Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system

Phenolic and nonphenolic (permethylated) synthetic [14C]lignins were depolymerized by Trametes villosa laccase in the presence of a radical mediator, 1-hydroxybenzotriazole (HOBT). Gel permeation chromatography of the treated lignins showed that approximately 10% of their substructures were cleaved. The system also cleaved a beta-O-4-linked model compound, 1-(4-ethoxy-3-methoxy-ring-[14C]phenyl)-2-(2-methoxyphenoxy)-propane- 1,3-diol, and a beta-1-linked model, 1, 2-bis-(3-methoxy-4-[14C]methoxyphenyl)-propane-1,3-diol, that represent nonphenolic substructures in lignin. High performance liquid chromatography of products from the oxidized models showed that they were produced in sufficient yields to account for the ability of laccase/HOBT to depolymerize nonphenolic lignin.     

Lignin as additive in polypropylene/coir composites: Thermal, mechanical and morphological properties

PP/CF composites were prepared and the effect of lignin incorporation in the presence and absence of compatibilizer (maleic anhydride grafted polypropylene, PP-g-MA) was investigated by means of mechanical, thermal and morphological properties. Lignin added to the composite was obtained by the Acetosolv process. The composites were prepared in a Haake torque rheometer and assessed by means of tensile testing, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The results showed that in the absence of PP-g-MA, incorporation of lignin did not affect tensile strength and in its presence this property was reduced. Thermal analysis revealed that incorporation of lignin in the composites resulted in increase in both the initial thermal decomposition temperatures and oxidation induction times.     

Effect of chemical modification of lignin on the gluebond performance of lignin-phenolic resins

Lignin obtained from eucalyptus wood by acetic acid pulping was methylolated or phenolated and used to prepare lignin-phenol-formaldehyde resins. The amount of formaldehyde consumed in the methylolation reaction, and supporting comparison of pre- and post-methylolation ¹H and ¹³C NMR spectra, showed the reactivity of the crude acetosolv lignin with formaldehyde to be relatively high. Pine and eucalyptus plywood boards manufactured using the resins prepared with the modified lignins complied with European Standard EN 314-1:1993 for WBP quality boards and gave knife test results similar to those of boards manufactured with a commercial phenol-formaldehyde resin.     

Comparison of different pretreatment methods for lignocellulosic materials. Part I: conversion of rye straw to valuable products

The conversion of lignocellulose to valuable products requires I: a fractionation of the major components hemicellulose, cellulose, and lignin, II: an efficient method to process these components to higher valued products. The present work compares liquid hot water (LHW) pretreatment to the soda pulping process and to the ethanol organosolv pretreatment using rye straw as a single lignocellulosic material. The organosolv pretreated rye straw was shown to require the lowest enzyme loading in order to achieve a complete saccharification of cellulose to glucose. At biomass loadings of up to 15% (w/w) cellulose conversion of LHW and organosolv pretreated lignocellulose was found to be almost equal. The soda pulping process shows lower carbohydrate and lignin recoveries compared to the other two processes. In combination with a detailed analysis of the different lignins obtained from the three pretreatment methods, this work gives an overview of the potential products from different pretreatment processes.     

Extraction and characterization of wax from sugarcane bagasse and the enzymatic hydrolysis of dewaxed sugarcane bagasse

Extraction of high-value products from agricultural wastes is an important component for sustainable bioeconomy development. In this study, wax extraction from sugarcane bagasse was performed and the beneficial effect of dewaxing pretreatment on the enzymatic hydrolysis was investigated. About 1.2% (w/w) of crude sugarcane wax was obtained from the sugarcane bagasse using the mixture of petroleum ether and ethanol (mass ratio of 1:1) as the extraction agent. Results of Fourier-transform infrared characterization and gas chromatography–mass spectrometry qualitative analysis showed that the crude sugarcane wax consisted of fatty fractions (fatty acids, fatty aldehydes, hydrocarbons, and esters) and small amount of lignin derivatives. In addition, the effect of dewaxing pretreatment on the enzymatic hydrolysis of sugarcane bagasse was also investigated. The digestibilities of cellulose and xylan in dewaxed sugarcane bagasse were 18.7 and 10.3%, respectively, compared with those of 13.1 and 8.9% obtained from native sugarcane bagasse. The dewaxed sugarcane bagasse became more accessible to enzyme due to the disruption of the outermost layer of the waxy materials.     

