Investigation of the dihydroxylignin ofAlthaea rhyticarpa

Summary 1. The developed semiempirical formula for the dioxane lignin from the stems ofAlthaea rhyticarpa has been found from the results of elementary and functional analyses.2. It has been shown that the lignin consists of high- and low-molecular-weight fractions.3. It has been shown by the nitrobenzene oxidation method that p-coumaryl, guaiacyl, and syringyl structural units are present in the lignin.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 242–246, March–April, 1976.     

The lignin of the algaFucus vesiculosus

Summary 1. The plant material ofFucus vesiculosus has been treated with metallic sodium in liquid ammonia. The total yield of products extractable by ether was 1.1%.2. In the products of the decomposition of the lignin ofFucus vesiculosus 12 phenols and acids have been identified by paper chromatography and gas-liquid chromatography and it has thereby been established that the lignin of algae consists of p-coumaryl, guaiacyl, and syringyl structural units.3. The composition of the decomposition products confirms the hypothesis expressed previously that the brown algaFucus vesiculosus contains lignin.S. M. Kirov Belorussian Technological Institute, Minsk. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 645–647, September–October, 1978.     

Lignin biodegradation by the ascomyceteChrysonilia sitophila

The lignin biodegradation process has an important role in the carbon cycle of the biosphere. The study of this natural process has developed mainly with the use of basidiomycetes in laboratory investigations. This has been a logical approach since most of the microorganisms involved in lignocellulosic degradation belong to this class of fungi. However, other microorganisms such as ascomycetes and also some bacteria, are involved in the lignin decaying process. This work focuses on lignin biodegradation by a microorganism belonging to the ascomycete class,Chrysonilia sitophila. Lignin peroxidase production and characterization, mechanisms of lignin degradation (lignin model compounds and lignin in wood matrix) and biosynthesis of veratryl alcohol are outstanding. Applications of C.sitophila for effluent treatment, wood biodegradation and single-cell protein production are also discussed.     

The first 85% kraft lignin-based thermoplastics

Interest in the development of lignin-containing polymeric materials has been upheld more or less continuously for the past 20 years. Tendencies toward high moduli and poorly defined thermal transitions have been regarded as imposing inevitable limitations upon the use of lignin derivatives for such purposes. Incorporation of more than 25–40% (w/w) lignin had usually resulted in materials that were brittle and weak. For the first time, however, from homogeneous blends containing 85% (w/w) underivatized industrial kraft lignin with poly(vinyl acetate) and two plasticizers, a series of thermoplastics has been fabricated with promising mechanical properties. The tensile behavior of these new polymeric materials depends directly upon the degree of association between the intrinsic kraft lignin components. In extending to values about 25 MPa and 1.5 GPa, respectively, the tensile strengths and Young's moduli vary linearly with the effective M̄_w for the kraft lignin species, under conditions where the proportions of the individual molecular components, both associated and discrete, do not change. Moreover, melt-flow index measurements indicate that these polymeric materials are amenable to thermal processing by extrusion molding. Thus a significant step has been taken toward developing a new generation of thermoplastics that are lignin-based in a very fundamental way. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 1899–1910, 1997     

Enzymes involved in cellulose and lignin degradation

A detailed presentation was given of the discovered and studied enzymes involved in degradation of cellulose and lignin by the white-rot fungus,Sporotrichum pulverulentum (Phanerochaete chrysosporium). The fungus utilizes, for the degradation of cellulose: (a) Five different endo-1,4-Β-glucanases (b) One exo-1,4-Β-glucanase (acting synergistically with the endoglucanases) (c) Two 1,4-Β-glucosidases The regulation, induction, and catabolite repression of the endoglucanases have been studied in depth and the results of these studies were also presented. In addition to the hydrolytic enzymes,S. pulverulentum also produces the oxidative enzyme cellobiose oxidase that is of importance for cellulose degradation. Another unconventional enzyme is cellobiose: quinone oxidoreductase, which is of importance for both cellulose and lignin degradation. It reduces quinones from the lignin under oxidation of cellobiose from the cellulose. It has recently been discovered thatS. pulverulentum produces two acidic proteases of importance for cellulose degradation since they enhance the endoglucanase activity, particularly in young cultures of the fungus grown on cellulose. The enzymes involved in lignin degradation are not known nearly as well as these involved in cellulose degradation. However, extracellular phenol oxidases, laccase, and peroxidase have been shown to be involved in and necessary for lignin degradation to take place. A phenol oxidase-less mutant ofS. pulverulentum cannot degrade lignin unless a phenol oxidase is added to the medium. Recently, an enzyme splitting the α—Β bond in the propane side chain has been discovered by Kirk and coworkers. Several enzymes involved in the metabolism of vanillic acid, always a metabolite in lignin degradation, have been discovered and studied in our laboratory. Presentations of the enzymes for decarboxylation, demethoxylation, methanol oxidation, ring cleavage, and intracellular quinone reduction by NAD(P)H: quinone oxidoreductase were given. A discussion of possibilities for a specific enzymic primary attack on the native lignin, as well as of the likeliness for an unspecific radical nature of this attack, was also given.     

