We thank the Department of Plant &#38; Soil Science and Cotton Inc. for their partial support of this study.

1. Preparation of plant samples
<ol>
	<li>Collect two-month-old cotton plants from the greenhouse (Figure 1A).</li>
	<li>Flip plant pots gently to separate soil and roots with intact lateral roots by loosening the soil around the plant (Figure 1B).</li>
	<li>Wash the collected plants thoroughly in trays filled with water to remove all the dirt (for root samples) (Figure 1C).</li>
	<li>Use paper towels to dry separated root, stem, and leaf tissues, and label them (Figure 1D). Air dry for 2 days at room temperature to prevent any fungal contamination (Figure 1E).</li>
	<li>Transfer sample tissues to labelled containers/aluminum foils and incubate in a temperature-controlled incubator at 49 &#176;C for 7 to 10 days (Figure 1F). 
	NOTE: Higher temperatures may alter the lignin structure. Alternatively, a freeze dryer can be used to dry samples for 1 to 2 days without causing any chemical changes to the plant biomass.</li>
	<li>Use a blade to cut the incubator dried tissue into 5 mm size pieces or alternatively employ a biomass grinder to grind the plant tissues (Figure 1G, Figure 1H). 
	NOTE: The biomass grinder/blade must be cleaned after each sample was cut/ground.</li>
	<li>Transfer the cut tissue/biomass grounded plant material into grinding vials and grind into fine powder of 1 mm size using a freezer mill or cryogenic grinder with liquid N2.</li>
	<li>Grind samples for three cycles at the rate of 10 CPS (each cycle span of 2 min) into a uniform powder (Figure 1I, 1J, 1K). 
	&#8203;NOTE: The experiment can be paused at this point and samples can be stored at room temperature in airtight containers for long-term storage.</li>
</ol>
2. Washing samples with water, PSB, and methanol
<ol>
	<li>Measure and record the weight of all empty 2 mL microfuge tubes used for lignin content estimation in the lab notebook.</li>
	<li>Transfer 20 mg of the ground sample powder to the pre-weighed tube. Weigh the tube with tissue and tissue powder and record these weights in the lab notebook.</li>
	<li>Incubate all 2 mL microfuge tubes (with open lids) with 20 mg of tissue powder in a heat block or oven at 60 &#176;C for 1 hour.</li>
	<li>After incubation, cool samples for 10 min at room temperature (RT).</li>
	<li>Add 1.8 mL of water to each microfuge tube and mix by vortexing. Then, centrifuge at 25,200 x g (15,000 rpm) for 10 min at RT and discard the supernatant (Figure 2).</li>
	<li>Add 1.8 mL of Protein Solubilization buffer (PSB) (Table 1) to each retained pellet and mix by vortexing. Centrifuge at 25,200 x g (15,000 rpm) for 10 min at RT and discard the supernatant.</li>
	<li>Repeat step 2.6 again for each sample.</li>
	<li>Add 1.8 mL of water to each pellet, mix by vortexing, and centrifuge at 25,200 x g (15,000 rpm) for 10 min. After centrifugation, save the pellet and discard the supernatant.</li>
	<li>To the retained pellet, add 1.8 mL of methanol and incubate in a 60 &#176;C heat block for 20 min. Then, centrifuge at 25,200 x g (15,000 rpm) for 10 min at RT. After centrifugation, discard the supernatant and retain the pellet (Figure 2).</li>
	<li>Repeat step 2.9 again for each sample.</li>
	<li>Air dry the pellet at RT or proceed immediately by vacuum drying. Vacuum dry using vacuum drier at 30 &#176;C for 2 to 3 hours or until the pellet is completely dry. 
	NOTE: The experiment can be paused at this point by air drying over-night or continue by vacuum drying.</li>
	<li>After drying, weigh sample tubes with the dried pellet and record the weight next to the respective empty tube weight in the lab notebook. Estimate the pellet weight by subtracting the two values. These weights will be used for lignin estimation at the end of lignin extraction process.</li>
	<li>At this point of lignin extraction, include the commercial bamboo lignin for generation of the lignin standard curve. Measure commercial bamboo lignin into separate tubes ranging from 0.5 mg to 5 mg in 0.5 mg increments (0.5 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, and 5.0 mg). Measure each concentration three times for three technical replicates. 
	&#8203;NOTE: From here on, the standards measured in the above step were processed in the same way as samples that were dried.</li>
</ol>
3. Treatment of pellet with TGA and acid to precipitate lignin
<ol>
	<li>Subject processed samples from the above step, along with measured standards, to TGA (thioglycolic acid) treatment.</li>
	<li>Add 1 mL of 3 N HCl (Table 1) and 100 &#181;L of TGA to each pellet.</li>
	<li>Vortex and incubate in an 80 &#176;C preheated heat block for 3 hours in a fume hood (Figure 2). 
	NOTE: The heating step at 80 &#176;C must be monitored. High pressure buildup may open the lids and can lead to chemical spills. Screw cap tubes are recommended but 2 mL tubes can be loosely capped during this step as an alternative to prevent such spills.</li>
	<li>After incubation, cool tubes at RT for 10-15 min and centrifuge at 25,200 x g (15,000 rpm) for 10 min at RT. 
	NOTE: Waste generated from acid and organic solvents must be separated and stored in glass containers with ventilated caps. Use separate glass containers for the collection of TGA-acid waste and acid waste.</li>
	<li>After centrifugation, discard the supernatant and retain the pellet. Add 1 mL of water, mix by vortexing, and centrifuge at 25,200 x g (15,000 rpm) for 10 min at RT.</li>
	<li>After centrifugation, discard the supernatant and mix the pellet in 1 N NaOH for 24 h at 37 &#176;C shaker/thermal mixer at low speed (Figure 2). 
	NOTE: This incubation time can be reduced to 1 hour7.</li>
	<li>After incubation, centrifuge the 2 mL microfuge tubes at 25,200 x g (15,000 rpm) for 10 min at RT. Retain the supernatant for the following step. 
	&#8203;NOTE: The procedure involves use of strong acids and other chemicals that are corrosive in nature. Hence, wearing proper PPE is recommended throughout the process of lignin estimation. TGA has a strong unpleasant smell and is corrosive in nature. Hence, it is recommended to use only in the fume hood.</li>
</ol>
4. Precipitation of lignin
<ol>
	<li>Transfer the supernatant to a fresh 2 mL microfuge tube and add 200 &#181;L of concentrated HCl. Incubate at 4 &#176;C for 4 hours or overnight (Figure 2). 
	NOTE: The extraction process can be paused at this point by extending the refrigeration step to overnight.</li>
	<li>Centrifuge at 25,200 x g (15,000 rpm) for 10 min at RT and dissolve the pellet in 1 mL of 1 N NaOH.</li>
	<li>Incubate in the shaker at RT for 10 min to suspend the pellet completely in NaOH.</li>
	<li>Finally, measure the absorbance of samples at 280 nm using a spectrophotometer and compare with the standard lignin curve.</li>
	<li>Measure the unknown concentration of lignin by using the calibration curve regression line values, and absorbances of extracted samples at 280 nm.</li>
</ol>
5. Standard curve preparation and lignin estimation in the sample
<ol>
	<li>Process lignin standards in the same way as experimental samples from TGA treatment.</li>
	<li>Measure commercial bamboo lignin standards in 0.5 mg increments starting from 0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg and 5 mg. Then, process by TGA, HCl, dissolve in 1 N NaOH followed by measuring absorbance at 280 nm (Figure 3A).</li>
	<li>Use values of lignin concentration and absorbance readings to generate a scattered plot of standard lignin curve (Figure 3B).</li>
	<li>Use the regression line, y = mx+c generated in the scattered plot, for the estimation of unknown lignin content of prepared samples using &#34;x&#34; values from average absorbances of extracted samples at 280 nm and &#34;m&#34; and &#34;c&#34; values from lignin standard curve regression line.</li>
	<li>Divide the lignin content in the resultant y value by total weight of the vacuum/air dried plant biomass sample after methanol extraction in mg (approximately 15 mg) to obtain lignin concentration per mg. Then, multiply this value by 100 to calculate lignin percentage per mg.</li>
</ol>