Immobilized methyltrioxo rhenium (MTO)/H2O2 systems for the oxidation of lignin and lignin model compounds

A convenient and efficient application of heterogeneous methylrhenium trioxide (MTO) systems for the selective oxidation of lignin model compounds and lignins is reported. Environmental friendly and low-cost H2O2 was used as the oxygen atom donor. Overall, the data presented and discussed in this paper point toward the conclusion that the immobilized heterogeneous catalytic systems based on H2O2/and MTO catalysts are able to extensively oxidize both phenolic and non-phenolic, monomeric, and dimeric, lignin model compounds. Condensed diphenylmethane models were found also extensively oxidized. Technical lignins, such as hydrolytic sugar cane lignin (SCL) and red spruce kraft lignin (RSL), displayed oxidative activity with immobilized MTO catalytic systems. After oxidation, these lignins displayed the formation of more soluble lignin fragments with a high degree of degradation as indicated by the lower contents of aliphatic and condensed OH groups, and the higher amounts of carboxylic acid moieties. Our data indicate that immobilized MTO catalytic systems are significant potential candidates for the development of alternative totally chlorine-free delignification processes and environmental sustainable lignin selective modification reactions.     

Thermal mobility of β-O-4-type artificial lignin

Several lignin model polymers and their derivatives comprised exclusively of β-O-4 or 8-O-4' interunitary linkages were synthesized to better understand the relation between the thermal mobility of lignin, in particular, thermal fusibility and its chemical structure; an area of critical importance with respect to the biorefining of woody biomass and the future forest products industry. The phenylethane (C6-C2)-type lignin model (polymer 1) exhibited thermal fusibility, transforming into the rubbery/liquid phase upon exposure to increasing temperature, whereas the phenylpropane (C6-C3)-type model (polymer 2) did not, forming a char at higher temperature. However, modifying the Cγ or 9-carbon in polymer 2 to the corresponding ethyl ester or acetate derivative imparted thermal fusibility into this previously infusible polymer. FT-IR analyses confirmed differences in hydrogen bonding between the two model lignins. Both polymers had weak intramolecular hydrogen bonds, but polymer 2 exhibited stronger intermolecular hydrogen bonding involving the Cγ-hydroxyl group. This intermolecular interaction is responsible for suppressing the thermal mobility of the C6-C3-type model, resulting in the observed infusibility and charring at high temperatures. In fact, the Cγ-hydroxyl group and the corresponding intermolecular hydrogen bonding interactions likely play a dominant role in the infusibility of most native lignins.     

Bioconversion of cellulose into ethanol by Clostridium thermocellum--product inhibition

Direct anaerobic bioconversion of cellulosic substances into ethanol by Clostridium thermocellum ATCC 27405 has been carried out at 60 degrees C and pH 7.0 (initial for 100 L) under continuous sparging of oxygen free nitrogen in a culture vessel. Raw bagasse, mild alkali-treated bagasse, and solka floc were used as substrates. The extent of conversion of raw bagasse (cellulose, 50%; hemicellulose, 25%; lignin, 19%) was observed as 52% (w/w) and 79% (w/w) in the case of mild alkali and steam-treated bagasse (cellulose, 72%; hemicellulose, 11%; lignin, 12%), respectively. Use of bagasse concentration above 10 g/L showed a decreased rate in ethanol production. An inoculum age between 28-30 h and cell mass content of 0.027-0.036 g/L (dry basis) were used. The results obtained with raw and pretreated bagasse have been compared with those of highly pure Solka Floc (hemicellulose, 10%). Studies on the product inhibition indicated a linear fall of the percent of survivors with time. An Arrhenius type correlation between the cell decay rate constant and the product concentration was predicted. Even at low levels, the inhibitory effects of products on cell viability, the specific growth rate, and extracellular cellulase enzyme were observed.     

