Consumption of water-insoluble phenolic products of lignin pyrolysis by the strain Penicillium tardum H-2

Physiological and biochemical properties of the strain Penicillium tardum H-2, a degrader of phenolic compounds formed during lignin pyrolysis, have been characterized. The micromycete P. tardum H-2 can consume phenol, catechol, p-cresol, vanillin, and guaiacum tar. When grown in a medium containing a water-soluble fraction of lignin pyrolysis waste at concentrations from 0.5 to 2%, it consumes 62–72% of the phenolic components of the waste. According to gas-liquid chromatography, cultivation of P. tardum H-2 in a medium with liquid pyrolysis products results in a complete consumption of the phenol-cresol fraction.     

Anaerobic treatability of waste water from pulp and paper industries

A black liquor evaporator condensate from a Kraft mill and a waste water from production of corrugating medium were anaerobically treated on a laboratory scale. The composition of the waste waters was determined before and after treatment in fixed bed reactors.

Toxicity studies by the Microtox-method showed that both waste waters were highly toxic and a slight decrease in toxicity was achieved by anaerobic treatment. Despite the toxicity efficient anaerobic treatment was obtained.

Major components of the condensate were methanol, ethanol, acetone, guaiacol, hydrogen sulfide and dimethyl disulfide. Anaerobic treatment reduced the concentration of the major components considerably with one exception. The concentration of hydrogen sulfide was unchanged.

Organic overloading of the fixed bed reactor or a temperature drop resulted in an accumulation of acetone, although methanol and ethanol were degraded.

Major components of the waste water from the production of corrugating medium were: Klason-lignin, acid-soluble lignin, carbohydrates, extractives and ash. When the fixed bed reactor was operated at a volumetric load of 1.6 kg COD/m³.d the following reductions were obtained:

Klason - lignin (solids fraction 84%; soluble and colloidal fraction 76%), acid-soluble lignin (solids fraction 56%; soluble and colloidal fraction 7%), carbohydrates (100%), extractives (71%), total-S (80%), COD (73%) and BOD₇ (78%).

Kinetic studies showed that condensate was more easily degraded anaerobically than corrugating medium waste water.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

Production of monomeric phenols by thermochemical conversion of biomass: a review

Biomass is a renewable and alternative source for the production of fuels and chemicals. This paper provides a brief survey of lignin precursors as well as thermogravimetric and pyrolysis studies of lignin with special reference to the production of phenols. Thermogravimetric analysis provides information on pyrolysis kinetics while thermogravimetry in combination with mass or infrared spectrometers allowed a rapid characterization of the vapours produced by thermal treatment. Pyrolysis enabled even greater insight into the thermal behaviour of lignin. Pyrolysis of single, dimeric and trimeric model lignin compounds can determine the thermal stability of the intermediate compounds formed and the origin of the pyrolysis products. A free radical mechanism has been suggested as a major route during the early lignin degradation stages followed by a combined free radical and concerted pathway at elevated temperatures. Pyrolysis of lignin in the presence of catalysts as additives was investigated. Significant differences in terms of yields of pyrolysis products and phenolic compounds were observed. The addition of salts resulted in a high weight loss at low temperature and yielded more char than untreated wood. Some metal catalysts such as transition metals and metal oxides such as Fe2O3 and Cu exhibited a better activity in terms of selectivity for the degradation of lignin.     

Preferential degradation of phenolic lignin units by two white rot fungi.

The differential biodegradation of phenolic and nonphenolic (C-4-etherified) lignin units in wheat straw treated with the white rot fungi Pleurotus eryngii and Phanerochaete chrysosporium was investigated under solid-state fermentation conditions. Two analytical techniques applied to permethylated straw were used for this purpose, i.e., alkaline CuO degradation and analytical pyrolysis (both followed by gas chromatography-mass spectrometry for product identification). Despite differences in the enzymatic machinery produced, both ligninolytic fungi caused a significant decrease in the relative amount of phenolic lignin units during the degradation process. Nevertheless, no differences in the biodegradation rates of phenolic and etherified cinnamic acids were observed. Changes in lignin composition and cinnamic acid content were also analyzed in the phenolic and nonphenolic lignin moieties. The results obtained are discussed in the context of the enzymatic mechanisms of lignin biodegradation.     