Molecular genetics ofPhanerochaete chrysosporium

InPhanerochaete chrysosporium ME446 lignin degradation is a secondary metabolic event triggered by carbon and nitrogen limitation. It is therefore possible to study lignin degradation at the level of gene expression by comparing mRNA populations produced during primary and secondary growth in both wild-type strains and in strains mutant in lignin degradation. We have isolated mutants deficient in phenol oxidase activity. These mutants fall into three phenotypic categories with respect to lignin degradation: (1) negative, (2) delayed onset after nitrogen starvation, (3) enhanced. Polyacrylamide gel electrophoresis of rabbit reticulocyte polypeptide translation products ofPhanerochaete mRNA shows differences between populations from primary and secondary growth. Differences in the range of polypeptides (and therefore of mRNA) have also been demonstrated between a mutant and its wild-type progenitor under identical conditions. A gene bank has been prepared from P.chrysosporium strain ME446 genomic DNA using a bacteriophage λ vector. This gene bank is being screened with labeled mRNA from secondary growth mycelium in the presence of excess competing cold RNA from primary growth mycelium. Using this method (and/or using labeled cDNA probes), we hope to isolate clones carrying sequences expressed only during lignin degradation. A gene bank has also been constructed fromSporotrichum pulverulentum (Novobranova), which is on morphological criteria considered to be the imperfect form ofP. chrysosporium. DNA probes from randomly chosen clones of both gene banks have been hybridized to restricted and electrophoresed total DNA of the two “gene bank” strains and of two other isolates ofP. chrysosporium on Southern blots. We found very strong DNA homology between the two “gene bank” strains, but far less homology between these two strains and the two others. These degrees of relationships were supported by the analysis of mitochondrial DNA from the four strains. We thank the Agricultural Research Council and the British Petroleum Venture Research Unit for support.     

PMR spectra of the lignins ofCystoseira barbata at different ages

The PMR spectra of the dioxane lignins from the brown algaCystoseira have been studied. It has been established that the lignins isolated from specimens ofCystoseira of different ages have different degrees of substitution of the C₃ side chain and differ in their degree of condensation. The most highly condensed is the dioxane lignin of young specimens ofCystoseira (age up to one year) and the least condensed the Björkman lignin ofCystoseira aged more than three years. All the PMR spectra of preparations of the lignin of the alga under investigation contain the signals of protons present in coumaran structures, their amount being the greatest in the lignins of the youngest samples and decreasing with increasing age of the alga.Odessa Institute of Structural Engineering. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 91–94, January–February, 1983.     

Profiles of extracellular proteins produced byCoriolus versicolor under ligninolytic and nonligninolytic growth conditions

The wood-rotting basidiomyceteCoriolus versicolor has been grown under a variety of conditions ranging from stationary cultures on spruce wood chips or milled-wood lignin, known to be actively ligninolytic, to agitated submerged cultures, with glucose or carboxymethylcellulose as the main carbon source, that had no ligninolytic activity. Extracellular proteins have been recovered from the growth medium by ammonium sulfate precipitation and fractionated into their polypeptide components by a combination of ion exchange, affinity column chromatography, and polyacrylamide gel electrophoresis, thus providing a “fingerprint” technique for different growth conditions. Characterization of some of the polypeptide components on the PAGE plates can be made by the use of selected staining techniques for proteins, glycoproteins, peroxidase activity, and heme-containing polypeptides. Variations in the “fingerprints” from different cultures can be demonstrated, in addition to showing the development of the extracellular protein population in an actively ligninolytic culture during the change from primary to secondary growth phases. The effect of some of the extracellular enzymes on polymeric lignin has been demonstrated. A crude protein extract isolated from rotting wood chips was incubated with milled-wood lignin extracted from spruce sapwood. Analysis of the lignin after 48 h incubation by UV and NMR spectroscopy showed there to be an increase in aromatic hydroxyl groups with a decrease in aliphatic hydroxyl groups in comparison with sound milled-wood lignin. There was also a small reduction in the mean molecular weight of the lignin, analyzed by HPLC size-exclusion chromatography. By contrast, lignin that had been incubated with purified laccase A showed a considerable increase in the mean molecular weight, almost doubling over a 48-h period of incubation.     

