Mild acetosolv process to fractionate bamboo for the biorefinery: structural and antioxidant properties of the dissolved lignin

Fractionation of lignocellulosic material into its constitutive components is of vital importance for the production of biofuels as well as other value-added chemicals. The conventional acetosolv processes are mainly focused on the production of pulp from woody lignocelluloses. In this study, a mild acetosolv process was developed to fractionate bamboo under atmospheric pressure to obtain cellulosic pulp, water-soluble fraction, and acetic acid lignin. The structural features of the lignins obtained under various conditions were characterized with elemental analysis, sugar analysis, alkaline nitrobenzene oxidation, gel permeation chromatography (GPC), (1)H nuclear magnetic resonance ((1)H NMR), and heteronuclear single-quantum coherence (HSQC) spectroscopy. As compared to milled wood lignin (MWL) of bamboo, acetic acid lignins had low impurities (carbohydrates 2.48-4.56%) mainly due to the cleavage of linkages between lignin and carbohydrates. In addition, acetic acid lignins showed a low proportion of syringyl (S) units. Due to the cleavage of linkages between lignin units, acetic acid lignins had weight-average molecular weights ranging from 4870 to 5210 g/mol, less than half that of MWL (13000 g/mol). In addition, acetic acid lignins showed stronger antioxidant activity mainly due to the significant increase of free phenolic hydroxyls. The lignins obtained with such low impurities, high free phenolic hydroxyls, and medium molecular weights are promising feedstocks to replace petroleum chemicals.     

Detection and determination of p-coumaroylated units in lignins.

The derivatization followed by reductive cleavage (DFRC) method cleaves alpha- and beta-ethers in lignins but leaves lignin gamma-esters intact. When applied to grasses, which contain p-coumarate esters on their lignins, esterified monolignol derivatives are released. Saturation of the p-coumarate double bond occurs during DFRC, so the released products are 4-acetoxycinnamyl 4-acetoxyphenylpropionates. Synthesis of the esters allowed determination of response factors for the released products. Maize and bamboo lignins released 221 and 38 micromol/g of p-coumarate-derived esters. The sinapyl ester was much more abundant than the coniferyl one. The bamboo and maize lignin S/G ratios in the conjugates were 12 and 38 times greater than those of the normal monomers released by DFRC, evidence of a strong selectivity for acylation of syringyl units. Of three possible biochemical mechanisms for incorporating p-coumarates into lignin, evidence is mounting that the process involves incorporation of preacylated monolignols into the normal lignification process.     

Fate of lignins in soils: A review

Lignins are amongst the most studied macromolecules in natural environments. During the last decades, lignins were considered as important components for the carbon cycle in soils, and particularly for the carbon storage. Thus, they are an important variable in many soil-plant models such as CENTURY and RothC, and appeared determinant for the estimation of the soil organic matter (SOM) pool-size and its stabilization. Recent studies challenged this point of view. The aim of this paper was to synthesise the current knowledge and recent progress about quantity, composition and turnover of lignins in soils and to identify variables determining lignin residence time. In soils, lignins evolve under the influence of various variables and processes such as their degradation or mineralization, as well as their incorporation into SOM. Lignin-derived products obtained after CuO oxidation can be used as environmental biomarkers, and also vary with the degree of degradation of the molecule. The lignin degradation is related to the nature of vegetation and land-use, but also to the climate and soil characteristics. Lignin content of SOM decreases with decreasing size of the granulometric fractions, whereas its level of degradation increases concomitantly. Many studies and our results suggest the accumulation and potential stabilization of a part of lignins in soils, by interaction with the clay minerals, although the mechanisms remain unclear. Lignin turnover in soils could be faster than that of the total SOM. Different kinetic pools of lignins were suggested, which sizes seem to be variable for different soil types. The mechanisms behind different degradation kinetics as well as their potential stabilization behaviour still need to be elucidated.     

Phenoxyacetic acid residue incorporation in cell walls of soybean (Glycine max.)

