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.     

Comparison of the acetyl bromide spectrophotometric method with other analytical lignin methods for determining lignin concentration in forage samples

Present analytical methods to quantify lignin in herbaceous plants are not totally satisfactory. A spectrophotometric method, acetyl bromide soluble lignin (ABSL), has been employed to determine lignin concentration in a range of plant materials. In this work, lignin extracted with acidic dioxane was used to develop standard curves and to calculate the derived linear regression equation (slope equals absorptivity value or extinction coefficient) for determining the lignin concentration of respective cell wall samples. This procedure yielded lignin values that were different from those obtained with Klason lignin, acid detergent acid insoluble lignin, or permanganate lignin procedures. Correlations with in vitro dry matter or cell wall digestibility of samples were highest with data from the spectrophotometric technique. The ABSL method employing as standard lignin extracted with acidic dioxane has the potential to be employed as an analytical method to determine lignin concentration in a range of forage materials. It may be useful in developing a quick and easy method to predict in vitro digestibility on the basis of the total lignin content of a sample.     

Toward a better understanding of the lignin isolation process from wood

The recently developed protocol for isolating enzymatic mild acidolysis lignins (EMAL) coupled with the novel combination of derivatization followed by reductive cleavage (DFRC) and quantitative (31)P NMR spectroscopy were used to better understand the lignin isolation process from wood. The EMAL protocol is shown to offer access at lignin samples that are more representative of the overall lignin present in milled wood. The combination of DFRC/(31)P NMR provided a detailed picture on the effects of the isolation conditions on the lignin structure. More specifically, we have used vibratory and ball milling as the two methods of wood pulverization and have compared their effects on the lignin structures and molecular weights. Vibratory-milling conditions cause substantial lignin depolymerization. Lignin depolymerization occurs via the cleavage of uncondensed beta-aryl ether linkages, while condensed beta-aryl ethers and dibenzodioxocins were found to be resistant to such mechanical action. Condensation and side chain oxidations were induced mechanochemically under vibratory-milling conditions as evidenced by the increased amounts of condensed phenolic hydroxyl and carboxylic acid groups. Alternatively, the mild mechanical treatment offered by ball milling was found not to affect the isolated lignin macromolecular structure. However, the overall lignin yields were found to be compromised when the mechanical action was less intense, necessitating longer milling times under ball-milling conditions. As compared to other lignin preparations isolated from the same batch of milled wood, the yield of EMAL was about four times greater than the corresponding milled wood lignin (MWL) and about two times greater as compared to cellulolytic enzyme lignin (CEL). Molecular weight distribution analyses also pointed out that the EMAL protocol allows the isolation of lignin fractions that are not accessed by any other lignin isolation procedures.     

Changes in the elemental composition of lignin during humification

Lignin was extracted with aqueous dioxane in the genetic series of humification: dead nonhumified plants → plant residues at the initial stage of humification (during the first 1–3 annual cycles) → weakly decomposed peat → highly decomposed peat. In the course of the humification and peat formation, the lignin was subjected to profound redox transformation; therefore, the elemental composition of the dioxane lignin in the humified peat-forming plants differed from that in the original plants by the higher contents of hydrogen and nitrogen and the lower contents of oxygen and carbon. As a result of the redox reactions, the lignin was partly oxidized and converted to humic substances during the humification of the dead plants. Therefore, it could not be extracted with aqueous dioxane. The reduced part of the lignin remained in the humified materials. The proportion of aromatic fragments in the molecules of the remaining lignin was smaller and that of aliphatic fragments larger than in the lignin of the original plants.     

Ligno-aliphatic complexes in soils revealed by an isolation procedure: implication for lignin fate

For the last decades, the fate of lignins in soil was analyzed mainly with cupric oxide (CuO) oxidation, which is traditionally used to quantify soil lignin content and characterize its state of degradation. This method presents limitations due to incomplete depolymerization of the lignin structure. In this study, we used a physicochemical soil lignin isolation procedure, which permits recovery of a milled wall enzymatic lignin (MWEL) fraction. Elemental composition and chemical structure of MWEL isolated from plants and soil were characterized. Its incorporation rate into an agricultural loamy soil was studied using stable isotope analyses of MWEL isolated from soils after 0 to 9 years of maize cultivation after wheat. Comparison of MWEL isolated from maize tissues and soil provided information on evolution of the lignin structure once incorporated into soil. We observed aromatic–aliphatic complex formation, which could lead to its sequestration in soil evidenced by increasing MWEL content after 9 years of maize cultivation. The ¹³C natural abundance of isolated MWEL showed faster incorporation of MWEL (17.4 % of renewed lignins after 9 years) compared to total soil organic matter (9 % of total soil organic carbon (SOC) was renewed). This faster incorporation rate of MWEL compared to bulk soil organic matter is in agreement with lignin turnover observed by CuO oxidation. Radiocarbon dating of MWEL suggested a mean age of around 50 years. We conclude that lignin isolation allows (1) access to a different fraction compared to CuO oxidation and (2) a detailed characterization of lignin transformation in soil. We suggest that interaction with aliphatic compounds could be one possible pathway of lignin preservation in soil.     

