质素在前20d降解的很快,之后降解减缓,在50d内,纤维素被降解34．02％,半纤维素被降解56．29％,木质素被降解61．65％。     

Cellulase stimulation during biodegradation of lignocellulosic residues at increased biomass loading

Microbial degradation of lignocellulosic biomass is primarily affected by the composition and structure of biomass, as well as enzyme activities that are influenced by the presence of in-process degradation products. This study focuses on the latter, and demonstrates that cellulase activity of Neurospora discreta is stimulated in the presence of in-process soluble lignin degradation products. Two types of biomass - cocopeat and sugarcane bagasse, with contrasting lignin content and cellulose structure were tested at two biomass loadings each. At the higher biomass loading, cocopeat showed the highest amount of hydrolyzed cellulose and cellulase activity, despite its low cellulose content and recalcitrant cellulose structure. A strong positive correlation was revealed between the amount of in-process degraded lignin and cellulase activity, indicating a stimulatory effect on cellulase, which contradicts most previous literature. Furthermore, the causal relationship between the amount of degraded lignin and cellulase activity was established in a model system of commercial cellulase and standard soluble lignin. This work could pave the way for using biomass loading as a process lever to enhance cellulose hydrolysis in microbial conversion of lignocellulosic biomass.     

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs.     

Lignin and cellulose concentrations in roots of Douglas fir and European beech of different diameter classes and soil depths

Comparing two tree species, we tested the effects of root diameter (up to 30 mm) and soil depth (down to 1.2 m) on the concentrations of lignin, cellulose and nitrogen (N) in roots of approximately 50-year-old Douglas fir and European beech growing in a temperate forest in South-western Germany. Fine roots (diameter 0.5–2 mm) exhibited significantly higher lignin concentrations, but lower cellulose concentrations than medium or coarse roots (diameter >5 mm). The cellulose and lignin concentrations of the roots as well as their lignin:cellulose ratios did not differ significantly among soil depths. In the Douglas fir, there was a tendency of decreasing N concentrations and increasing lignin:N ratios with increasing soil depth. This trend was absent or less pronounced in the beech. Beech roots displayed significantly higher cellulose and N concentrations and lower lignin:cellulose and lignin:N ratios than roots of the Douglas fir. Generally, the lignin concentrations of the roots did not differ between the tree species. Cellulose and lignin concentrations exhibited a significantly negative correlation. As several studies have demonstrated that plant litter decomposition is governed by the lignin:cellulose and lignin:N ratios more than by the lignin concentration of the detritus, the fraction of individual tree species in the stand composition might affect the decomposability of roots in beech–Douglas fir forests, and might also have an influence on soil carbon sequestration.     

四川2种丛生竹木质素和纤维含量的研究

以四川4个地区2种丛生竹(慈竹和硬头黄)为研究对象,对其木质素和综纤维含量进行研究.结果表明,不同地区竹种间综纤维素含量差异显著;除青神地区外,其它几个地区木质素含量差异不大.纬度对木质素积累的作用因竹种而异,对慈竹木质素含量的作用呈极明显的正效应,对硬头黄的作用则呈不明显的正效应,对综纤维含量的影响不大.竹龄影响木质素和综纤维素含量的积累,对慈竹木质素和综纤维的积累呈显著的正效应,但对硬头黄的作用却不明显.研究认为,青神、长宁和绵阳地区的硬头黄以及长宁地区的慈竹是造纸的较好原料.     

The use of natural abundance stable isotopic ratios to indicate the presence of oxygen-containing chemical linkages between cellulose and lignin in plant cell walls

Qualitative and quantitative understanding of the chemical linkages between the three major biochemical components (cellulose, hemicellulose and lignin) of plant cell walls is crucial to the understanding of cell wall structure. Although there is convincing evidence for chemical bonds between hemicellulose and lignin and the absence of chemical bonds between hemicellulose and cellulose, there is no conclusive evidence for the presence of covalent bonds between cellulose and lignin. This is caused by the lack of selectivity of current GC/MS-, NMR- and IR-based methods for lignin characterisation as none of these techniques directly targets the possible ester and ether linkages between lignin and cellulose. We modified the widely-accepted “standard” three-step extraction method for isolating cellulose from plants by changing the order of the steps for hemicellulose and lignin removal (solubilisation with concentrated NaOH and oxidation with acetic acid-containing NaClO₂, respectively) so that cellulose and lignin could be isolated with the possible chemical bonds between them intact. These linkages were then cleaved with NaClO₂ reagent in aqueous media of contrasting ¹⁸O/¹⁶O ratios. We produced cellulose with higher purity (a lower level of residual hemicellulose and no detectable lignin) than that produced by the “standard” method. Oxidative artefacts may potentially be introduced at the lignin removal stage; but testing showed this to be minimal.Cellulose samples isolated from processing plant-derived cellulose–lignin mixtures in media of contrasting ¹⁸O/¹⁶O ratios were compared to provide the first quantitative evidence for the presence of oxygen-containing ester and ether bonds between cellulose and lignin in Zea mays leaves. However, no conclusive evidence for the presence or lack of similar bonds in Araucaria cunninghamii wood was obtained.     

