Determination Of Lignin Content Research Articles

Determination of the lignin content of flax fiber with the acetyl bromide-ultraviolet visible spectrophotometry method

The conventional Klason lignin (KL) method has some shortcomings in the determination of lignin content, such as large sample consumption, long analysis time, and a large number of errors. Therefore, a rapid and accurate method for lignin content determination was studied. In this paper, flax fibers in three forms, namely, flax raw, flax noil and flax rove, were selected for the study, and the acetyl bromide-ultraviolet spectrophotometric lignin (ABUL) method was optimized for quantitative testing of the lignin content in flax fibers. The effect of acetyl bromide concentration, perchloric acid dosage, reaction temperature, and reaction time on the absorbance value was tested systematically. The test results indicated that the maximum characteristic absorption peak of lignin in flax fiber was determined at 276 nm. The optimized reaction conditions were as follows: acetyl bromide concentration of 35%, perchloric acid dosage of 0.3 mL, reaction temperature of 70°C, and reaction time of 80 min. The standard curve of the ABUL method was y = 23.75714 x – 0.00636, R2 = 0.99913. By comparing the lignin content of flax fiber tested by the KL method and the ABUL method, it was found that the lignin content of flax raw, flax noil, and flax rove tested by the ABUL method was higher than that of the KL method. In addition, the ABUL method has the advantages of simplicity, rapidity, accuracy, and small sample consumption compared with the KL method.

Transcriptome Analysis of Chinese Cabbage Provides Insights into the Basis of Understanding the Lignin Affected by Low Temperature.

Chinese cabbage, which is a cold season crop, can still be damaged at an overly low temperature. It is crucial to study the mechanism of the resistance to low temperature of Chinese cabbage. In this study, the Chinese cabbage 'XBJ' was used as the material, and nine different low temperatures and control samples were treated. Using RNA-seq and lignin content determination, we analyzed 27 samples, and the stained sections of them were observed. A total of 8845 genes were screened for the WGCNA analysis, yielding 17 modules. The GO and KEGG analyses of the modules was highly associated with a low-temperature treatment. The pathways such as 'starch and sucrose metabolism' and 'plant hormone signal transduction' were enriched in modules related to low temperature. Interestingly, L-15DAT-associated MEcoral2 was found to have 14 genes related to the 'lignin biosynthetic process' in the GO annotation. The combination of the determination of the lignin content and the treatment of the stained sections showed that the lignin content of the low-temperatures samples were indeed higher than that of the control. We further explored the expression changes of the lignin synthesis pathway and various genes and found that low temperature affects the expression changes of most genes in the lignin synthesis pathway, leading to the speculation that the lignin changes at low temperature are a defense mechanism against low temperatures. The 29 BrCOMT gene sequence derived from the RNA-seq was non-conserved, and eight BrCOMT genes were differentially expressed. This study provides a new insight into how lignin is affected by low temperature.

RELATIONSHIP BETWEEN CELL LUMEN AREA AND LIGNIN CONTENT OF ALKALINE-TREATED DENSIFIED TIMBER OF PARASERIANTHES FALCATARIA

In this study, low-density plantation timber, Paraserianthes falcataria was pretreated with 3%, 6% and 9% NaOH before densification process. Alkaline pretreatment leads to lignin reductions and cell wall structure becomes more porous. Densification was done by crushing the cell wall with hot-press machine, resulting in reduction of thickness to about 60%. Scanning electron microscopy images were captured and processed through ImageJ software. As to support the data, lignin content determination was conducted according to TAPPI T222 and the correlation coefficient between cell lumen areas and lignin content were studied statistically.

Insights into the Molecular Regulation of Lignin Content in Triploid Poplar Leaves.

After polyploidization, plants usually undergo some morphological and physiological changes, including the lignin content of polyploids usually becoming lower than that of diploids. However, the regulatory mechanism of the variation of lignin content in polyploid plants remains unclear. Therefore, in this research, we used full-sib poplar triploids and diploids to explore the molecular regulatory basis of lignin content in poplar triploid leaves through the determination of lignin content, the observation of xylem cells, and transcriptome sequencing. The results showed that the lignin content of triploid leaves was significantly lower than that of diploid leaves. The xylem cells of triploid leaves were significantly larger than those of diploids. Transcriptome sequencing data show that most lignin biosynthesis genes were significantly downregulated, and genes related to cell growth were mostly upregulated in triploid leaves compared with diploid leaves. In addition, co-expression network analysis showed that several transcription factors might be involved in the regulation of lignin biosynthesis. Consequently, the altered expression of genes related to lignin might lead to the reduced lignin content in triploids. These results provide a theoretical basis for further exploring the molecular mechanism of the variation of polyploid lignin content and the utilization of polyploid lignocellulosic resources.