The metabolism of [(14)C] phenoxyacetic acid (POA) and the formation of bound residues were studied in soybean leaves and stems. POA was metabolized to 4-HO-POA and to 4-HO-POA glucoside, and a significant fraction of the radioactivity was incorporated in the cell walls (CW). An extraction procedure of CW polymers was developed to specifically isolate the radioactivity associated with each of them. In leaves, the radioactivity showed a preferential distribution into the hemicelluloses and lignins, while pectins and lignins were the most radioactive CW polymers in stems. The identified bound metabolites were 4-HO-POA, POA, and phenolic residues. The latter and POA were essentially incorporated into the lignin fractions and were linked to the benzylic carbons of lignin monomers. 4-HO-POA, the major bound residue, was more evenly distributed in CW polymers. It was esterified to noncellulosic polysaccharides and lignins, but in the latter, contrary to other POA residues, it was mainly linked at the gamma-carbon of propanoid side-chains of lignin monomers. That type of linkage suggested an enzymatic incorporation of 4-HO-POA in CW, contrary to others residues which have an opportunistic lignin incorporation. That incorporation of 4-HO-POA in CW polymers looks like that of endogenous hydroxycinnamic acids.     

Highly acylated (acetylated and/or p-coumaroylated) native lignins from diverse herbaceous plants

The structure of lignins isolated from the herbaceous plants sisal ( Agave sisalana), kenaf ( Hibiscus cannabinus), abaca ( Musa textilis) and curaua ( Ananas erectifolius) has been studied upon spectroscopic (2D-NMR) and chemical degradative (derivatization followed by reductive cleavage) methods. The analyses demonstrate that the structure of the lignins from these plants is highly remarkable, being extensively acylated at the gamma-carbon of the lignin side chain (up to 80% acylation) with acetate and/or p-coumarate groups and preferentially over syringyl units. Whereas the lignins from sisal and kenaf are gamma-acylated exclusively with acetate groups, the lignins from abaca and curaua are esterified with acetate and p-coumarate groups. The structures of all these highly acylated lignins are characterized by a very high syringyl/guaiacyl ratio, a large predominance of beta- O-4' linkages (up to 94% of all linkages), and a strikingly low proportion of traditional beta-beta' linkages, which indeed are completely absent in the lignins from abaca and curaua. The occurrence of beta-beta' homocoupling and cross-coupling products of sinapyl acetate in the lignins from sisal and kenaf indicates that sinapyl alcohol is acetylated at the monomer stage and that, therefore, sinapyl acetate should be considered as a real monolignol involved in the lignification reactions.     

NMR characterization of lignins isolated from fruit and vegetable insoluble dietary fiber

Compositional information for lignins in food is rare and concentrated on cereal grains and brans. As lignins are suspected to have important health roles in the dietary fiber complex, the confusing current information derived from nonspecific lignin determination methods needs to be augmented by diagnostic structural studies. For this study, lignin fractions were isolated from kiwi, pear, rhubarb, and, for comparison, wheat bran insoluble dietary fiber. Clean pear and kiwi lignin isolates allowed for substantive structural profiling, but it is suggested that the significance of lignin in wheat has been overestimated by reliance on nonspecific analytical methods. Volume integration of NMR contours in two-dimensional (13)C-(1)H correlation spectra shows that pear and wheat lignins have comparable guaiacyl and syringyl contributions and that kiwi lignins are particularly guaiacyl-rich (approximately 94% guaiacyl) and suggest that rhubarb lignins, which could not be isolated from contaminating materials, are as syringyl-rich (approximately 96% syringyl) as lignins from any known natural or transgenic fiber source. Typical lignin structures, including those newly NMR-validated (glycerols, spirodienones, and dibenzodioxocins), and resinols implicated as possible mammalian lignan precursors in the gut are demonstrated via their NMR correlation spectra in the fruit and vegetable samples. A novel putative benzodioxane structure appears to be associated with the kiwi lignin. It is concluded that the fruits and vegetables examined contain authentic lignins and that the detailed structural analysis exposes limitations of currently accepted analytical methods.     

Structural characterization of the lignin from the nodes and internodes of Arundo donax reed

Milled wood lignin (MWL) and dioxane lignin (DL) from different morphological regions (nodes and internodes) of Arundo donax reed were subjected to a comprehensive structural characterization by (13)C, (1)H NMR, FTIR, and UV spectroscopies and functional analysis. The permanganate and nitrobenzene oxidation methods were also applied to the in situ lignins. Both node and internode lignins are HGS-type lignins, with a significant amount of H units (including p-coumaric acid type structures). The S/G ratio (1.13-1.32), the weight-average molecular weight (20,400-24,500), the methoxyl group content (0.90-0.98), the phenolic hydroxyl group content (0.23-0.27), and the aliphatic hydroxyl group content (1.00-1.09) are not very different in the lignins from nodes and internodes. However, some structural differences between node and internode lignins were observed. The former has much more phenolic acids (p-coumaric and ferulic), 8.8% in node versus 1.2% in internode and less beta-O-4 (0. 32 and 0.49 per aromatic unit in node and internode, respectively). In situ node lignin is more condensed than internode lignin.     

Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method.

Lignin extracted with acidic dioxane was investigated as a possible standard for quantitatively determining lignin content in plant samples using the spectrophotometric method employing acetyl bromide. Acidic dioxane lignins were analyzed for carbohydrate, total protein, nitrobenzene oxidation products, and UV spectral characteristics. Total carbohydrate content of isolated lignins ranged from 2.21 to 5.70%, while protein ranged from 0.95 to 6.06% depending upon the plant source of the original cell wall sample. Nitrobenzene analysis indicated differences in the amount of guaiacyl and syringyl units making up the lignins, but this did not alter the UV spectrum of lignin solubilized in acetyl bromide. Regression equations developed for the acetyl bromide method using the isolated lignins for all the plant samples were similar to each other. Lignin values obtained by the acetyl bromide method were similar to the lignin values obtained as acid insoluble residues following a Klason lignin procedure.     

Determination of arylglycerol-beta-aryl ethers and other linkages in lignins using DFRC/(31)P NMR

An analytical method for lignins has been developed that involves derivatization followed by reductive cleavage (DFRC), depolymerization, and quantitative (31)P NMR spectroscopy. This technique detects and quantifies the various ether linkages present in softwood residual kraft lignins (RKL) and milled wood lignins (MWL). In addition, the technique supplies new quantitative information about beta-aryl ethers linked to condensed and noncondensed aromatic moieties, including dibenzodioxocins. Within RKL, beta-aryl ether bonds connected to condensed phenolic moieties predominated over those connected to noncondensed phenolic moieties. In addition, the amount of DFRC monomers determined by gas chromatography was minute in the RKL but large in the MWL. This indicates that almost all noncondensed beta-aryl ether linkages were cleaved during kraft pulping. The method offers new avenues for the detailed investigation of the bonding patterns of native and technical lignins.     

ACIDIC PROPERTIES OF PLANT LIGNINS AND HUMIC MATERIALS OF SOILS

The acidic properties of lignins from the native vegetation of a virgin Ella loamy sand and the humic acids, the 1, 4-dioxane extractable humic fraction and residual humic acids from the virgin and cultivated soils were investigated by determining solid-state cation exchange capacities for Cu and Ca and by non-aqueous potentiometric titration, using potassium methoxide in a benzene-methanol mixture as titrant. The lignins and soil dioxane extracts were titrated in pyridine; the humic acids in dimethylformamide. Platinized Pt and CH₃OH modified calomel electrodes formed the electrode combination. The CEC of the humic acids were much higher than the CEC of lignins with both cations but the Cu: Ca absorption ratios were less for humic acids than for lignins. Titration curves of lignins showed two potential breaks with total acidities of 960 to 1,190 me/100 g. Titration curves of the humic acids showed three to six potential breaks while curves of the soil dioxane extracts showed three and four. The total acidities of the two types of humic isolates were 1,100-1,200 me/100 g. The equivalent weights of most lignins and humic materials were very similar although the acid groups might be different with respect to types and acid strength. Lignins from other plant materials and humic isolates from other soils were also investigated by non-aqueous titration to determine the general applicability of the results obtained with isolates from the Ella soils.     

Characterization of dietary fiber lignins from fruits and vegetables using the DFRC method

Insoluble fiber fractions from 11 fruits and vegetables were investigated for their lignin composition using the derivatization followed by reductive cleavage (DFRC) methodology. To enrich lignin contents and to minimize polysaccharide excess that led to nonanalyzable DFRC chromatograms, the insoluble fibers were degraded by a carbohydrolases mixture. The residues that were found to be representative for the insoluble fiber lignins were analyzed. The investigated fibers differ considerably in their lignin contents and also in their lignin compositions. With the exception of radish fiber, only trace amounts (or none) of the products resulting from p-hydroxyphenyl units were detected. Lignins noticeably differed in the ratio of the DFRC products resulting from syringyl units (S) and guaiacyl (G) units (G/S ratios ranged from approximately 39 to 0.2). The insoluble fiber lignins were classified as G-rich lignins (G/S ratio > 3; carrot, spinach, kiwi, curly kale, radish, and asparagus), S-rich lignins (S/G ratio > 3; rhubarb), or balanced lignins (0.3 < G/S ratio < 3; pear, apple, small radish, and kohlrabi). Information about further structural characteristics, for example, cinnamyl endgroups, was obtained from the analysis of DFRC minor products.     

