Chemo-enzymatic synthesis and characterization of graft copolymers from lignin and acrylic compounds

Chemo-enzymatic initiation of graft copolymerization of acrylic compounds onto different technical lignosulfonates (LS) was compared to a Fenton-like system (ferrous ion, t-BHP). The enzyme tested was a phenoloxidase laccase (EC 1.10.3.2) from the white rot basidomycete Trametes versicolor. Most applied lignins were successfully grafted, resulting in a polymer yield of more than 90%. The effect of initiator concentration and the lignin/monomer ratio on the yield and M(w) of enzymatically grafted polymers were studied. The homopolymer proportion in the enzymatically produced grafts of Ca-LS and acrylic acid was 5 to 6x lower than those initiated by the Fenton-like reagent; no such differences were observed for Na-LS.     

Enzymatic treatment of lignochemicals; adhesive and resin uses

The possibility of upgrading lignochemicals for use in the adhesives industry through the application of biotechnology was investigated. It was hypothesized that ligninolytic organisms or isolated ligninolytic enzymes might modify lignin in such a manner as to make it more reactive as a copolymer in adhesive manufacturing. The following discussion takes the form of three sections; the first discusses some background on lignin degradation and describes the isolation and characterization of fungal and recombinant ligninolytic enzymes. The second section describes the making of PVA/lignochemical glues and the results of modifying, both chemically and enzymatically, the lignochemicals used in those glues. Finally, the last section describes the effects observed upon treatment of a variety of lignochemicals, kraft lignins and lignosulfonates, with recombinant H8 (rH8), a lignin peroxidase whose gene was cloned from Phanerochaete chrysosporium.     

Enzymatic demethylation of lignin for potential biobased polymer applications

Lignin is a highly methylated, recalcitrant biopolymer available aplenty in nature, and is highly heteropolymer in nature, but yet it has been an under-utilized biopolymer. Modifying it chemically, biologically or enzymatically could render it a good candidate for phenol formaldehyde resin or into fine chemicals, fuels, and plastics applications. Lignin demethylation is facilitated by the enzymes called the O-demethylases, which are able to strip-off of the –OCH₃ group in lignin, that give rise to the more widely accessible phenolic hydroxyls groups. Biological demethylation of lignins can be accomplished by means of the microorganisms, such as the white-rot, soft-rot and brown-rot fungi, besides some species of bacteria. Although the enzymes responsible for the lignin demethylation process have not been identified and purified adequately, it is perhaps possible that the O-demethylases, which have the ability to remove the O-methyl groups at the C-3 and (or) C-4 positions of the benzyl ring of low molecular weight lignin-like model compounds (LMCs) and lignin makes them the suitable candidate. These LMCs resemble the aromatic moieties inherent in the molecular structure of lignins, such as the vanillate, syringate, and veratrate. Thus, these enzymes are known as vanillate-O-demethylases, syringate O-demethylases, veratrate O-demethylases and Tetrahydrofolate (THF)-dependent O-demethylase (LigM), respectively. Whereas, some ligninolytic enzymes are known to cause damage to the structure of lignins (e.g., laccases, manganese-dependent peroxidase and lignin peroxidases). The O-demethylase enzymes are believed to be capable of removing the O-methyl groups from the lignins without affecting the complex backbone structure of the lignins. The mechanism of action of O-demethylases on lignin degradation is still largely unexplored, and their ability to remove the O-methyl groups from lignins has not been elucidated sufficiently. In this review, the recent advances made on the molecular approaches in the lignin demethylation (O-demethylases and ligninolytic enzymes), degradation and the probable strategies to tone up the lignin quality have been discussed in detail. The demethylation process of lignins by means of enzymes is envisaged to open up new vistas for its application as a biopolymer in various bioprocess and biorefinery process.     

Comparative study of lignins isolated from Alfa grass (Stipa tenacissima L.)