Kinetics of alkaline delignification of hemp and determination of lignin content by thermogravimetry

Raw hemp (Cannabis sativa L.) was subjected to alkaline delignification with the application of NaOH ranging from [0.125M] to [1M] for times from 5 to 480 min. Lignin contents of the original and delignified samples were determined. The samples were subjected to thermogravimetric analysis (TGA) under a N2 atmosphere with temperatures ramped from 25 to 600 °C at 10 °C min-1. Lignosulfonates samples of low, medium and high molecular weight were subjected to the same TGA and showed that lignin decomposed over the entire temperature range. The contributions of lignin to the mass loss during thermal decomposition of hemp and kinetics of lignin content and the most relevant TGA results were determined. Regression models estimating the lignin content of the fibers based on TGA results were formulated. These models allow for the prediction of mean lignin content within ± 0.1% (based on dry weight of pulp) in the central range of the experimental field.

Reliable and realistic models for lignin content determination in poplar wood based on FT-Raman spectroscopy

Studying lignin content is critical for analyzing the inherent constitution, performance, and application of lignocellulosic materials. However, the traditional methods (wet chemical methods) for the determination of lignin content have several limitations such as labor intensive, time-consuming preparations, and the use of toxic reagents. To address these shortcomings, based on 140 groups of poplar samples, a strategy of 1064 nm FT-Raman spectroscopy combined with several algorithms was proposed in our study. Before modeling, smoothing algorithm (Savitzky-Golay), baseline correction algorithm (adaptive iteratively reweighted penalized least squares), and deconvolution algorithm (Gaussian and Lorentzian) were applied to extract the Raman information. Also, several peaks including 1095, 1378, and 2895 cm−1 were selected as the candidates of internal standard peak for normalizing the lignin-related peaks. Subsequently, lignin content predictive models were established based on data extracted from FT-Raman spectra combined with regression algorithms, including principal components regression (PCR), partial least square regression (PLSR), ridge regression (RR), lasso regression (LR), and elastic net regressions (ENR). Consequently, credible models were obtained based on data normalized by peak of 2895 cm−1 combined with RR, LR, and ENR algorithms (Pearson’s R > 0.88, RMSE < 0.62, MAE < 0.46, and MAPE < 1.92%). Thus, the lignin quantitative models based on Raman spectroscopy developed here can be used to predict the lignin content in poplar clones accurately and efficiently.

Comparison of calibration models for rapid prediction of lignin content in lignocellulosic biomass based on infrared and near-infrared spectroscopy

Lignocellulosic biomass has great potential as a renewable energy source due to abundance both as vegetation and in residues from agriculture and forestry. It can be utilized for biofuels and for value-added chemicals and materials. The challenge concerning lignocellulosic biomass lies in its heterogenous nature. The chemical composition may vary according to species, location, harvest season and botanic fractions. Therefore, it is crucial to assess the composition prior to any biofuel conversion method. Vibrational spectroscopy has been used extensively for rapid predictions of the chemical properties of biomass. In this study, calibration models based on infrared and near-infrared spectra has been compared using the same calibration set, 36 samples comprising wood, bark, needles, and twigs of three different wood species. PLS was performed to correlate EMSC pretreated infrared and near-infrared spectra to the chemical contents of the samples. R2 for the standard curve of the infrared model is 0,896 and for the near-infrared standard curve 0,921, using 3 PLS components. The effect of heterogeneity was tested by comparing calibration models based on finely and coarsely ground sample, where the R2 of the coarsely ground sample was 0,825, lower than 0,896, but still significant. The results also show that there is more variation in the lignin content between the fractions from a single tree than between similar fractions from different species. The calibration models that have been developed will be useful for frequent, rapid determination of lignin content in wood biorefining feedstocks.