Gene Expression in Secondary Metabolism and Metabolic Switching Phase of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Ligninolytic enzymes are well-known to play the crucial roles in lignin biodegradation and have potential applications in industrial processes. The filamentous white-rot fungus, Phanerochaete chrysosporium, has been widely used as a model organism for studying these ligninolytic enzymes that are able to degrade the lignin during the secondary metabolism. To study the gene expression in secondary metabolism and metabolic switching phase of P. chrysosporium, we constructed a metabolic-switching phase suppression subtractive hybridization (SSH) cDNA library and a secondary metabolic phase SSH cDNA library to compare their mRNA expression profiles. We isolated the genes that are specially expressed and subsequently identified four genes that specially expressed during metabolic-switching phase while 22 genes in secondary metabolic phase. Accordingly, these specially expressed genes might play key roles in different metabolic stages, which would offer more new insights into the shift from nitrogen to lignin metabolism.     

Radical grafting on lignin. Part I. Radiation-induced grafting of styrene onto hydrochloric acid lignin

By irradiation with gamma rays styrene was grafted onto hydrochloric acid lignin. When the graft polymers were subjected to nitrobenzene oxidation, the vanillin yields indicated two kinds of reaction occurring in the grafting. Polystyrene branches were separated from the graft polymers, and their M?_n were determined osmometrically. At grafting ratios of up to 100 the vanillin yields diminished proportionately with increasing grafting, and the M?_n of the branches, 5000, was unchanged. At grafting ratios of more than 100 the vanillin yields were constant, independent of the ratios, but the M?_n values of the branches increased with grafting. Paper chromatography of the aromatic acids obtained by oxidation of methylated lignin and the graft polymer indicated that isohemipic and metahemipic acids were more abundant in the acid fraction of the graft polymer than in the lignin itself. A qualitative mass analysis of the gaseous products evolving from the irradiated lignin showed the presence of hydrogen molecules only. Gamma-ray radiation brought about no change in the yields of vanillin. It was therefore concluded that radiation grafting on lignin at grafting ratios of less than 100 proceeded through the addition of the styrene polymer radicals to the aromatic nuclei of the lignin and that then branches propagated from the aliphatic part of the lignin, where C? H bond scission had been caused by the irradiation. The grafting sites of lignin would be C-5 and C-6 of the guaiacyl nucleus and, probably the β and γ carbon atoms of the aliphatic side chain of the lignin.     

Can delignification decrease cellulose digestibility in acid pretreated corn stover?

It has previously been shown that the improved digestibility of dilute acid pretreated corn stover is at least partially due to the removal of xylan and the consequent increase in accessibility of the cellulose to cellobiohydrolase enzymes. We now report on the impact that lignin removal has on the accessibility and digestibility of dilute acid pretreated corn stover. Samples of corn stover were subjected to dilute sulfuric acid pretreatment with and without simultaneous (partial) lignin removal. In addition, some samples were completely delignified after the pretreatment step using acidified sodium chlorite. The accessibility and digestibility of the samples were tested using a fluorescence-labeled cellobiohydrolase (Trichoderma reesei Cel7A) purified from a commercial cellulase preparation. Partial delignification of corn stover during dilute acid pretreatment was shown to improve cellulose digestibility by T. reesei Cel7A; however, decreasing the lignin content below 5% (g g⁻¹) by treatment with acidified sodium chlorite resulted in a dramatic reduction in cellulose digestibility. Importantly, this effect was found to be enhanced in samples with lower xylan contents suggesting that the near complete removal of xylan and lignin may cause aggregation of the cellulose microfibrils resulting in decreased cellulase accessibility.     