Soda lignin, dioxane lignin and milled lignin were isolated from Alfa grass (Stipatenacissima L.). The physico-chemical characterization of three different lignins: one industrial lignin precipitated from soda spent liquor and two lignin preparations isolated under laboratory conditions from Alfa grass (also know as Esparto grass) was performed. The structures of lignins were studied by three non-destructive (FT-IR, solid state ¹³C NMR and UV/visible spectroscopy) and two destructive (nitrobenzene oxidation and thermogravimetric analysis) methods. Elemental analysis and the methoxyl content determination were performed in order to determine the C₉ formulae for the studied lignins. The total antioxidant capacity of the studied lignins has been determined and compared to commercial antioxidants commonly used in thermoplastic industry.     

Preparation and evaluation of lignosulfonates as a dispersant for gypsum paste from acid hydrolysis lignin

In order to effectively utilize a by-product of the acid saccharification process of woody materials, the chemical conversion of guaiacyl sulfuric acid lignin (SAL), one of the acid hydrolysis lignins, into water-soluble sulfonated products with high dispersibitity was investigated. At first, SAL was phenolated (P-SAL) to enhance the solubility and reactivity. Lignosulfonates were prepared from P-SAL by three methods of hydroxymethylation followed by neutral sulfonation (two-step method), sulfomethylation (one-step method) and arylsulfonation. Surprisingly, all prepared lignosulfonates possessed 30 to 70% higher dispersibility for gypsum paste than the commercial lignosulfonate. Evaluation of the preparations for gypsum paste suggested that the higher molecular weights and sulfur contents of the preparations increased their dispersibility.     

Lignin genetic engineering

Although lignins play important roles in plants, they often represent an obstacle to the utilization of plant biomass in different areas: pulp industry, forage digestibility. The recent characterization of different lignification genes has stimulated research programmes aimed at modifying the lignin profiles of plants through genetic engineering (antisense and sense suppression of gene expression). The first transgenic plants with a modification of monomeric composition of lignins and lignin content have been recently obtained. Down regulation of the OMT gene induces dramatic reduction of syringyl units. CAD down regulated plants exhibit a unusual red phenotype associated with the developing xylem and several chemical modifications of their lignins including an increase in cinnamaldehydes in the polymer structure. Interestingly this novel lignin is removed more easily during the pulping process. In both OMT and CAD down regulated plants no changes in phenotypic characteristics such as growth architecture and morphology were observed. More recent experiments have shown that a reduction of CCR activity determines specific changes in the coloration of the xylem area suggesting significant chemical modifications which are currently being studied.These different results show that it is possible to manipulate lignins through targeted genetic transformation of plants and that lignins exhibit a relative flexibility of their chemical structure. Future developments should probe the impact of down regulating the expression of other recently characterized lignification genes such as F5H and CCoAOMT and also of a combination of genes in order to tailor lignins more adapted to specific purposes. In addition to biotechnological applications which should provide important economical benefits for utilization of wood in the pulp industry, genetic engineering of lignins offer very promising perspectives for the understanding of lignin synthesis, structure and properties.     

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.     

Structural characterization of alkaline hydrogen peroxide pretreated grasses exhibiting diverse lignin phenotypes

Background

For cellulosic biofuels processes, suitable characterization of the lignin remaining within the cell wall and correlation of quantified properties of lignin to cell wall polysaccharide enzymatic deconstruction is underrepresented in the literature. This is particularly true for grasses which represent a number of promising bioenergy feedstocks where quantification of grass lignins is particularly problematic due to the high fraction of p-hydroxycinnamates. The main focus of this work is to use grasses with a diverse range of lignin properties, and applying multiple lignin characterization platforms, attempt to correlate the differences in these lignin properties to the susceptibility to alkaline hydrogen peroxide (AHP) pretreatment and subsequent enzymatic deconstruction.