Количественное определение лигнина в растительных материалах с помощью ИК-спектроскопии

A methodical approach for determining the content of lignin in plant materials by the FT-IR method in the mid-infrared range has been developed. The technique for determining lignin content is based on the use of a cali- bration graph reflecting the dependence of the intensity of the 1510 cm-1 analytical absorption band on the lignin con- tent in model samples. This method was tested on a wide range of lignocellulosic biomass samples. The suitability of the proposed method for the determination of lignin content has been shown.

A facile spectroscopic method for measuring lignin content in lignocellulosic biomass

A spectroscopic method was developed for the determination of lignin contents in lignocellulosic biomass, based on a full dissolution of the biomass sample in a cysteine–sulfuric acid solution at room temperature.

Improving crops genome through genetic engineering of the key metabolic pathways

Soybean loss due to pests and pathogens is a serious problem worldwide. Soybean producers have few options to manage diseases caused by general pathogens where major genes for full resistance have not been discovered. The innate defense of soybean plants could be enhanced by improving content and composition of lignin by genetic engineering of the phenylpropanoid pathway. We used a novel technique of germ-line genetic transformation of soybean plants via natural pollen tubes as vectors. This technique uses Agrobacterium tumefaciens to mediate transfer of genes of interest to the zygote to introduce the key lignification genes (PtMYB4, PAL5, F5H, CAD1) into soybean genome. We observed 5.6% average transformation efficiency in the first generation of transgenic plants and in the second generation the presence of the transgene constructs was confirmed in more than 50% (for CsVMV/PtMYB4sens, 35SVTM/PAL5, C4H/F5H, CsVMV/CAD1 constructs) transgenic soybean lines. We confirmed the expression of the introduced genes at transcriptional level using RT-PCR and Northern blot. Functional analysis using lignin content determination and the activity of PAL5 and CAD1 enzymes demonstrated that the transgenes perform their function in planta. The proposed technique is effective and inexpensive and can be used to create novel stress and disease resistant soybean genotypes.

Cell wall remodeling under salt stress: Insights into changes in polysaccharides, feruloylation, lignification, and phenolic metabolism in maize.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.

Characterization of composite material from the copolymerized polyphenolic matrix with treated cassava peels starch

Conventional binders in the particleboards formulation involve use of formaldehyde resins. Epidemiologic studies show that formaldehyde is carcinogenic. Efforts to reduce formaldehyde emissions by use of scavengers has not been proven to reduce the emission. Molecular bonding of biobased adhesive molecules with lignocellulose materials provides an alternative way of producing composite material. In this study, maize stalk (MS), rice husks (RH) and sugarcane bagasse (SB) were used as sources of lignocellulose materials for particleboard formulation. SB, MS and RH were collected from their respective sites, sorted and dried. MS and RH were ground. Lignin content determination was done by drying lignocellulose material at 105 °C. Lignocellulose materials were prepared by hydrolysis of dried lignocellulose material with sodium hydroxide. Oxidized starch was prepared by oxidation of cassava peel starch using alkaline hydrogen peroxide. Particleboards were formulated through starch-lignocellulose polymerization at 60 °C compressed with 6.5 Nmm−2 pressure. Characterization of raw materials and formulated particleboards was done using XRD for mineralogical analysis, FTIR and NMR for elucidation of functional groups transformation. The results showed that esterification is the main process of chemical bonding in the particleboard formulation due to reaction between –COOH from starch and and OH- from lignocellulose. Etherification between hydroxyl groups from starch with hydroxyl groups from lignocellulose material. RH combined more through silication process with cassava peels starch than RH and SB showing materials containing high cellulose and hemicellulose content are more compatible. Composite materials formulated were used to produce medium density particleboards that can be used for making furniture and room partitioning.