Litter decay rates are determined by lignin chemistry

Litter decay rates are often correlated with the initial lignin:N or lignin:cellulose content of litter, suggesting that interactions between lignin and more labile compounds are important controls over litter decomposition. The chemical composition of lignin may influence these interactions, if lignin physically or chemically protects labile components from microbial attack. We tested the effect of lignin chemical composition on litter decay in the field during a year-long litterbag study using the model system Arabidopsis thaliana. Three Arabidopsis plant types were used, including one with high amounts of guaiacyl-type lignin, one with high aldehyde- and p-hydroxyphenyl-type lignin, and a wild type control with high syringyl-type lignin. The high aldehyde litter lost significantly more mass than the other plant types, due to greater losses of cellulose, hemicellulose, and N. Aldehyde-rich lignins and p-hydroxyphenyl-type lignins have low levels of cross-linking between lignins and polysaccharides, supporting the hypothesis that chemical protection of labile polysaccharides and N is a mechanism by which lignin controls total litter decay rates. 2D NMR of litters showed that lignin losses were associated with the ratio of guaiacyl-to-p-hydroxyphenyl units in lignin, because these units polymerize to form different amounts of labile- and recalcitrant-linkages within the lignin polymer. Different controls over lignin decay and polysaccharide and N decay may explain why lignin:N and lignin:cellulose ratios can be better predictors of decay rates than lignin content alone.     

iTRAQ-based quantitative secretome analysis of Phanerochaete chrysosporium

The basidiomycete fungi such as Phanerochaete chrysosporium secrete large amount of hydrolytic and oxidative enzymes and degrade lignocellulosic biomass. The lignin depolymerizing proteins were extensively studied, but cellulose, hemicellulose and pectin hydrolyzing enzymes were poorly explored. In this study P. chrysosporium was grown in cellulose, lignin and mixture of cellulose and lignin, and secretory proteins were quantified by isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS). An iTRAQ quantified 117 enzymes comprising cellulose hydrolyzing endoglucanases, exoglucanases, beta-glucosidases; hemicelluloses hydrolyzing xylanases, acetylxylan esterases, mannosidases, mannanases; pectin-degrading enzymes polygalacturonase, rhamnogalacturonase, arabinose and lignin degrading protein belonging to oxidoreductase family. Under cellulose and cellulose with lignin culture conditions, enzymes such as endoglucanases, exoglucanases, β-glucosidases and cellobiose dehydrogenase were significantly upregulated and iTRAQ data suggested hydrolytic and oxidative cellulose degradation. When lignin was used as a major carbon source, enzymes such as copper radical oxidase, isoamyl oxidase, glutathione S-transferase, thioredoxin peroxidase, quinone oxidoreductase, aryl alcohol oxidase, pyranose 2-oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase were expressed and significantly regulated. This study explored cellulose, hemicellulose, pectin and lignin degrading enzymes of P. chrysosporium that are valuable for lignocellulosic bioenergy.     

Reconstitution of cellulose and lignin after [C2mim][OAc] pretreatment and its relation to enzymatic hydrolysis

Although the effects of cellulose crystallinity and lignin content as two major structural features on enzymatic hydrolysis have been extensively studied, debates regarding their effects still exist. In this study, reconstitution of cellulose and lignin after 1‐ethyl‐3‐methylimidazolium acetate ([C₂mim][OAc]) pretreatment was proposed as a new method to study their effects on enzymatic digestibility. Different mechanisms of lignin content for reduction of cellulose hydrolysis were found between the proposed method and the traditional method (mixing of cellulose and lignin). The results indicated that a slight change of the crystallinity of the reconstituted materials may play a minor role in the change of enzyme efficiency. In addition, the present study suggested that the lignin content does not significantly affect the digestibility of cellulose, whereas the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase plays an important role when an ionic liquid pretreatment of biomass was conducted. Biotechnol. Bioeng. 2013; 110: 729–736. © 2012 Wiley Periodicals, Inc.     

The cellulose/lignin assembly assessed by molecular modeling. Part 2: Seeking for evidence of organization of lignin molecules at the interface with cellulose.

We have extended our previous computational investigation of the cellulose lignin assembly by considering more complex systems. Surface coverage of cellulose, structural parameters such as molecular mass and structural features of the lignin models and the presence of an explicit hydrated environment have been taken into account to examine their influence on the associative interactions between cellulose and lignin. To this end, different lignin molecular models, from beta-O-4 dimers up to a 20-units oligomer, were considered. Independently of the system studied, the key feature of the adsorption is globally preserved: aromatic rings of lignin adopt a preferential parallel orientation relative to the cellulose surface. Such structural order appears to be limited to the first shell of lignin units adsorbed on the cellulose. The pre-organization of the lignin monolayer at the surface of cellulose is not significantly changed at the interface with water. However, adsorption significantly depends on the molecular mass and the structure of lignin. The structural order is significantly hindered by the presence of branching or some particular inter-units linkages in the structure of lignin. Such results rationalize the apparent contradiction between the available experimental results.     

Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments

The occurrence of covalent bonds between residual lignin and polysaccharides in birch and pine kraft pulps was investigated by specific enzymatic treatments. Pure enzymes degrading cellulose, xylan and mannan were used both separately and in combination. Comparison of the molar masses of polysaccharides and lignin in the orginal pulps and in the residual pulps after enzymatic treatments showed that residual lignin in birch kraft pulp is linked at least to xylan. A minor portion may also be linked to cellulose. In pine kraft pulp some of the residual lignin appears to be linked to cellulose, glucomannan and xylan. The linkages between lignin and cellulose and hemicelluloses may be either native or formed during pulp processing. The results also provided new information on the synergistic action of cellulose- and hemicellulose-degrading enzymes on pulp fibres. The synergism appears to be mainly due to the structure of the pulp fibres, with different layers of cellulose sheets, hemicelluloses and lignin. On the other hand the results also provided information about fibre structure. The degradation of xylan clearly enhanced the action of enzymes on cellulose, suggesting that xylan partially covers the cellulose. A similar phenomenon was not observed in the simultaneous hydrolysis of glucomannan and cellulose. However, the results suggest that glucomannan does interact with cellulose, possibly by non-covalent linkages. Received: 8 July 1998 / Received revision: 7 October 1998 / Accepted: 11 October 1998     

氮钾对慈竹纤维素和木质素动态积累的调控效应

以慈竹为材料,通过施用不同比例的氮、钾肥,研究其对慈竹木质素和纤维素分布和动态积累的调控效应,为慈竹优质栽培提供理论依据。研究结果表明,氮钾肥处理并没有改变木质素和纤维素动态积累趋势,不同处理的慈竹木质素的动态积累呈先上升后下降的趋势,而纤维素的动态积累呈上升的趋势;慈竹不同部位木质素和纤维素的分布呈下部＞中部＞上部的趋势。施用氮、钾肥可以降低慈竹木质素含量,降低幅度为1.05%~3.97%,提高纤维素含量,提高幅度为1.25%～3.36%。N、K肥和时间对慈竹木质素和纤维素含量总变异的贡献都是N×K＞N＞TIME＞K＞N×K×TIME＞N×TIME＞K×TIME。慈竹纤维素和木质素含量之间呈不明显的正相关关系（r=0.020 4）。     

Effect of manganese on preferential degradation of lignin by Pleurotus ostreatus during solid-state fermentation.

Practical utilization of the polysaccharides in the lignocellulosic complex is limited because of the high lignin content of the complex. In this study we focused on the effect of Mn on lignin and cellulose biodegradation during solid-state fermentation by the edible mushroom Pleurotus ostreatus. Preferential degradation of lignin was enhanced by the addition of Mn(II) to cotton stalks at concentrations ranging from 30 to 620 micrograms of Mn per g. This effect was most apparent when we compared mineralization rates of [14C] lignin with mineralization rates of [14C] cellulose. Enhanced selectivity was also observed when we analyzed residual organic matter at the end of the fermentation period by using crude fiber analysis. The cellulose fraction in the original material was 1.8 times larger than the cellulose fraction of lignin. The cellulose/lignin ratio increased during 32 days of solid-state fermentation from 2.5 in the control to 3.3 following the addition of Mn to the medium. The in vitro digestibility value for fermented cotton stalks was 53% of the dry matter. Addition of 600 micrograms of Mn per g to the cotton stalks resulted in a digestibility value of 65.4%. Enhancement of preferential lignin degradation could be result of either increased activity of the ligninolytic enzymes or production of Mn (III), which might preferentially degrade aromatic structures in the lignocellulosic complex.     

Biodegradation of rice stumps by soil mycoflora

Summary Cellulose and lignin contents in left-overs of rice stump decreased due to decay caused by soil mycoflora. The loss of cellulose and lignin was considerable in presence of Curvularia and Fusarium respectively. Other tested mycoflora could also destroy cellulose and lignin to some extent. The amount of loss of cellulose and lignin increased with time of incubation of the tested mycoflora.     

四川2种丛生竹理化特性及纤维形态研究

以四川不同地区两种丛生竹—慈竹和梁山慈竹为研究对象,通过测定纤维素和木质素含量以及纤维形态,对其变异规律进行研究。结果表明,同一地区不同竹种间纤维素和木质素含量差异显著,同一竹种不同地区间纤维素和木质素含量、纤维长度、宽度和纤维长宽比因地域差异而异,且与竹龄有关。从纸浆用竹和纤维原料综合考虑,竹海地区的慈竹（纤维素含量在45%左右,木质素含量低于28%）、梁山慈竹（纤维素含量在50%以上,木质素含量低于20%）比较适合竹浆造纸。