Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula × alba)

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula × alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 l l(-1)). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.     

The cellulose/lignin assembly assessed by molecular modeling. Part 1: adsorption of a threo guaiacyl beta-O-4 dimer onto a Ibeta cellulose whisker.

The assembly of the two major cell wall components, cellulose and lignin, were investigated at the atomistic scale using molecular dynamics simulations. To this end, a molecular model of a cellulose crystal corresponding to the allomorph Ibeta and exhibiting different surfaces was considered to mimic the carbohydrate matrix present in native wood cell wall. The lignin model compound considered here is a threo guaiacyl beta-O-4 dimer. The dynamical process of adsorption of the lignin dimer onto the different surfaces of the cellulose crystal was examined. The modes of association between the two constituents were analyzed; energies of adsorption of the dimer are calculated favorable and of the same order of magnitude on all sides of the cellulosic model, suggesting that the deposition of lignin precursors onto cellulose fibers is non-specific from an enthalpic point of view. Interestingly, geometrical characteristics and energetical details of the adsorption are surface-dependent. Computed data have underlined the predominant contribution of van der Waals interactions for adsorption onto the (200) face, as well as the major influence of H-bonding interactions in the dynamical process of adsorption onto (110) and (1-10) faces. A large number of adsorption sites have been identified and a noticeable "flat" geometry of adsorption of the lignin dimer has been observed, as a consequence of the stacking interactions between lignin aromatic rings and C-H groups of cellulose. Importantly, these dispersive interactions lead to a preferential parallel orientation of lignin aromatic rings relative to the cellulose surface, notably on the (200) face. Such a parallel orientation is consistent with previously reported experimental observations.     

Cellulose depolymerization to glucose and other water soluble polysaccharides by shear deformation and high pressure treatment

The simultaneous action of shear deformation and high pressure (SDHP) creates changes in the structure of wood and its main components (cellulose, hemicelluloses, lignin). The formation of water and alkali soluble polysaccharides under SDHP action, proceeds in seconds in the solid state, without the use of any reagents and solvents. Therefore, SDHP seems to be a technologically safe method and friendly to the environment. The amorphization of cellulose crystallites and depolymerization of cellulose chains were observed under a wide range of pressures (1–6 GPa), both for cellulose samples and the cellulose part of wood. Similar depolymerization occurs in the hemicellulose part of wood. The decomposition of polysaccharides under SDHP causes the formation of the water soluble part, whose content increases with pressure and the applied shear deformation. A maximum solubility of 40% and 55% was registered at 6 GPa following treatment of cellulose and birch wood samples. A higher output in the case of wood can be explained by a specific role of lignin under SDHP, which acts as a ‘grinding stone’ during cellulose and hemicelluloses destruction. As shown by high-performance size exclusion chromatography, the water soluble fraction obtained from cellulose contained glucose (2.6%), cellobiose (9.6%), cellotriose (16.6%) and other higher water soluble oligomers (71%). Almost complete dissolution (98%) of the treated cellulose sample can be achieved by extraction with 10% NaOH solution. The SDHP treated birch wood was subjected to submerged fermentation (with Trichoderma viride), and a 13% output of proteins was obtained. In this case, the water soluble part played the role of the so called ‘start sugars’. Abbreviations: ASF, alkali soluble fraction; DP, degree of polymerization; EC, energy consumption; HP, high pressure; LMWS, low molecular weight sugars; MC, moisture content; MCC, microcrystalline cellulose; SD, shear deformation, SDHP, shear deformation under high pressure; SS, shear strength; WSF, water soluble fraction This revised version was published online in November 2006 with corrections to the Cover Date.     

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.     

Degradation of the Pine Wood Structure in Ozonolytic Delignification

Russian Journal of Bioorganic Chemistry - Transformations of pine wood during exposure to ozone were studied. The content of lignin and cellulose in the cellulose-containing material (CCM) from...     

Degradation of lignin by combined chemical and biological treatment

It has been demonstrated that ozone dosages of 0.06 lb and 0.78 lb per pound of lignin can reduce the optical density (in the visible range) of the latter by 82% and 92%, respectively. The reduction in color is accompanied by a shift in the molecular weight distribution of lignin from a broad peak of between 20,000 and 70,000 to lower molecular weight, species including substantial amounts possessing a molecular weight of 1000 or less. The cost of decolorizing a typical kraft paper bleach effluent is estimated to be under 50¢/1000 gal which compares favorably with competitive decolorizing processes. Lignin ozonation results in the production of a series of decolorized products which can serve as the sole source of carbon for a variety of microorganisms. Feasibility studies indicated that at least 40% of the ozonated material can be transferred into microbial biomass (protein) as well as other products of commercial interest such as fumaric acid and penicillin.     