Degradation of non-phenolic lignin by the white-rot fungus Pycnoporus cinnabarinus

High-molecular-weight lignin was methylated with diazomethane. The lignin (i.e., phenolic lignin) and methylated lignin (i.e., non-phenolic lignin) were mixed with fully bleached softwood pulp. Degradation of the lignin preparations by the white rot fungus Pycnoporus cinnabarinus was studied. After a 3-month incubation with the fungus, over 40% of the non-phenolic lignin and about 70% the phenolic lignin were degraded. The presence of phenolic hydroxyl groups in lignin greatly enhanced the degradation rate of lignin. This study reveals that P. cinnabarinus, an exclusively laccase-producing fungus, is capable of oxidatively degrading both phenolic and non-phenolic lignins. The ability of the fungus to degrade non-phenolic lignin suggests that a laccase/mediator system is involved in the complete degradation of lignin. After the fungal degradation of lignins, the content of carboxylic acids substantially increased for both phenolic and non-phenolic lignins.     

The relationship of brown humus to lignin

Summary A fraction of organic matter called brown humus was studied in relation to plant lignins. Infrared analysis, methoxy content, and presence of certain phenolic groups showed that this material contains a lignin structure. Decomposition of corn roots showed that one of the first parts of the lignin molecule to be decomposed is by the loss of methoxy groups probably from 2,6-dimethoxyphenol part of the molecule. Studies withP. ochro chloron. Showed two transformations occurred in the decomposition of the lignin molecule: — (1) A demethylation and (2) a formation of carbonyl groups.     

Solubilization of lignin by the ruminal anaerobic fungus Neocallimastix patriciarum.

The ability of the ruminal anaerobic phycomycete Neocallimastix patriciarum to digest model lignin compounds and lignified structures in plant material was studied in batch culture. The fungus did not degrade or transform model lignin compounds that were representative of the predominant intermonomer linkages in lignin, nor did it solubilize acid detergent lignin that had been isolated from spear grass. In a stem fraction of sorghum, 33.6% of lignin was apparently solubilized by the fungus. Solubilization of ester- and either-linked phenolics accounted for 9.2% of the lignin released. The amounts of free phenolic acids detected in culture fluid were equivalent to the apparent loss of ester-linked phenolics from the sorghum substrate. However, the fungus was unable to cleave the ether bond in hydroxycinnamic acid bridges that cross-link lignin and polysaccharide. It is suggested that the majority of the solubilized lignin fraction was a lignin carbohydrate complex containing ether-linked hydroxycinnamic acids. The lignin carbohydrate complex was probably solubilized through dissolution of xylan in the lignin-xylan matrix rather than by lignin depolymerization.     

Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

Eight experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher (P less than 0.01) cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose (Solka Floc), or 6.0% arenaceous flour. NADPH cytochrome c reductase activity was not affected by treatment. Chicks fed 12.0% wood molasses had a higher (P less than 0.06) cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced (P less than 0.05) cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic (bacitracin:neomycin sulfate, 2:1), had a higher (P less than 0.01) cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower (P less than 0.01) intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower (P less than 0.05) rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.     