Nondestructive determination of lignin content in Korla fragrant pear based on near-infrared spectroscopy

The lignin content is a key index affecting the quality of Korla fragrant pear. This study applied a method of near-infrared diffuse reflectance spectroscopy combined with chemometric method to establish a predictive model of lignin content. Among them, moving average, baseline, standard normal variate transformations and multiplicative scatter correction were used to preprocess the collected near-infrared spectra. then, the backward interval partial least-squares, synergy interval partial least squares and uninformative variable elimination algorithms were used to select characteristic wavelengths from the whole spectral range to establish a partial least-squares prediction model of lignin content. The partial least-squares model based on uninformative variable elimination selected characteristic wavelengths was simplified, and the prediction of lignin content in Korla pear was more accurate. Multiple determination coefficient values, standard prediction errors and residual prediction errors for prediction were 0.87, 1.36 and 2.03%, respectively. Compared with partial least squares based on the whole spectral range and partial least squares based on Uninformative variable elimination selection, the number of characteristic wavelength variables was reduced from 289 to 51, thereby improving the prediction accuracy of the model. The experimental results showed that the uninformative variable elimination by partial least-squares model was the best model. The combination of near-infrared spectroscopy and uninformative variable elimination optimization can effectively determine the lignin content in Korla fragrant pear. The near-infrared diffuse reflectance spectroscopy proved to be a good tool for nondestructive determination of lignin content in Korla fragrant pear. The selection of characteristic wavelengths and appropriate pretreatment methods can improve the accuracy of near-infrared spectroscopy in actual detection.

A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification.

One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.

Non-isothermal pyrolysis of grape marc

The non-isothermal decomposition behavior of grape marc (GM) residues from wine industry was investigated by using different thermal analysis techniques, including: (1) thermogravimetric analysis/differential thermogravimetry (TGA-DTG) to study the thermal decomposition kinetics; (2) thermogravimetry (TG-DTG) coupled with Fourier transform infrared spectroscopy (FTIR) to investigate the nature of the gas-phase products released during the pyrolytic breakdown; and (3) simultaneous thermogravimetry/differential scanning calorimetry (TGA–DSC) analysis to obtain information on the heat flows associated with the thermal decomposition of grape marc. Thermogravimetric measurements at five different heating rates (i.e., 2.5, 5, 10, 20, 40 K min−1) were performed for the kinetic computations, which were carried out by adopting a “model-free” approach based on the application of isoconversional methods. In more details, two different integral methods, i.e., the linear Ozawa–Flynn–Wall (OFW) method and the nonlinear Vyazovkin incremental method, were comparatively used in order to obtain a set of kinetic parameters useful for the conceptual design of thermochemical processes involving grape marc. The reliability of the obtained parameters was confirmed by the successful application of the same data to reproduce experimental TG curves not included in the kinetic computations. The effect of the heating rate on the nature of the gas-phase products arising from grape marc decomposition as well as on the heat flows associated with the pyrolytic process was also investigated. Finally, the study was complemented with an extensive investigation on chemical and physical properties of grape marc residues (i.e., ultimate analysis, proximate analysis, calorific values determination, FTIR analysis and cellulose, hemicellulose and lignin content determination), which provides useful input data for modeling grape march conversion processes.

Proteomic analysis of the key mechanism of exocarp russet pigmentation of semi-russet pear under rainwater condition

This study aimed to explore the mechanism of exocarp russet pigmentation of semi-russet ‘Cuiguan’ sand pears at the proteome level following rainwater. Exocarp russeting was characterized in semi-russet sand pears cultivated in the rain shelter and control (rainwater) groups via microscopy and ultra-microscope observations, followed by determination of lignin content and enzyme activities. The differentially expressed proteins between different cultivation conditions were analyzed using the isobaric tags for relative or absolute quantitation (iTRAQ) technology, followed by confirmation by real-time quantitative polymerase chain reaction (qPCR). The rust color of the outer pericarp of ‘cuiguan’ was rapidly accumulated on 60–80 days after full bloom (DAFB) in both two groups. Under the shelter condition, most stratum corneum cracks were shallow and had less suberization. The lignin content as well as the enzyme activities of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), catechol-O-methyltransferase (COMT), cinnamoyl-CoA reductase (CCR) and peroxidase (POD) in exocarp from the rain shelter group were all lower than that in control group. Additionally, 309 differentially expressed proteins were identified in the rain shelter group, and eight down-regulated proteins associated with lignin monomer synthesis were identified, such as PAL (E9JWF1), CCR1 (F4Y9E7), CAD (U3PGC0), two peroxidases (K9URQ0 and M5W3Y0, respectively), two COMT (S5TPC3 and Q09K02, respectively) and omega-hydroxypalmitate O-feruloyl transferase (HHT1) (M5VMR7), and their expression was confirmed by qPCR. Moreover, many defense proteins were significantly down-regulated. These findings suggest that the rain shelter treatment may affect phenylalanine lignin monomer synthesis and subsequent defense metabolism by regulating corresponding proteins, thus decreasing exocarp russet accumulation in semi-russet pear. This study provides a further understanding of pear russeting and plant defense mechanisms.