Solution and solid state13C-NMR of barley straw lignins

Grass lignins are formed by the polymerization of phenoxy radicals and contain a variety of carbon-carbon and carbon-oxygen bonds. They are similar to the hardwood lignins, but differ by containing a substantial proportion of esterified cinnamic acids. Detailed nuclear magnetic resonance studies in conjunction with chemical analysis have given new information on the structure of grass lignins. Milled straw lignins (MSL) from barley were examined by both solution and solid-state (CP/MAS) NMR before and after acetylation. The assignment of the carbon-13 (100 MHz) solution spectra was achieved using model compound data, nuclear Overhauser enhancement (NOE) suppression, and insensitive nuclei enhanced by polarization transfer (INEPT) techniques. The NOE suppression permitted quantitative analysis of lignin, giving information on the ratio of specific carbon atoms. Use of the relaxation agent, chromium acetylacetonate, enabled accumulation of sufficient spectral data to give a spectrum suitable for integration after 90 h. The INEPT technique, which had not previously been used for lignin analysis, was successfully applied to acetylated MSL. This technique increased signal intensities 3–4-fold and simplified the spectrum by inverting methylene carbons and eliminating or inverting quaternary carbons. Comparison of this spectrum with the normal spectrum permitted accurate assignment of quaternary and methine carbons. The solid-state carbon-13 CP/MAS NMR was used to examinein situ lignin and the isolated MSL. The¹³C-CP/MAS spectrum ofin situ lignin shows that cellulose and hemicellulose resonances dominate with little evidence ot the aromatic structure of lignin. the¹³C-CP/MAS of MSL shows reduced carbohydrate resonances and increased aromatic resonances. The extent of modification to the barley straw was estimated and results indicate the presence oflignin-carbohydrate complexes. Detailed information on the nature of the linkages between lignin components and between lignin and carbohydrate components has been obtained from these spectra.     

Fourier-transform Raman spectroscopic study of a Neolithic waterlogged wood assemblage

The use of Fourier-transform Raman spectroscopy for characterising lignocellulosics has increased significantly over the last twenty years. Here, an FT-Raman spectroscopic study of changes in the chemistry of waterlogged archaeological wood of Pinus sp. and Quercus sp. from a prehistoric assemblage recovered from northern Greece is presented. FT-Raman spectral features of biodeteriorated wood were associated with the depletion of lignin and/or carbohydrate polymers at various stages of deterioration. Spectra from the archaeological wood are presented alongside spectra of sound wood of the same taxa. A comparison of the relative changes in intensities of spectral bands associated with lignin and carbohydrates resulting from decay clearly indicated extensive deterioration of both the softwood and hardwood samples and the carbohydrates appear to be more deteriorated than the lignin. The biodeterioration of the archaeological timbers followed a pattern of initial preferential loss of carbohydrates causing significant loss of cellulose and hemicellulose, followed by the degradation of lignin.     

Beech Lignin—Proposal of a Constitutional Scheme

The high molecular weight material lignin consists of phenylpropane units linked together by a variety of bond types. During the past eight years, two newly developed degradation procedures have permitted the first direct determinations of the nature of these bonds. The first procedure affords a very mild partial hydrolysis of benzyl ether bonds. Eleven dimeric, trimeric, and tetrameric degradation products were obtained in this way from spruce and beech lignin: they exhibited three different kinds of bonds between the C₉ structural units, and their structures have all been elucidated. In the second procedure, the most important kind of bond in lignin, i. e. the arylglycerol-β-aryl ether bond, can be subjected to directed cleavage under mild conditions after introduction of a suitable neighboring group. On application to beech lignin, 91 % of the material was degraded giving monomeric to tetrameric phenols. Complete structural elucidation of the twenty dimeric phenols isolated and a knowledge of their relative yields and the yields of the other fractions obtained by gel filtration permitted a structural scheme to be set up for beech lignin in which the C₉ structural units are linked together by no less then ten different kinds of bonds. The structural scheme, which can be readily explained biogenetically, has the same elemental composition as natural beech lignin. Further support for the structural scheme comes from a comparison of the ¹³C-NMR spectrum of natural beech lignin and a ¹³C-NMR spectrum calculated for the proposed structure on the basis of about fifty lignin model substances.     