Fractionation and characterization of chlorophyll and lignin from de-juiced Italian ryegrass (Lolium multifolrum) and timothy grass (Phleum pratense)

In this study, de-juiced Italian ryegrass and timothy grass were successfully fractionated into one chlorophyll rich fraction, two lignin fractions, two hemicellulosic preparations, and one cellulosic fraction by sequential processes with 80% ethanol containing 0.2% NaOH, 2.5% H₂O₂–0.2% EDTA containing 1.5% NaOH, and 2.5% H₂O₂–0.2% TAED containing 1.0% NaOH at 75 °C for 3 h, respectively. The yields of chlorophyll rich fraction and total lignin fraction were found to be 6.1 and 11.0% from de-juiced Italian ryegrass, and 12.7 and 13.2% from de-juiced timothy grass. It should be noted that the two chlorophyll rich fractions contained noticeable amounts of contaminated hemicelluloses (17.1–18.8%) and lignin, whereas the four lignin fractions contained only small amounts of bound hemicelluloses (5.6–8.6%). Nitrobenzene oxidation and NMR spectra of the lignin fraction revealed that the presence of both guaiacyl and syringyl units, as well as p-hydroxyphenyl structures. Small amounts of esterified p-coumaric acids and mainly etherified ferulic acids were also identified in the lignin fractions. It was found that the lignin fractions obtained from the two different grasses had very similar structural composition. They are distinguished by rather low amounts of β-O-4 structures, resinol units (β-β), and of condensed units (β-5 and 5-5). This trait is even more pronounced in the production of phenolic compounds for chemical industry from grass.     

Analysis of Lignin-Polysaccharide Complexes Formed during Grass Lignin Degradation by Cultures of Pleurotus Species

A brown material, precipitable with ethanol, was formed during wheat straw and lignin degradation by liquid cultures of different species of Pleurotus. Fourier transform infrared spectroscopy and cross-polarization and magic-angle-spinning (sup13)C nuclear magnetic resonance spectroscopy showed that most of the precipitable material was formed from exopolysaccharide secreted by the fungus but it also contained an aromatic fraction. The results of acid hydrolysis, methylation analysis, and Smith degradation indicated that the major exopolysaccharide produced by these fungi is a (1(symbl)3)-(beta)-glucan branched at C-6 every two or three residues along the main chain. The presence of lignin or straw in the culture medium had little effect on the composition and structure of the extracellular polysaccharide. Cross-polarization and magic-angle-spinning (sup13)C nuclear magnetic resonance spectroscopy provided an estimation of the aromatic content of the lignin-polysaccharide complexes, assigning 20% of the total (sup13)C signal in the material recovered from cultures of Pleurotus eryngii in lignin medium to aromatic carbon. Analytical pyrolysis indicated that the aromatic fractions of the lignin-polysaccharide complexes were derived from lignin, since products characteristic of pyrolytic breakdown of H (p-hydroxyphenylpropane), G (guaiacylpropane), and S (syringylpropane) lignin units were identified. These complexes cannot be fractionated by treatment with polyvinylpyrrolidone or extraction with lignin solvents, suggesting that the two polymers were chemically linked. Moreover, differences in composition with respect to the original lignin indicated that this macromolecule was modified by the fungi during the process of formation of the lignin-polysaccharide complexes.     

Degradation of Phenols Formed during Lignin Pyrolysis by Microfungi of Genera <Emphasis Type="Italic">Trichoderma</Emphasis> and <Emphasis Type="Italic">Penicillium</Emphasis>

This work presents the data on the rate of utilization of phenols formed during lignin pyrolysis by micromycetes of the genera Trichoderma and Penicillium. Trichoderma strains utilized phenols at the concentration of liquid pyrolytic products of 0.5–1%, while Penicillium strains degraded over 50% phenols at the concentration of pyrolytic products of 2%. Micromycete cultures completely utilized phenol and the cresol-xylenol fraction. Penicillium strains demonstrated a more active growth on media containing phenol, p-cresol, or guaiacum tar as the source of carbon as compared to Trichoderma. m-Cresol and m-xylenol in micromycete growth media containing products of lignin pyrolysis seem to be utilized by co-oxidation.     