Distribution of lignin and its coniferyl alcohol and coniferyl aldehyde groups in Picea abies and Pinus sylvestris as observed by Raman imaging

Wood cell wall consists of several structural components, such as cellulose, hemicelluloses and lignin, whose concentrations vary throughout the cell wall. It is a composite where semicrystalline cellulose fibrils, acting as reinforcement, are bound together by amorphous hemicelluloses and lignin matrix. Understanding the distribution of these components and their functions within the cell wall can provide useful information on the biosynthesis of trees. Raman imaging enables us to study chemistry of cell wall without altering the structure by staining the sample or fractionating it. Raman imaging has been used to analyze distributions of lignin and cellulose, as well as the functional groups of lignin in wood. In our study, we observed the distribution of cellulose and lignin, as well as the amount of coniferyl alcohol and aldehyde groups compared to the total amount of lignin in pine (Pinus sylvestris) and spruce (Picea abies) wood samples. No significant differences could be seen in lignin and cellulose distribution between these samples, while clear distinction was observed in the distribution of coniferyl alcohols and coniferyl aldehyde in them. These results could provide valuable insight on how two similar wood species control biosynthesis of lignin differently during the differentiation of cell wall.     

The bioconversion of mountain pine beetle-killed lodgepole pine to fuel ethanol using the organosolv process

Lodgepole pine (Pinus contorta) killed by mountain pine beetle (Dendroctonus ponderosae) (BLP) was compared with healthy lodgepole pine (HLP) for bioconversion to ethanol and high-value co-products. The BLP and HLP chips were pretreated using an ethanol organosolv process at a variety of severities. It was shown that the BLP was easier to pretreat and delignify than were the HLP chips. The resulting pretreated BLP substrate had a lower residual lignin, lower degree of polymerization of cellulose, lower cellulose crystallinity, smaller fiber size and thereby a better enzymatic hydrolysability than did the HLP substrates. However, under the same conditions, the BLP showed lower substrate yield and cellulose recovery than did the HLP, which likely resulted from the excessive hydrolysis and subsequent decomposition of the cellulose and hemicellulose during the pretreatment. The BLP wood yielded more ethanol organosolv lignin than was obtained with the HLP material. The HLP lignin had a lower molecular weight and narrower distribution than did the BLP lignin. It appears that the beetle killed LP is more receptive to organosolv pretreatment other than a slightly lower recovery of carbohydrates.     

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.     

Molecular characterization of PoGT8D and PoGT43B, two secondary wall-associated glycosyltransferases in poplar

Dicot wood is mainly composed of cellulose, lignin and glucuronoxylan (GX). Although the biosynthetic genes for cellulose and lignin have been studied intensively, little is known about the genes involved in the biosynthesis of GX during wood formation. Here, we report the molecular characterization of two genes, PoGT8D and PoGT43B, which encode putative glycosyltransferases, in the hybrid poplar Populus alba x tremula. The predicted amino acid sequences of PoGT8D and PoGT43B exhibit 89 and 75% similarity to the Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9, respectively, both of which have been shown to be required for GX biosynthesis. The PoGT8D and PoGT43B genes were found to be expressed in cells undergoing secondary wall thickening, including the primary xylem, secondary xylem and phloem fibers in stems, and the secondary xylem in roots. Both PoGT8D and PoGT43B are predicted to be type II membrane proteins and shown to be targeted to Golgi. Overexpression of PoGT43B in the irx9 mutant was able to rescue the defects in plant size and secondary wall thickness and partially restore the xylose content. Taken together, our results demonstrate that PoGT8D and PoGT43B are Golgi-localized, secondary wall-associated proteins, and PoGT43B is a functional ortholog of IRX9 involved in GX biosynthesis during wood formation.     

Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state ¹³C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS ¹³C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.     