Results

We were able to determine that the enzymatic hydrolysis of cellulose to to glucose (i.e. digestibility) of four grasses with relatively diverse lignin phenotypes could be correlated to total lignin content and the content of p-hydroxycinnamates, while S/G ratios did not appear to contribute to the enzymatic digestibility or delignification. The lignins of the brown midrib corn stovers tested were significantly more condensed than a typical commercial corn stover and a significant finding was that pretreatment with alkaline hydrogen peroxide increases the fraction of lignins involved in condensed linkages from 88?C95% to ~99% for all the corn stovers tested, which is much more than has been reported in the literature for other pretreatments. This indicates significant scission of ??-O-4 bonds by pretreatment and/or induction of lignin condensation reactions. The S/G ratios in grasses determined by analytical pyrolysis are significantly lower than values obtained using either thioacidolysis or 2DHSQC NMR due to presumed interference by ferulates.

Conclusions

It was found that grass cell wall polysaccharide hydrolysis by cellulolytic enzymes for grasses exhibiting a diversity of lignin structures and compositions could be linked to quantifiable changes in the composition of the cell wall and properties of the lignin including apparent content of the p-hydroxycinnamates while the limitations of S/G estimation in grasses is highlighted.     

On the propensity of lignin to associate: a size exclusion chromatography study with lignin derivatives isolated from different plant species

Despite evidence that lignin associates under both aqueous and organic media, the magnitude and nature of the underlying driving forces are still a matter of discussion. The present paper addresses this issue by examining both solution properties and size exclusion behaviour of lignins isolated from five different species of softwoods, as well as from the angiosperms Eucalyptus globulus and wheat straw. This investigation has used the recently described protocol for isolating enzymatic mild acidolysis lignin (EMAL), which offers lignin samples highly representative of the overall lignin present in the wood cell wall. The molecular weight distributions of these EMALs were found to be dependent upon the wood species from which they were isolated and upon the incubation conditions used prior to size exclusion chromatography. While the chromatograms of EMALs isolated from softwoods displayed a bimodal behaviour, the elution profiles of EMAL from E. globulus and straw were nearly unimodal. A marked tendency to dissociate prevailed under incubation at room temperature for all examined species with the exception of the straw lignin preparation; furthermore, lignin solutions incubated at 4 degrees C showed an associative behaviour manifested by an increase in the weight and number average molecular weights for some species. The extent of such association/dissociation, as well as the time needed for the process to reach completion, was also found to depend upon the wood species, i.e. lignins from softwoods were found to associate/dissociate to a greater extent than lignins from E. globulus and straw. The origin of such effects within the lignin structure is also discussed.     

Steam explosion lignins; their extraction,structure and potential as feedstock for biodiesel and chemicals

In the present study, a steam explosion wood pre-treatment process, optimized earlier with respect to ethanol production, has been applied to both softwoods (Picea abies and Pinus sylvestris) and hardwoods (Betula verrucosa and Populus tremula). The alkaline extractable lignins have then been isolated to investigate lignin separation efficiency and lignin structure and to evaluate their potential for producing value-added products, such as biodiesel components or chemicals, in terms of the purity, molecular size, functional groups, β-O-4′ inter-unit linkage content, and degradability in a subsequent processing treatment. The mechanism of lignin modification and possible improvements to the steam explosion pre-treatment process are discussed.     

Rapid Degradation of Isolated Lignins by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded purified Kraft lignin, alkali-extracted and dioxane-extracted straw lignin, and lignosulfonates at a similar rate, producing small-molecular-weight (~1,000) soluble products which comprised 25 to 35% of the original lignins. At concentrations of 1 g of lignin liter⁻¹, 90 to 100% of the acid-insoluble Kraft, alkali straw, and dioxane straw lignins were degraded by 1 g of fungal mycelium liter⁻¹ within an active ligninolytic period of 2 to 3 days. Cultures with biomass concentrations as low as 0.16 g liter⁻¹ could also completely degrade 1 g of lignin liter⁻¹ during an active period of 6 to 8 days. The absorbance at 280 nm of 2 g of lignosulfonate liter⁻¹ increased during the first 3 days of incubation and decreased to 35% of the original value during the next 7 days. The capacity of 1 g of cells to degrade alkali-extracted straw lignin under optimized conditions was estimated to be as high as 1.0 g day⁻¹. This degradation occurred with a simultaneous glucose consumption rate of 1.0 g day⁻¹. When glucose or cellular energy resources were depleted, lignin degradation ceased. The ability of P. chrysosporium to degrade the various lignins in a similar manner and at very low biomass concentrations indicates that the enzymes responsible for lignin degradation are nonspecific.     