Structural characterization of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems

The structure of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems was studied both in situ and in isolated milled "wood" lignins by several analytical methods. The presence of p-coumarate and ferulate in the cortex and pith, as well as in their isolated lignins, was revealed by pyrolysis in the presence of tetramethylammonium hydroxide, and by 2D NMR, and indicated that ferulate acylates the carbohydrates while p-coumarate acylates the lignin polymer. 2D NMR showed a predominance of alkyl aryl ether (β-O-4') linkages (82% of total interunit linkages), with low amounts of "condensed" substructures, such as resinols (β-β'), phenylcoumarans (β-5'), and spirodienones (β-1'). Moreover, the NMR also indicated that these lignins are extensively acylated at the γ-carbon of the side chain. DFRC analyses confirmed that p-coumarate groups acylate the γ-OHs of these lignins, and predominantly on syringyl units.     

INFRA-RED SPECTRA AND DIFFERENTIAL THERMOGRAMS OF LIGNINS AND SOIL HUMIC MATERIALS SATURATED WITH DIFFERENT CATIONS

Comparison of infra-red spectra of lignins and humic materials obtained before and after saturation with Cu showed that Cu saturation induced changes in the shape of the OH absorption bands and pronounced shifts in the C=O (1700 cm^-1) bands to lower frequencies. Differential thermograms indicated that Cu and Ca saturation reduced the thermal stabilities of lignins and humic materials. There was more reduction in stability with Cu than with Ca saturation and with lignins than with humic materials.     

In search of a maize ideotype for cell wall enzymatic degradability using histological and biochemical lignin characterization

Grass cell wall degradability is conventionally related to the lignin content and to the ferulic-mediated cross-linking of lignins to polysaccharides. To better understand the variations in degradability, 22 maize inbred lines were subjected to image analyses of Fasga- and M?ule-stained stem sections and to chemical analyses of lignins and p-hydroxycinnamic acids. For the first time, the nearness of biochemical and histological estimates of lignin levels was established. Combination of histological and biochemical traits could explain 89% of the variations for cell wall degradability and define a maize ideotype for cell wall degradability. In addition to a reduced lignin level, such an ideotype would contain lignins richer in syringyl than in guaiacyl units and preferentially localized in the cortical region rather than in the pith. Such enrichment in syringyl units would favor wall degradability in grasses, contrary to dicots, and could be related to the fact that grass syringyl units are noticeably p-coumaroylated. This might affect the interaction capabilities of lignins and polysaccharides.     

Quantitative (13)C NMR analysis of lignins with internal standards

Novel protocols for acquiring quantitative (13)C NMR spectra of lignins have been developed using the internal reference compounds 1,3,5-trioxane and pentafluorobenzene. Trioxane offers a convenient internal standard for collecting inverse gated proton decoupled (13)C NMR spectra for lignins, whereas pentafluorobenzene can be used to provide information on the amount of methine carbon using the DEPT experiment. In each case, the internal reference compounds provide single, un-overlapped sharp signals in the middle of the spectral region, permitting facile integration. These integrals could be used to determine the amounts of different structural features of lignins, expressed in absolute units of millimoles per gram. The optimum parameters for these experiments were validated for a variety of spectrometer platforms, and standard errors were determined for different spectral areas using lignin model compounds and "standard" lignins. In addition, the data derived for the International Round Robin "standard" lignins showed good agreement with the data from quantitative (31)P NMR spectroscopy and published data, obtained by independent laboratories using independent methods of analysis.     