Destruction of aspen wood by the fungusPhanerochaete sanguinea quantitative1H and13C NMR spectroscopies of biologically degraded lignin

The lignin of mechanically ground aspen wood and lignins isolated from aspen wood attacked by the fungusPhanerochaete sanguinea have been investigated by quantitative¹H and¹³C NMR spectroscopies. It has been shown that the biodestruction of the lignin takes place through the cleavage of alkyl-aryl and aryl-aryl bonds and is accompanied by demethylation (demethoxylation) reactions, and the oxidation of C and C atoms. In addition to reactions in which the C—C bonds are cleaved, the formation of ether bonds has been observed. An interconnection has been shown between the variations in the amount of functional groups, fragments, and the bonds in biolignins and the loss in mass of the wood. A method is proposed for evaluating the carbohydrate content in lignin preparations using the NMR method.Wood Chemistry Branch of the Institute of Organic Chemistry, Siberian Division of the Russian Academy of Sciences, Irkutsk. Irkutsk State University. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 547–557, July–August, 1994.     

Polymerization of lignin model compounds with formaldehyde in acidic aqueous medium

The reaction of formaldehyde with lignin model compounds in acidic medium was shown to give fast crosslinking of alkyl-substituted phenolic and etherified phenolic lignin model compounds at positions meta to the aromatic hydroxy groups. This reaction differs from the reaction of formaldehyde with phenolic lignin model compounds in alkaline conditions, where the reaction with formaldehyde always occurs at positions ortho/para to the aromatic hydroxy group., The reaction of formaldehyde with lignin in acidic medium have considerable potential for the crosslinking of lignin, particularly heavily condensed alkali lignin, for use in polymeric products.     

Studies of grass forages and their fibrous fractions by pyrolysis gas chromatography – ion trap detection

Grass forage samples with different fiber content and composition, and their fibrous fractions, have been analyzed by pyrolysis – gas chromatography with ion trap detection. The identities of the pyrolysis fragments indicated their origin to be carbohydrates, lignin, and nitrogen compounds. Comparison of the resulting quantitative data by factorial variance revealed pyrogram peaks attributable to neutral detergent fiber, cellular contents, and lignin. Quantitative pyrolysis data from these components showed the same trends as those recorded by the method of Goering and Van Soest [1].     

Inhibiting properties of lignin

Inhibitory efficacy has been determined by the method of the weight losses of steel in acid with native lignin, hydrolysis lignin, ammoniated native lignin, chlorinated hydrolysis lignin, and ammoniated hydrolysis lignin, and without these additives. It has been established that the efficacy of lignin and its modifications as inhibitors of acid corrosion increases with a rise in the number of carboxy groups in the macromolecule, and therefore the ammoniated hydrolysis lignin is the most effective.Central Scientific-Research and Planning Technological Institute for the Mechanization and Electrification of Animal Husbandry of the Southern Zone of the USSR. Zaporozh'e Astrakhan Technical Institute of the Fisheries Industry and Economy, Astrakhan'. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 378–380, May–June, 1983.     

Degradation of lignin by ozone. I. The kinetics of lignin degradation of ozone

The ozonolysis of spruce periodate and cuoxam lignins and protolignin in spruce wood has been studied in 45% aqueous acetic acid at room temperature. Stirring affected the rate of reaction and a tentative explanation is given. Degradation followed first-order kinetics characterized by a rate constant K with values of 6.96 × 10^?4s^?1 for periodate lignin, 5.10 × 10^?4s^?1 for cuoxam lignin, and 5.09 × 10^?4s^?1 for protolignin in spruce wood. The similarity of the rate constants shows (1) that periodate and cuoxam lignins are good models for wood lignin and (2) that the carbohydrate matrix has an insignificant effect on the rate of delignification of the protolignin by ozone. The average rate of ozone consumption per C₉ unit for periodate lignin was determined as 0.12 mol/min and 0.08 mol/min for cuoxam lignin. The implications of the various results are discussed.     

Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds

Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer, it has been a challenge to effectively depolymerize lignin and produce high‐value chemicals with high selectivity. In this study, a highly efficient lignin‐to‐monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC‐H) and monomeric phenolic acid compounds (MPC‐A) including 4‐hydroxy‐2‐methoxyphenol, p‐hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4‐dihydroxybenzoic acid. The maximized MPC yields obtained were 18 and 22 % based on the initial weight of the lignin in SESPL and DACSL, respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47 %. The key reaction steps and main mechanisms involved in this new lignin‐to‐MPC valorization pathway were investigated and elucidated.