Comparative evaluation of acid and alkaline sulfite pretreatments for enzymatic saccharification of bagasses from three different sugarcane hybrids

Sugarcane bagasses from three experimental sugarcane hybrids and a mill‐reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and β‐glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70–100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH, and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:944–951, 2018     

Catalytic Alkaline Oxidation of Lignin and its Model Compounds: a Pathway to Aromatic Biochemicals

Catalytic oxidation via the application of molecular oxygen and copper complexes is a useful pathway toward valuable low molecular mass compounds from in situ or waste stream lignins. In this study, two dimeric β-ether model compounds, one β-ether oligomer, and a milled wood lignin sample from Loblolly pine were catalytically oxidized. Yields and stability of the aromatic aldehyde and acid products were measured. Nuclear magnetic resonance spectroscopy and gel permeation chromatography were used to monitor structure/composition and molecular mass changes of the lignin before and after catalytic oxidation to study the degree of depolymerization and structure of the residual lignin. Oxidized units appear to be derived from β-aryl ether, phenylcoumaran, and biphenyl ether components. To date, this method breaks down the lignin polymeric structure reasonably effectively, producing low molecular mass products; this work also highlights some of the issues that need to be overcome to optimize this approach.     

Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity

Transgenic poplars (Populus tremula x Populus alba) were obtained by introduction of a sense homologous transgene encoding caffeic acid O-methyltransferase (COMT) under the control either of the cauliflower mosaic virus double 35S promoter or of the eucalyptus cinnamyl alcohol dehydrogenase promoter. Although these constructs conferred a moderate overexpression of COMT in some lines, a transgenic line with the double 35S promoter was found where COMT activity in woody tissues was close to zero due to a gene-silencing phenomenon. For the first time in COMT down-regulated trees, this alteration substantially reduced lignin level in 6-month-old trees (17% decrease). Lignin structure was found to be strongly altered, with a two times higher content in condensed bonds, an almost complete lack of syringyl units, and the incorporation of 5-hydroxyguaiacyl units to the most remarkable extent reported so far. Consistent with the higher cellulose content and with the higher condensation degree of the lignin, the impact of the transformation on the kraft-pulping performances of the poplar trees positively affected the pulp yield (10% relative increase), but made lignins less amenable to industrial degradations.     

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 effect of metal salts on the decomposition of sweet sorghum bagasse in flow-through liquid hot water

The impact of the metal salts NaCl, KCl, CaCl(2), MgCl(2), FeCl(3), FeCl(2), and CuCl(2), particularly the latter, on the decomposition of hemicellulose and lignin from sweet sorghum bagasse in liquid hot water pretreatment processing was studied in an attempt to enhance the recovery of sugars. Transition metal chlorides significantly enhanced the hemicellulose removal compared to the alkaline earth metal chlorides and alkaline metal chlorides, contributing to the formation of a saccharide-metal cation intermediate complex. FeCl(2) greatly increased xylose degradation and about 60% xylan was converted into non-saccharide products. In contrast, an excellent total and monomeric xylose recovery was obtained after the CuCl(2) pretreatment. Most of the lignin was deposited on the surface of the residual solid with droplet morphologies after this pretreatment, and about 20% was degraded into monomeric products. The total recovery of sugars from sweet sorghum bagasse with 0.1% CuCl(2) solution pretreatment and 48 h enzymatic digestibility, reached 90.4%, which is superior to the recovery using hot water pretreatment only.     

Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment

Plant cell walls are composed primarily of cellulose, hemicelluloses, lignins, and pectins. Of these components, lignins exhibit unique chemistry and physiological functions. Although lignins can be used as a product feedstock or as a fuel, lignins are also generally seen as a barrier to efficient enzymatic breakdown of biomass to sugars. Indeed, many pretreatment strategies focus on removing a significant fraction of lignin from biomass to better enable saccharification. In order to better understand the fate of biomass lignins that remain with the solids following dilute acid pretreatment, we undertook a structural investigation to track lignins on and in biomass cell walls. SEM and TEM imaging revealed a range of droplet morphologies that appear on and within cell walls of pretreated biomass; as well as the specific ultrastructural regions that accumulate the droplets. These droplets were shown to contain lignin by FTIR, NMR, antibody labeling, and cytochemical staining. We provide evidence supporting the idea that thermochemical pretreatments reaching temperatures above the range for lignin phase transition cause lignins to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls. This decompartmentalization and relocalization of lignins is likely to be at least as important as lignin removal in the quest to improve the digestibility of biomass for sugars and fuels production.     