Conversion of native lignin to a highly phenolic functional polymer and its separation from lignocellulosics

An original reaction system (the phase separative reaction system) has been designed for derivatizing native lignins to highly phenolic, functional polymers. This system is composed of a phenol derivative and concentrated acid, which are not miscible at room temperature. The key point of the lignin functionalization process, including the phase separative system, is that lignin and carbohdrates, which are totally different in structures and reactivitie, are modified individually in the different phases: lignin is present in the organic phase and carbohydrates in the aqueous phase. Through the process, lignin was modified selectively at Calpha-positions of side chains, the most reactive sites, to give highly phenolic, light-colored, diphenylmethane-type materials which still retained original interunit linkages formed by the dehydrogenative polymerization during the biosynthesis. The carbohydrates were swollen, followed by partial hydrolysis and dissolution in the acid solution, resulting in the perfect decomposition of interpenetrating polymer network structures in the cell wall. Therefore, the functionalization of lignin and the separation of resulting lignin from carbohydrates were quickly achieved at room temperature, independent of wood species. This process would be a powerful tool for estimating strutures and reactivities of lignins as well as the functionalization of lignins, because of the selective structural modifications. (c) 1995 John Wiley & Sons, Inc.     

CCoAOMT suppression modifies lignin composition in Pinus radiata

A cDNA clone encoding the lignin‐related enzyme caffeoyl CoA 3‐O‐methyltransferase (CCoAOMT) was isolated from a Pinus radiata cDNA library derived from differentiating xylem. Suppression of PrCCoAOMT expression in P. radiata tracheary element cultures affected lignin content and composition, resulting in a lignin polymer containing p‐hydroxyphenyl (H), catechyl (C) and guaiacyl (G) units. Acetyl bromide‐soluble lignin assays revealed reductions in lignin content of up to 20% in PrCCoAOMT‐deficient transgenic lines. Pyrolysis‐GC/MS and 2D‐NMR studies demonstrated that these reductions were due to depletion of G‐type lignin. Correspondingly, the proportion of H‐type lignin in PrCCoAOMT‐deficient transgenic lines increased, resulting in up to a 10‐fold increase in the H/G ratio relative to untransformed controls. 2D‐NMR spectra revealed that PrCCoAOMT suppression resulted in formation of benzodioxanes in the lignin polymer. This suggested that phenylpropanoids with an ortho‐diphenyl structure such as caffeyl alcohol are involved in lignin polymerization. To test this hypothesis, synthetic lignins containing methyl caffeate or caffeyl alcohol were generated and analyzed by 2D‐NMR. Comparison of the 2D‐NMR spectra from PrCCoAOMT‐RNAi lines and synthetic lignins identified caffeyl alcohol as the new lignin constituent in PrCCoAOMT‐deficient lines. The incorporation of caffeyl alcohol into lignin created a polymer containing catechyl units, a lignin type that has not been previously identified in recombinant lignin studies. This finding is consistent with the theory that lignin polymerization is based on a radical coupling process that is determined solely by chemical processes.     