Evaluation of lignin and lignin‐nitrogen fractionation following alternative detergent fiber pre‐treatment methods

Abstract

Decomposition of plant material, compost, or mulch may be monitored or predicted by measuring certain key parameters in live or senescent material. Lignin is one of the key parameters to measure as it provides an indication of soil quality. This study evaluates the most promising detergent clean‐up procedures for pre‐treating plant material to provide a more rapid method for lignin determination. Performance was tested with a variety of plant material, typical of vegetation analyzed in decomposition, nutrient cycling and natural ecosystem studies. Lignin‐nitrogen (N) and fiber‐N residues were also determined to assess the efficiency of the clean‐up processes. Good agreement was obtained for lignin on a range of plant material between the time‐consuming classical Klasson method and either modified neutral detergent fiber (NDF) excluding sodium sulphite and Dekalin, or the acidified detergent fiber (ADF) pre‐treatments. We recommend either of the detergent pre‐treatment procedure as they provide rapid and effective preliminary steps for precise lignin determination. Residual lignin‐N following NDF and ADF detergent pre‐treatment methods were on average respectively 31% and 24% of the plant nitrogen.     

Occurrence of naturally acetylated lignin units

This work examines the occurrence of native acetylated lignin in a large set of vascular plants, including both angiosperms and gymnosperms, by a modification of the so-called Derivatization Followed by Reductive Cleavage (DFRC) method. Acetylated lignin units were found in the milled wood lignins of all angiosperms selected for this study, including mono- and eudicotyledons, but were absent in the gymnosperms analyzed. In some plants (e.g., abaca, sisal, kenaf, or hornbeam), lignin acetylation occurred at a very high extent, exceeding 45% of the uncondensed (alkyl-aryl ether linked) syringyl lignin units. Acetylation was observed exclusively at the gamma-carbon of the lignin side chain and predominantly on syringyl units, although a predominance of acetylated guaiacyl over syringyl units was observed in some plants. In all cases, acetylation appears to occur at the monomer stage, and sinapyl and coniferyl acetates seem to behave as real lignin monomers participating in lignification.     

木质素对木质纤维素降解性能的影响

木质素影响木质纤维素降解性能,明确木质素影响木质纤维素降解的程度和机理,对于植物基因改造、纤维素酶基因改造/筛选、预处理工艺优化均具有重大意义。但是由于木质素和木质纤维素结构的复杂性,木质素对木质纤维素影响的程度和机理尚无定论。该文综述了关于目前研究主要集中在木质素的含量和结构对木质纤维素降解性能的影响上,初始木质素含量和残留木质素含量对同物种和不同物种木质纤维素降解性能的影响;木质素单体比例(syringyl units/guaiacyl units)、键连方式、官能团对木质纤维素降解性能的影响;纯化木质素对木质纤维素降解性能的影响。该文为木质素对纤维素降解性能的影响的相关研究工作提供指导。     

Accurate and reproducible determination of lignin molar mass by acetobromination

The accurate and reproducible determination of lignin molar mass by using size exclusion chromatography (SEC) is challenging. The lignin association effects, known to dominate underivatized lignins, have been thoroughly addressed by reaction with acetyl bromide in an excess of glacial acetic acid. The combination of a concerted acetylation with the introduction of bromine within the lignin alkyl side chains is thought to be responsible for the observed excellent solubilization characteristics acetobromination imparts to a variety of lignin samples. The proposed methodology was compared and contrasted to traditional lignin derivatization methods. In addition, side reactions that could possibly be induced under the acetobromination conditions were explored with native softwood (milled wood lignin, MWL) and technical (kraft) lignin. These efforts lend support toward the use of room temperature acetobromination being a facile, effective, and universal lignin derivatization medium proposed to be employed prior to SEC measurements.     