In vitro depolymerization of lignin by manganese peroxidase of Phanerochaete chrysosporium

Homogeneous manganese peroxidase catalyzed the in vitro partial depolymerization of four different 14C-labeled synthetic lignin preparations. Gel permeation profiles demonstrated significant depolymerization of 14C-sidechain-labeled syringyl lignin, a 14C-sidechain-labeled syringyl-guaiacyl copolymer (angiosperm lignin), and depolymerization of 14C-sidechain- and 14C-ring-labeled guaiacyl lignins (gymnosperm lignin). 3,5-Dimethoxy-1,4-benzo-quinone, 3,5-dimethoxy-1,4-hydroquinone, and syringylaldehyde were identified as degradation products of the syringyl and syringyl-guaiacyl lignins. These results suggest that manganese peroxidase plays a significant role in the depolymerization of lignin by Phanerochaete chrysosporium.     

Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium.

We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic 14C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.     

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose

Douglas-fir was SO₂-steam pretreated at different severities (190, 200, and 210 °C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and ¹³C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins.     

Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium.

We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic 14C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.     

Delignification of sugarcane bagasse using glycerol-water mixtures to produce pulps for saccharification

This paper describes the organosolv delignification of depithed bagasse using glycerol–water mixtures without a catalyst. The experiments were performed using two separate experimental designs. In the first experiment, two temperatures (150 and 190 °C), two time periods (60 and 240 min) and two glycerol contents (20% and 80%, v/v) were used. In the second experiment, which was a central composite design, the glycerol content was maintained at 80%, and a range of temperatures (141.7–198.3 °C) and time (23–277 min) was used. The best result, obtained with a glycerol content of 80%, a reaction time of 150 min and a temperature of 198.3 °C, produced pulps with 54.4% pulp yield, 7.75% residual lignin, 81.4% delignification and 13.7% polyose content. The results showed that high contents of glycerol tend to produce pulps with higher delignification and higher polyoses content in relation to the pulps obtained from low glycerol content reactions. In addition, the proposed method shows potential as a pretreatment for cellulose saccharification.     

Study of lignin biotransformation by Aspergillus fumigatus and white-rot fungi using (14)C-labeled and unlabeled kraft lignins

The biodegradation of lignin by fungi was studied in shake flasks using (14)C-labeled kraft lignin and in a deep-tank fermentor using unlabeled kraft lignin. Among the fungi screened, A. fumigatus-isolated in our laboratories-was most potent in lignin biotransformation. Dialysis-type fermentation, designed to study possible accumulation of low MW lignin-derived products, showed no such accumulation. Recalcitrant carbohydrates like mi-crocrystalline cellulose supported higher lignolytic activity than easily metabolized carbohydrates like cellobiose. An assay developed to distinguish between CO(2) evolved from lignin and carbohydrate substrates demonstrated no stoichiometric correlation between the metabolism of the two cosubstrates. The submerged fermentations with unlabeled lignin are difficult to monitor since chemical assays do not give accurate and true results. Lignolytic efficiencies that allowed monitoring of such fermentations were defined. Degraded lignins were analyzed for structural modifications. A. fumigatus was clearly superior to C. versicolor in all aspects of lignin degradation; A. fumigatus brought about substantial demethoxylation and dehydroxylation, whereas C. versicolor degraded lignins closely resembled undegraded kraft lignin. There was a good agreement among the different indices of lignin degradation, namely, (14)CO evolution, OCH(3) loss, OH loss, and monomer and dimer yield after permanganate oxidation.