Lignin incorporation combined with electron-beam irradiation improves the surface water resistance of starch films

We investigated the potential of an electron-beam post-treatment to tailor the properties of 70/30 and 80/20 wt. extruded starch-lignin films. The effect of a 400 kGy radiation on films differing essentially by the kind of lignins incorporated (lignosulfonates/alkali lignins) was assessed both at the macroscopic and the molecular levels. Changes in the polymer molecular structure were studied by IR spectroscopy, by thioacidolysis as well as by model compound experiments. Electron beam-irradiation at 400 kGy, a rather high dose for processing natural polymers, alters to some extent the mechanical resistance of the starch-based materials. However this treatment substantially reduces the hydrophilic surface properties of the films, while not harming their biodegradability. Involved in radical cross-coupling reactions, lignin phenolic compounds are likely to play a primary role in the formation of a hydrophobic condensed network. This study suggests that lower irradiation doses might yield biomaterials with improved usage properties.     

Elicitor-Induced Spruce Stress Lignin (Structural Similarity to Early Developmental Lignins)

Suspension cultures of Picea abies (L.) Karst released polymeric material into the culture medium when treated with an elicitor preparation from the spruce needle pathogen Rhizosphaera kalkhoffii. The presence of lignin (about 35%, w/w) was demonstrated by phloroglucinol/HCI reactivity and quantitation with thioglycolic acid. Carbohydrate (about 14%, w/w) and protein (about 32%, w/w) were also detected. Amino acid analysis revealed that hydroxyproline and proline predominated. Thioacidolysis and subsequent Raney nickel desulfurization allowed the analysis of lignin-building units and interunit bonds. Compared with spruce wood lignin, an approximately 20-fold higher relative amount of p-hydroxyphenyl units was determined. A high content of p-hydroxyphenyl units is typical for certain developmental lignins, such as conifer compression wood and middle lamella lignins, as well as all induced cell culture lignins so far analyzed. Cross-linkages of the pinoresinol type ([beta]-[beta]) in the excreted cell culture lignin were markedly increased, whereas [beta]-1 interunit linkages were decreased relative to spruce wood lignin. The amount and nature of cross-linkages were shown to be intermediate between those in wood lignin and in enzymatically prepared lignins. In summary, the elicitor-induced stress lignin was excreted as a lignin-extensin complex that closely resembled early developmental lignins.     

Macromolecular lignin replication: A mechanistic working hypothesis

At the time of the first realization that the last step in lignin biosynthesis involves lignol radical coupling, it was difficult to envisage how such a process could be regiospecifically controlled. It was thus natural to expect that lignin macromolecules should have random primary structures. This has now been the prevailing assumption for almost fifty years, but of its correctness there has been no clear proof. Rather there have been occasional but insistent indications that lignins cannot just be products of random monolignol dehydropolymerization. Thus the present article seeks to apprehend the mechanistic implications of a situation where lignin primary structure would be determined by the sequence of interunit linkages along each biopolymer chain. The ramifications of a simple working hypothesis, that macromolecular lignin replication might occur directly through a template polymerization mechanism, are explored in detail. The manner in which the fidelity of the process could be maintained, through specific π-orbital interactions between the lignol radical precursors and characteristic substructures in the pre-existing lignin macromolecules, is explicitly described. The consequences of template polymerization are shown to be consistent with the absence of both optical activity and crystallinity in macromolecular lignin domains. It is proposed that the inherent primary structures of lignins are encoded in contiguous ‘dirigent’ arrays of lignol radical coupling sites distributed along individual polypeptide chains within lignifying plant cell walls. This revised version was published online in June 2006 with corrections to the Cover Date.     