废次烟叶提取液源木质素降解菌Bacillus subtilis SM降解特性

【目的】目前造纸法再造烟叶工艺已经成为我国重要的废烟叶处理和利用方式,该工艺中烟梗中高木质素的降解是个挑战性的需解决问题。从废次烟叶提取液(Tobacco waste extract,TWE)中筛选木质素的降解微生物用来直接处理烟梗或烟末提取液,可实现对木质素含量的调控。【方法】将废次烟叶提取液(TWE)浓缩液中分离出的Bacillus subtilis SM接种到以Kraft木质素为唯一碳源的无机盐培养基中,在pH 7.0、30°C培养基中培养4 d来检测菌株对木质素的降解效果。通过HPLC、TOC、GPC和色度来表征SM对木质素的降解,并采用烟梗无机盐培养基在pH 7.0、30°C培养4 d检测SM对烟梗木质素的降解。【结果】HPLC结果显示SM在以木质素磺酸钠为唯一碳源的无机盐培养基中可全部降解分子质量为534.5的木质素磺酸钠,而对Kraft木质素降解不明显,仅观察到组分的变化。脱色结果显示脱色率达到40.7%,但在对Kraft木质素矿化方面矿化率只能达到5.4%。SM在烟梗无机盐培养基中可使烟梗失重率分别达到50%以上(对照组为18.9%),烟梗中木质素含量减少了70%左右。【结论】来源于废次烟叶提取液(TWE)的Bacillus subtilis SM能够以Kraft木质素为唯一碳源生长,也能够有效降解烟梗中的木质素,可应用于烟草废弃物原料中木质素的降解。     

Effect of nitrogen deficiency on the ion-exchange properties of cell wall polymers from wheat roots

The ion-exchange properties of cell wall polymers isolated from the roots of wheat (Triticum aestivum L.) plants grown on either nitrate-free (N-deficient) or nitrate-containing (+N) hydroponic nutrient medium have been investigated. Irrespective of the nitrogen nutrition regimen, the studied cell walls contained four types of ion-exchange groups: primary amino groups of structural proteins (pK_a < 3), carboxyl groups of polygalacturonic acid in pectin (pK _a ~4.7), carboxyl groups of hydroxycinnamic acids (pK _a ~7.3), and phenolic OH-groups of lignin (pK_a ~10.2). The quantitative ratio between these types of ion-exchange groups, the mass fraction of cell walls in the dry weight of roots, and the swelling coefficient of cell walls depended on the nitrate presence in the growing medium. Compared to the +N variant, the N-deficient variant was characterized by a 2.4 times higher content of phenolic OH-groups in cell walls and 1.24 times higher mass fraction of cell walls; at the same time, the swelling coefficient for this variant was lower by 10%. The obtained data indicate that nitrogen deficiency results in a formation of thicker root cell walls with a higher degree of polymer cross-linking that may be caused by the increased lignin content.     

Soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum f.sp. cubense

The accumulation of soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to cell wall-derived elicitor from the pathogen, Fusarium oxysporum, f.sp. cubense, race four, was investigated. The root tissue from the banana cultivar "Goldfinger" was found to respond strongly and rapidly towards the elicitor through the increased synthesis of phenolic compounds. Following elicitation, the conjugated and non-conjugated phenolic metabolites in the induced root tissue were extracted and quantified. Induced phenolic synthesis was rapid and reached near maximum values after 16 h. High-performance liquid chromatography revealed both compositional and quantitative differences between induced phenolics (p-coumaric, ferulic, and sinapic acids) and those constitutively present (p-coumaric- and ferulic acid). In addition, vanillic acid was found in the ester-bound fraction and protocatechuic acid in the cell-wall bound fraction of elicited tissue. The deposition and accumulation kinetics of polymerized phenolic monomers as lignin and lignin-like polymers was quantified over a time period of 0-36 h and found to reach maximum values after 24 h. Ionization difference UV spectra of lignin indicated compositional differences in the newly synthesized lignin fraction and correlated with increased concentrations of ferulic acid and sinapic acids esters. The results show that the increased flux through the phenylpropanoid pathway resulted in the synthesis of cinnamic acid and benzoic acid derivatives that were esterified and incorporated into the cell wall fraction as part of the anti-microbial defenses activated in the root tissue.     