Rapid, microscale, acetyl bromide-based method for high-throughput determination of lignin content in Arabidopsis thaliana

The acetyl bromide method has been modified to enable the rapid microscale determination of lignin content in Arabidopsis with the goal of determining the genes that control lignin in plants. Modifications include reduction in sample size, use of a microball mill, adoption of a modified rapid method of extraction, use of an ice-bath to stabilize solutions and reduction in the volume of solutions. The microscale method was shown to be rapid, accurate and precise with values in agreement with those determined by the full-scale acetyl bromide method. The extinction coefficient for Arabidopsis lignin, dissolved using acetyl bromide, was determined to be 23.35 g(-1) L cm(-1) at 280 nm. This value is independent of the Arabidopsis accession, environmental growth conditions and is insensitive to lignin structure. The newly developed method can be used to determine lignin content in the inflorescence stems of Arabidopsis for mapping of lignin-related genes.     

Influences of lignin from paper mill sludge on soil properties and metal accumulation in wheat

The influences of lignin application on soil properties of three different soils, Jiangxi soil (Ultisol, Hapludult), Heilongjiang soil (Alfisol, Entioboralf) and Beijing soil (Alfisol, Haplustalf), and metal accumulation in wheat (Triticum aestivum L.) were studied in a pot experiment. By lignin amendment, soil pH, organic matter (OM) and cation exchange capacity (CEC) increased, except for CEC in the Beijing soil. Analysis showed that available P and K in lignin-amended soils were also elevated, except for P in the Jiangxi soil. A three-step sequential extraction procedure proposed by the Standards, Measurements and Testing Programme (formerly BCR) of the European Commission was used to investigate the fraction redistribution of heavy metals in soils with lignin application. The fractions were specified as B1: water soluble, exchangeable and carbonate bound, and weakly adsorbed; B2: Fe-Mn oxide bound; and B3: organic matter and sulfide bound. Generally, the heavy metal content of the B2 fraction decreased whereas that of the B3 fraction increased. Lignin application to arable soils can not only improve plant growth in vitro, but also reduce the accumulation of the heavy metals Cu, Zn, Cd, Pb, Cr and Ni in wheat plants.     

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.     

Microwave-assisted lignin isolation using the enzymatic mild acidolysis (EMAL) protocol

The use of microwaves is explored in an effort to further improve the recently developed lignin isolation protocol termed EMAL (enzymatic mild acidolysis lignin). Because the presence of the lignin-carbohydrate linkages seems to be rather pronounced within wood, a microwave reactor was used to replace traditional refluxing during the mild acidolysis step. This was done in an attempt to augment the selectivity of this step toward cleaving lignin-carbohydrate bonds as well as reducing the overall intensity of this step toward inducing changes in the lignin structure, thus affording lignin in greater yields and purities. Consequently, in this study the yields, purities, and structures of lignins isolated from spruce (softwood) by the EMAL protocol under various microwave conditions were examined. The variables studied included microwave power, microwave heating time, hydrochloric acid concentration and water content of the reaction medium. Microwave heating afforded EMAL samples of high purity (90%, comparable to the conventional protocol) but in significantly greater gravimetric yields. Quantitative (31)P NMR and SEC data confirmed that the structure of lignin was similar to that obtained by traditional EMALs, with comparable contents of beta-aryl ether bonds, phenolic hydroxyls (condensed and uncondensed), and carboxylic acids.     

Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes

The acetyl bromide assay was developed to provide a rapid and sensitive method for quantifying lignin in woody plant species. The original procedure cautioned against prolonged reaction times and advised keeping the reaction temperature at 70 degrees C to prevent excessive carbohydrate degradation that would skew the absorption spectra. Characterization of the reaction conditions revealed that the acetyl bromide reagent readily degrades xylans, a prominent polysaccharide group within all lignified plants. This degradation results in increased absorbance in the 270-280 nm region that is used to quantify lignin. The degradation of xylans is temperature dependent and is exacerbated by the addition of perchloric acid. Lowering the reaction temperature to 50 degrees C and increasing the reaction time from 2 to 4 h allows complete lignin solubilization but minimizes degradation of the xylans.     