Purified lignin: Nutritional and health impacts on farm animals—A review

Lignin, the second most abundant natural compound after cellulose (Boudet and Grima-Pettenati, 1996), is a high-molecular weight polymer of phenolic compounds that occurs naturally in plants. It is mostly present in the cell wall, conferring structural support, impermeability and resistance to microbial attack. Commercial purified lignin is produced as a by-product of the paper industry, separated from wood by chemical pulping processes. These purified lignins are low molecular weight mono-phenolic fragments that have biological characteristics that differ from those of native lignin. Different chemical treatments during wood-pulping processes yield diverse types of purified lignin, such as Alcell lignin and Kraft lignin. Although these phenolic fragments may potentially have important applications in animal agriculture, research with purified lignin has not received much attention and there are few published results. In contrast to native lignin, purified lignin does not represent a barrier to digestion in monogastric or ruminant animals. Several in vitro and in vivo studies have demonstrated antimicrobial properties of the phenolic fragments in purified lignin. Recently, purified Alcell lignin has been shown to exhibit prebiotic effects in chickens, favouring growth of beneficial bacteria and improving the morphological structures of the intestines, as measured by increased villi height and goblet cell number. These findings suggest that purified lignin may exert health benefits in monogastric animals and could potentially be considered as a natural feed additive. Based on the few published studies, animal responses to purified lignin seem dependent on dosage, animal species and type and source of the lignin product. More research is required before establishing conclusive benefits of purified lignin on animal performance and health.     

Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw

Wheat and rye straws were pretreated with ozone to increase the enzymatic hydrolysis extent of potentially fermentable sugars. Through a 2(5-1) factorial design, this work studies the influence of five operating parameters (moisture content, particle size, ozone concentration, type of biomass and air/ozone flow rate) on ozonization pretreatment of straw in a fixed bed reactor under room conditions. The acid insoluble lignin content of the biomass was reduced in all experiments involving hemicellulose degradation. Near negligible losses of cellulose were observed. Enzymatic hydrolysis yields of up to 88.6% and 57% were obtained compared to 29% and 16% in non-ozonated wheat and rye straw respectively. Moisture content and type of biomass showed the most significant effects on ozonolysis. Additionally, ozonolysis experiments in basic medium with sodium hydroxide evidenced a reduction in solubilization and/or degradation of lignin and reliable cellulose and hemicellulose degradation.     

Ozone pretreatment to increase digestibility of lignocellulose

Treatment of lignocellulose with ozone yielded products which included acids, sugars and a fibrous material rich in cellulose. Ozone attacks lignin and hemicelluloses in preference to cellulose, and is effective in disrupting the association between the various components so as to produce a substrate with enhanced reactivity to hydrolytic enzymes.     

Enhancement of enzymatic hydrolysis by simultaneous attrition of cellulosic substrates

It has been shown that simultaneous attrition of cellulose in an attritor containing stainlesssteel beads results in a substantial enhancement of the enzymatic hydrolysis. The attrition exerts two opposing effects, continuous delamination and comminution of the substrate with formation of new reactive sites and a gradual denaturation and inactivation of the enzyme. Consequently, the hydrolysis proceeds very rapidly at first and levels off at about 70% saccharification of the substrate. Accumulation of hydrolysis products is also responsible for inhibition of the enzyme. The attrition method is effective for the saccharification of cottonwood in which the cellulosic microfibrils are embedded in a matrix of lignin and hemicelluloses. A comparison between the saccharification of wood, lignocellulose, holocellulose, and cellulose with simultaneous attrition showed that the lignin component provided more hindrance toward the saccharification process than hemicelluloses, which are themselves subject to enzymatic hydrolysis.     

Biodegradation of hardwood lignocellulosics by the western poplar clearwing borer, Paranthrene robiniae (Hy. Edwards)

Lignin in plant cell wall is a source of useful chemicals and also the major barrier for saccharification of lignocellulosic biomass for producing biofuel and bioproducts. Enzymatic lignin degradation/modification process could bypass the need for chemical pretreatment and thereby facilitate bioprocess consolidation. Herein, we reveal our new discovery in elucidating the process of hardwood lignin modification/degradation by clearwing borer, Paranthrene robiniae . The wood-boring clearwing borer, P. robiniae , effectively tunnels hardwood structures during the larval stage; its digestion products from wood components, however, has not yet been investigated. A series of analysis conducted in this study on tunnel walls and frass produced provided evidence of structural alterations and lignin degradation during such hardwood digestion process. The analysis included solid state (13)C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy, attenuated total reflectance Fourier transform infrared (ATR-FTIR), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and thermogravimetric (TG) analysis; the results strongly suggest that the structural alteration of lignin primarily involved a preferential degradation of syringyl units accompanied by oxidation on the side chains of lignin guaiacyl moieties. This study also further indicated that unlike the wood-feeding termite the clearwing borer does not target cellulose as an energy source, and thus its lignin degradation ability should provide potential information on how to disassemble and utilize hardwood lignin. Overall, this biological model with an efficient lignin disruption system will provide the new insight into novel enzyme system required for effective plant cell wall disintegration for enhanced cellulose accessibility by enzymes and production of value-added lignin derived products.     