Analysis of technical lignins by two- and three-dimensional NMR spectroscopy

Modern multidimensional NMR spectroscopic methods were applied to investigate the effects of kraft pulping and oxygen delignification on lignin side-chain structures. In addition to the two-dimensional HSQC measurements, the three-dimensional HSQC-TOCSY technique was utilized to elucidate the (1)H-(1)H and (1)H-(13)C correlations of individual spin systems and thus indicate a certain lignin side-chain structure. Unlike earlier, nonlabeled samples were used for 3D measurements. According to 2D and 3D NMR spectra, most of the structures identified in milled wood lignin (MWL) are still present in technical lignins after kraft pulping and oxygen delignification. Although the main reaction during kraft pulping is the cleavage of beta-O-4 linkages, these structures are still left in spent liquor lignin as well as in residual lignin. The amount of coniferyl alcohol and dihydroconiferyl alcohol end groups, as well as some unidentified saturated end groups, is higher in technical lignins than in MWL. Contrary to our earlier observations, no diphenylmethane structures were observed in any technical lignins. Vinyl aryl ether structures could not be detected in technical lignins either.     

Organosolv ethanol lignin from hybrid poplar as a radical scavenger: relationship between lignin structure, extraction conditions, and antioxidant activity

Twenty-one organosolv ethanol lignin samples were prepared from hybrid poplar (Populus nigra xP. maximowiczii) under varied conditions with an experimental matrix designed using response surface methodology (RSM). The lignin preparations were evaluated as potential antioxidants. Results indicated that the lignins with more phenolic hydroxyl groups, less aliphatic hydroxyl groups, low molecular weight, and narrow polydispersity showed high antioxidant activity. Processing conditions affected the functional groups and molecular weight of the extracted organosolv ethanol lignins, and consequently influenced the antioxidant activity of the lignins. In general, the lignins prepared at elevated temperature, longer reaction time, increased catalyst, and diluted ethanol showed high antioxidant activity. Regression models were developed to enable the quantitative prediction of lignin characteristics and antioxidant activity based on the processing conditions.     

Pyrolysis of lignin in the presence of tetramethylammonium hydroxide: a convenient method for S/G ratio determination

Pyrolysis-gas chromatography in the presence of tetramethylammonium hydroxide (TMAH) was applied to the determination of the ratio of the abundances of the syringyl beta-aryl ether subunits to those of the guaiacyl equivalents (S/G) in lignin. Diazomethane-methylated kenafs (Hibiscus cannabinus and Hibiscus sabdariffa) and beech (Fagus crenata) in situ lignins were employed. Relative abundances of pyrolysis products derived from the guaiacyl and syringyl beta-aryl ether subunits were determined. The S/G ratios for in situ lignins were obtained with average 3.1% relative standard deviation for a minimum of six repeated runs. The S/G ratios determined by pyrolysis in the presence of TMAH agreed well with those determined by thioacidolysis, with a significant linear regression (R(2) = 0.9867). The results showed that pyrolysis with TMAH is an effective tool for obtaining information on the S/G ratio for in situ lignins.     

Are lignins optically active?

The accepted derivation of lignins from non(enzymatically)-controlled radical coupling reactions has been recently challenged, and it is relevant to ascertain unequivocally whether lignins are or are not (as normally assumed) optically active. Two approaches were used. First, DFRC (derivatization followed by reductive cleavage) dimers derived from beta-5- and beta-beta-units in pine lignins, which certainly retain unaltered chiral centers (as well as beta-1- and beta-O-4-units where the intactness may be debated), were shown to be optically inactive by circular dichroism (CD) and chiral high-performance liquid chromatography. CD of beta-5-derived dimers following enantiomeric separation readily demonstrated the sensitivity of the method. Second, no optical activity could be detected (above 250 nm to avoid carbohydrate contributions) by CD of lignin isolates from pine, kenaf, maize, or a CAD-deficient pine mutant. Representative lignins are therefore not, within limits of detection by these methods, optically active.     

19F nuclear magnetic resonance spectroscopy for the quantitative detection and classification of carbonyl groups in lignins

A novel method that permits the quantitative detection and classification of various carbonyl groups in lignins has been developed. The proposed method was optimized with the quantitative trifluoromethylation of a series of carbonyl-containing lignin-like model compounds. This effort was followed by (19)F NMR spectral analyses of the resulting fluorine derivatives allowing for a thorough understanding of their structure/(19)F chemical shift relationships. The various carbonyl groups present in lignins were also investigated by trifluoromethylating them in the presence of catalytic amounts of tetramethylammonium fluoride (TMAF), followed by hydrolysis with TMAF in tetrahydrofuran. By using a variety of selective reactions, it became possible to assign a number of prominent (19)F NMR signals to a variety of carbonyl groups present in lignins. These studies demonstrated that the proposed method can be applied to the quantitative determination of carbonyl groups that are present in soluble native and technical lignins.