Macromolecular replication during lignin biosynthesis

Lignins play a crucial role in the cell-wall architecture of all vascular plants. They are composed of p-hydroxyphenylpropanoid units interconnected through covalent bonds formed during lignol radical coupling between six different pairs of atomic centers. For 50 years, the primary structures of lignins have been thought to be random, but for a number of reasons such an assumption is not tenable. For example, it has been reported that, by simple physicochemical means, the rather recalcitrant lignins in spruce wood can be decisively separated into two fractions containing quite dissimilar biopolymer chains. Thus, a paradigm shift should be imminent, and a detailed working hypothesis for the mechanism of lignin biosynthesis would be invaluable in delineating how the process of macromolecular lignin assembly can be properly investigated. In conjunction with an earlier experimental result, an explicit model for a template dehydropolymerization process has been developed that describes how lignin primary structure is replicated. The strengths of the powerful noncovalent interactions have been calculated that control the transient placement of lignol radicals about to undergo coupling on a double-stranded lignin template. These elementary steps engender, in the growing daughter chain, a primary structure identical to that of the distal template strand. The interactions are governed by dynamical electron correlation in the π-orbitals of each immobilized lignol radical and the complementary aromatic ring in the antiparallel proximal strand. The resulting noncovalent forces are computed to be stronger than those stabilizing GC/CG base pairs in DNA double-helices, but the mechanism of replication is fundamentally different from that of any other biopolymer.     

Exogenous chalcone synthase expression in developing poplar xylem incorporates naringenin into lignins

Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of noncanonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentata) to express chalcone synthase 3 (MdCHS3) derived from apple (Malus domestica) in lignifying xylem. Transgenic trees displayed an accumulation of the flavonoid naringenin in xylem methanolic extracts not inherently observed in wild-type trees. Nuclear magnetic resonance analysis revealed the presence of naringenin in the extract-free, cellulase-treated xylem lignin of MdCHS3-poplar, indicating the incorporation of this flavonoid-derived compound into poplar secondary cell wall lignins. The transgenic trees also displayed lower total cell wall lignin content and increased cell wall carbohydrate content and performed significantly better in limited saccharification assays than their wild-type counterparts.

Expressing exogenous, apple-derived chalcone synthase in actively lignifying poplar xylem tissue results in less total lignin, improved saccharification rates, and incorporation of naringenin into lignins.     

Polymer properties of softwood organosolv lignins produced in two different reactor systems

Lignin, the second most abundant biopolymer on earth and with a predominantly aromatic structure, has the potential to be a raw material for valuable chemicals and other bio-based chemicals. In industry, lignin is underutilized by being used mostly as a fuel for producing thermal energy. Valorization of lignin requires knowledge of the structure and different linkages in the isolated lignin, making the study of structure of lignin important. In this article, lignin samples isolated from two types of reactors (autoclave reactor and displacement reactor) were analyzed by FT-IR, size exclusion chromatography, thermogravimetric analysis (TGA), and Py-GC-MS. The average molecular mass of the organosolv lignins isolated from the autoclave reactor decreased at higher severities, and FT-IR showed an increase in free phenolic content with increasing severity. Except for molecular mass and molecular mass dispersity, there were only minor differences between lignins isolated from the autoclave reactor and lignins isolated from the displacement reactor. Carbohydrate analysis, Py-GC–MS and TGA showed that the lignin isolated using either of the reactor systems is of high purity, suggesting that organosolv lignin is a good candidate for valorization.     

Hydroxymethylation and oxidation of Organosolv lignins and utilization of the products

Organosolv lignins obtained from Eucalyptus grandis, sugarcane bagasse and Picea abies by Acetosolv, Formacell and Organocell processes were characterized, fractionated and converted to hydroxymethylated and oxidized products. The reactivity of lignins with formaldehyde did not improve significantly with the fractionation. Both eucalyptus Acetosolv (EAc) and eucalyptus Formacell (EFo) lignins retained high heterogeneity in relation to the molecular weight distribution but not in relation to structural units. The temperatures of the exothermic peaks and the apparent activation energies for the cross-linking are different for hydroxymethylated lignins and phenol, with similar cure temperatures of the resols. Chemical oxidation using cobalt(II) and manganese(II) salts furnished oxidized lignins with improved chelating properties. These chelating agents can remove up to 14% of Mn present in pulps, decreasing the peroxide consumption in the bleaching process. The products obtained can be also used as oxidized phenols and controlled-release matrices. Oxidation of Acetosolv bagasse lignin with polyphenol oxidase furnishes lignins with chelating capacity 110% higher than that of original lignin.