Influence of prolonged arable cropping on lignin compounds in sandy soils of the South African Highveld

The preservation of plant residues is important for sustainable arable cropping. Lignin is a marker for plant residues in soils. We have investigated influences of the length of cultivation on the dynamics of lignin. Composite samples were taken from the top 20 cm of soils that have been cropped for periods varying from 0 to 98 years in each of three different agro‐ecosystems in the Free State Province of South Africa. Lignin‐derived phenols were determined in the <2 µm (clay), 2–20 µm (silt), 20–250 µm (fine sand) and 250– 2000 µm (coarse sand) size separates. With increasing length of cultivation, the concentration of such phenols decreased to 36% of that in the grassland. The lignin contents as proportions of the total carbon did not change during cultivation, suggesting that there was no selective enrichment of lignin moieties as C was lost as a result of cultivation. The loss rate constants of lignin concentrations in particle‐size fractions increased in the order clay (0.17 year^?1) ≤ silt (0.18 year^?1) < fine sand (0.20 year^?1) < coarse sand (0.22 year^?1). Increasing ratios of phenolic acids to aldehydes in bulk soil, silt and fine sand fractions with increasing length of cultivation indicated that side chains were being oxidized. The ratios in the silt fraction, however, decreased after 10–20 years. We attribute this to a loss of lignin together with silt by wind erosion, resulting in a rejuvenation of lignin compounds in the remaining silt‐sized pools of C.     

Estimation and decomposition pattern of the lignin component in forest humus layers

Lignin is one of the most abundant polymeric organic constituents of forest litter. Due to its molecular structure and heterogeneity the isolation of lignin in an unchanged form and its exact determination in forest humus have not proved possible. The oxidative degradation with CuO provides a specific method for the characterization of intact lignin structures in forest humus layers. The sum of phenolic CuO oxidation products gives an overall pattern of lignin decomposition. The degree of alteration of the remnant lignin is described by the acid-to-aldehyde ratio (Ac:Al) of syringyl and vanillyl units for angiosperm and gymnosperm lignin and the ratio of syringyl-to-vanillyl units (S:V) for angiosperm lignin.The lignin component of three different forest humus layers investigated was partly decomposed. The residual lignin fraction had undergone extensive modification during microbial decomposition. The chemical changes occurring in the lignin molecule during decomposition in forest humus layers suggest similar mechanisms of lignin degradation (“white-rot”) are shared by wood and forest litter.     

NMR evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into Lignins of O-methyltransferase-deficient poplars

Benzodioxane structures are produced in lignins of transgenic poplar plants deficient in COMT, anO-methyltransferase required to produce lignin syringyl units. They result from incorporation of 5-hydroxyconiferyl alcohol into the monomer supply and confirm that phenols other than the three traditional monolignols can be integrated into plant lignins.     

Inhomogeneities in the chemical structure of sugarcane bagasse lignin

Sequential treatments of dewaxed bagasse with distilled water, 0.5 M NaOH, 0.5, 1.0, 1.5, 2.0, and 3.0% H(2)O(2) at pH 11.5, and 2 M NaOH at 55 degrees C for 2 h solubilized 2.8, 52.5, 14.9, 3.3, 5.5, 5.0, 2.8, and 2.2% of the original lignin, respectively. The eight isolated lignin fractions were subjected to a comprehensive structural characterization by UV, FT-IR, and (1)H and (13)C NMR spectroscopies and thermal analysis. The nitrobenzene oxidation method was also applied to the in situ lignins. The seven lignin fractions, isolated successively with alkali and alkaline peroxide, were all SGH-type lignins, with a small amount of esterified p-coumaric acid and mainly etherified ferulic acid. No significant differences were found in the weight-average molecular weights (1680-2220 g/mol) of the seven alkali and alkaline peroxide dissolved lignins. However, the first four lignin fractions, isolated with 0.5 M NaOH and 0.5, 1.0, and 1.5% H(2)O(2) at pH 11.5, were rich in syringyl units and contained large amounts of noncondensed ether structures, whereas the last three lignin fractions, isolated sequentially with 2.0 and 3.0% H(2)O(2) at pH 11.5 and 2 M NaOH at 55 degrees C for 2 h, had a higher degree on condensation and were rich in guaiacyl lignins.     

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.     

Lignin in Wurzeln von Triticum

Lignin in roots of Triticum Lignin isolated from roots of T. vulgare was on the whole quite similar to that from straw. From all analytical methods used the fluorescence spectroscopy shows the greatest differences between the two lignins. But also the u. v.-spectra differentiated regarding the oxygen substituted benzene rings and the higher degree of condensation of root lignin. The latter had also ～ 25 % less conjugated aldehyde groups and between 1720–1620, about 1365 and from 720–630 cm^?1 slight modifications in the region of infrared absorption. The free radicals of the lignin and their oxidation in NaOH had only insignificant deviations. The content of lignin in roots was ～ 22 % and in straw ～ 25 %.