Extraction of lignin from tobacco stem using ionic liquid

Lignin only accounts for about 6% of total mass in tobacco stem, but it influences the harmful substances in the side stream smoke of cigarette in a significant way. Traditional researches focus only on the determination of lignin content. In the present work, we investigate four typical imidazolium-based ionic liquids for efficient extraction of lignin under mild conditions and 1-ethyl-3-methylimidazolium diethylphosphate ([Emim][DEP]) shows the best results. The pretreatment of stem using water at 80 °C for 30 min can not only remove most of the sugars but also loose the microfibers. The extractive rate of lignin reaches 85.38% at 150 °C for 4 h and the purity of lignin is 90.21%.

High-Throughput Method for Determining the Sugar Content in Biomass with Pyrolysis Molecular Beam Mass Spectrometry

There is an important need to assess biomass recalcitrance in large populations of both natural and transgenic plants to identify promising candidates for lignocellulosic biofuel production. In order to properly test and optimize parameters for biofuel production, the starting sugar content must be known to calculate percent sugar yield and conversion efficiencies. Pyrolysis molecular beam mass spectrometry (py-MBMS) has been used as a high-throughput method for determination of lignin content and structure, and this report demonstrates its applicability for determining glucose, xylose, arabinose, galactose, and mannose content in biomass. Biomass from conifers, hardwoods, and herbaceous species were used to create a 44 sample partial least squares (PLS) regression models of py-MBMS spectra-based sugar estimates on high-performance liquid chromatography (HPLC) sugar content data. The total sugar py-MBMS regression model had a R 2 of 0.91 with a 0.17 mg/mg root mean square error of validation indicating accurate estimation of total sugar content for a range of biomass types. Models were validated using eight independent biomass samples from multiple species, with predictions falling within errors of the HPLC data. With a data collection time of 1.5 min per sample, py-MBMS serves as a rapid high-throughput method for quantifying sugar content in biomass.

Quantitative Examination of Pre-Extraction Treatment on the Determination of Lignin Content in Leaves

It has been suggested that the Klason method overestimates the lignin content of non-wood tissues of plants. To evaluate the effect of pre-extraction treatments on lignin determination in leaves, nine kinds of pre-extraction treatment were applied to ginkgo leaves and zelkova leaves. The apparent lignin contents (lignin determination by the Klason method) of ginkgo and zelkova leaves without pre-extraction treatments were 30.7% and 42.6%, respectively. After the various pre-extraction treatments, the apparent lignin contents were still high. On the other hand, the yield of nitrobenzene oxidation products (NPs) per apparent lignin content was maintained at a very low level before and after pre-extraction treatments (maximum value was 6% for ginkgo leaf and 11% for zelkova leaf after extraction treatment) compared with the value from wood (25 to 60%). These results suggested that the Klason method overestimates the lignin content of leaves even after the pre-extraction treatments examined in this study. In addition, a considerable part of the sample from which NPs or neutral sugars originate was lost during these pre-extraction stages. These results implied that some parts of the cell wall components were also removed by these pre-extraction treatments.

Determination of lignin content in kraft black liquors by capillary zone electrophoresis (CZE)

Abstract Capillary zone electrophoresis (CZE) in an alkaline glycine buffer is suggested for the quantification of lignin content in black liquors (BL). The method was first tested by an external calibration with LignoBoost lignins. Then, the lignin content in BL was determined by means of a multivariate calibration with the application of a standard normal variate filter and partial least squares approach based on five principal components. The results are in agreement with those obtained by sulfuric acid lignin precipitation combined with ultraviolet measurement of the lignin in solution. The advantage of the CZE method is its independence from the knowledge of the exact absorptivity coefficient, which is needed for direct spectrophotometric lignin determination. Moreover, interfering substances and degradation products could be recognized and excluded from lignin determination; thus, the selectivity was increased significantly.