黑龙江省6种乔木叶片热解特性及气体释放特征

森林可燃物是森林火灾发生的基础,地表死可燃物是森林可燃物的重要组成部分,研究地表死可燃物的热解特性和气体释放对探究森林火灾对大气环境和碳平衡的影响及森林火灾的预防和扑救具有重要意义.本研究对黑龙江省6种乔木(樟子松、红皮云杉、水曲柳、胡桃楸、蒙古栎和白桦)地表凋落的叶片进行热重分析和气体释放分析,探究森林可燃物的热解过...     

Fate of Carbohydrates and Lignin during Composting and Mycelium Growth of Agaricus bisporus on Wheat Straw Based Compost

In wheat straw based composting, enabling growth of Agaricus bisporus mushrooms, it is unknown to which extent the carbohydrate-lignin matrix changes and how much is metabolized. In this paper we report yields and remaining structures of the major components. During the Phase II of composting 50% of both xylan and cellulose were metabolized by microbial activity, while lignin structures were unaltered. During A. bisporus’ mycelium growth (Phase III) carbohydrates were only slightly consumed and xylan was found to be partially degraded. At the same time, lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was found to be modified by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 during mycelium growth, while fewer decorations on the phenolic skeleton of both S and G units remained.     

Bacterial decomposition of synthetic 14C-labeled lignin and lignin monomer derivatives.

Nocardia sp. which was isolated from soil is capable of degrading synthetic lignin and utilizing its monomer derivatives. Decomposition was monitored by measuring the 14CO2 evolved and O2 consumed, when the bacterium was grown on a medium containing specifically 14C-labeled ligning or monomer phenolic compounds as major carbon source. The time course of the 14CO2 release and O2 uptake indicates a significant depolymerization and utilization of lignin by the Nocardia sp.     

Universal fractionation of lignin–carbohydrate complexes (LCCs) from lignocellulosic biomass: an example using spruce wood

It is of both theoretical and practical importance to develop a universally applicable approach for the fractionation and sensitive lignin characterization of lignin–carbohydrate complexes (LCCs) from all types of lignocellulosic biomass, both natively and after various types of processing. In the present study, a previously reported fractionation approach that is applicable for eucalyptus (hardwood) and flax (non‐wood) was further improved by introducing an additional step of barium hydroxide precipitation to isolate the mannan‐enriched LCC (glucomannan‐lignin, GML), in order to suit softwood species as well. Spruce wood was used as the softwood sample. As indicated by the recovery yield and composition analysis, all of the lignin was recovered in three LCC fractions: a glucan‐enriched fraction (glucan‐lignin, GL), a mannan‐enriched fraction (GML) and a xylan‐enriched fraction (xylan‐lignin, XL). All of the LCCs had high molecular masses and were insoluble or barely soluble in a dioxane/water solution. Carbohydrate and lignin signals were observed in ¹H NMR, ¹³C CP‐MAS NMR and normal‐ or high‐sensitivity 2D HSQC NMR analyses. The carbohydrate and lignin constituents in each LCC fraction are therefore believed to be chemically bonded rather than physically mixed with one another. The three LCC fractions were found to be distinctly different from each other in terms of their lignin structures, as revealed by highly sensitive analyses by thioacidolysis‐GC, thioacidolysis‐SEC and pyrolysis‐GC.     

Effect of lignin on growth and tyrosinase activity of fungi from the genus Aspergillus

The effect of lignin hydrolyzate (a waste product of the pulp and paper industry) on the growth and tyrosinase activity of several strains of fungi of the genus Aspergillus was studied. During fungal growth lignin was degraded with the formation of soluble aromatic products. Accumulation of these products in the growth medium leads to a 60-100-fold increase in tyrosinase synthesis. The intensity of tyrosinase synthesis depends on the composition of the growth medium. The ratio between lignin and glucose concentrations seems to be the main factor; the activity was maximal at the ratio 2:1.