Host responses and metabolic profiles of wood components in Dutch elm hybrids with a contrasting tolerance to Dutch elm disease

Background and Aims

Changes occurring in the macromolecular traits of cell wall components in elm wood following attack by Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), are poorly understood. The purpose of this study was to compare host responses and the metabolic profiles of wood components for two Dutch elm (Ulmus) hybrids, ‘Groeneveld’ (a susceptible clone) and ‘Dodoens’ (a tolerant clone), that have contrasting survival strategies upon infection with the current prevalent strain of DED.

Methods

Ten-year-old plants of the hybrid elms were inoculated with O. novo-ulmi ssp. americana × novo-ulmi. Measurements were made of the content of main cell wall components and extractives, lignin monomer composition, macromolecular traits of cellulose and neutral saccharide composition.

Key Results

Upon infection, medium molecular weight macromolecules of cellulose were degraded in both the susceptible and tolerant elm hybrids, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels, but rarely in the fibres. The ¹³C nuclear magnetic resonance spectra revealed that loss of crystalline and non-crystalline cellulose regions occurred in parallel. The rate of cellulose degradation was influenced by the syringyl:guaiacyl ratio in lignin. Both hybrids commonly responded to the medium molecular weight cellulose degradation with the biosynthesis of high molecular weight macromolecules of cellulose, resulting in a significant increase in values for the degree of polymerization and polydispersity. Other responses of the hybrids included an increase in lignin content, a decrease in relative proportions of d-glucose, and an increase in proportions of d-xylose. Differential responses between the hybrids were found in the syringyl:guaiacyl ratio in lignin.

Conclusions

In susceptible ‘Groeneveld’ plants, syringyl-rich lignin provided a far greater degree of protection from cellulose degradation than in ‘Dodoens’, but only guaiacyl-rich lignin in ‘Dodoens’ plants was involved in successful defence against the fungus. This finding was confirmed by the associations of vanillin and vanillic acid with the DED-tolerant ‘Dodoens’ plants in a multivariate analysis of wood traits.     

Recent advances in fungal cellobiose oxidoreductases

When grown on cellulose, the white-rot fungus Phanerochaete chrysosporium (Sporotrichum pulverulentum), produces two cellobiose oxidoreductases, i.e., cellobiose:quinone oxidoreductase (CBQ) and cellobiose oxidase (CBO). Similar cellobiose-oxidizing enzymes, capable of utilizing a wide variety of electron acceptors, have been detected in many other fungi. However, the role of the cellobiose oxidoreductases in white-rot fungi, or in any fungi for that matter, is still not known. The original role ascribed to CBQ was as a link between cellulose and lignin degradation. CBQ has been shown to reduce quinones and phenoxyradicals released during lignin degradation concomitantly oxidizing cellobiose and other cellodextrins released during cellulose degradation. Thus, one function proposed for the cellobiose oxidoreductases is to prevent repolymerization of phenoxyradicals formed when phenoloxidases (peroxidases and laccases) attack lignin and lignin degradation products. However, evidence obtained so far indicates that the presence of CBO/CBQ with lignin peroxidases and laccases actually reduces the rate of oxidation of lignin degradation products. CBQ has a molecular mass of about 60 kD and contains an FAD cofactor. CBO contains both heme and FAD, and has a mass of about 90 kD. It has recently been demonstrated that CBO can be proteolytically cleaved into FAD and heme domains. The FAD domain of CBO seems to have all the properties of CBQ, suggesting that CBQ is a cleavage product of CBO. Whether CBO is a precursor of CBQ is not yet known. CBO and CBQ can be distinguished not only by the differences in their spectral properties, but also by the ability of CBO, but not CBQ, to reduce cytochrome c. Both CBO and CBQ have a cellulose-binding domain (CBD), as do a large number of endoglucanases and cellobiohydrolases. The induction-repression patterns regulating cellobiose oxidoreductase genes are not known in any detail. Most reports point to induction during cellulose degradation, but repression has not been studied. Induction has also been suggested to occur by addition of lignosulfonate to the medium.