<ol>
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The authors declare that they have no conflict of interest.

Lignin plays a significant role in plant growth and development and recently has been extensively studied for biofuel, bioenergy and bioproduct applications. Lignin is rich in aromatic compounds that are stored in all vascular plant secondary cell walls. It has several industrial applications such as wood panel products, bio dispersants, flocculants, polyurethane foams and in resins of circuit boards29,30,31. Most of the lignin ...

Lignin is one of the vital load-bearing components of plant cell walls and the second most abundant polymer on Earth1. Chemically, lignin is a crosslinked heteropolymer made up of high molecular weight complex phenolic compounds that form a natural renewable source of aromatic polymers and synthesis of biomaterials2,3. This natural polymer plays significant roles in plant growth, development, survival, mechanical support, cell wall rigidity, water transport, mineral transport, lodging resistance, tissue and organ development, deposition of energy, and protection from biotic and abiotic stresses4,5,6,7. Lignin is primarily composed of three different monolignols: coniferyl, sinapyl and p-coumaryl alcohols that are derived from the phenyl propanoid pathway8,9. The amount of lignin and the composition of monomers vary based on the plant species, the tissue/organ type, and different stages of plant development10. Lignin is broadly classified into softwood, hardwood, and grass lignin based on the source and monolignol composition. Softwood is primarily composed of 95% coniferyl alcohol with 4% p-coumaryl and 1% sinapyl alcohols. Hardwood has coniferyl and sinapyl alcohols in equal proportions, while grass lignin is composed of various proportions of coniferyl, sinapyl and p-coumaryl alcohols11,12. The composition of monomers is critical as it determines the lignin strength, decomposition, and degradation of the cell wall as well as determining molecular structure, branching, and crosslinking with other polysaccharides13,14.
Lignin research is gaining importance in foraging, textile industries, paper industries, and for bioethanol, biofuel, and bio-products due to its low cost and high abundance15,16. Various chemical methods (e.g., acetyl bromide, acid detergents, Klason, and permanganate oxidation) along with instrumental methods (e.g., near infrared (NIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and ultraviolet (UV) spectrophotometry) were used for lignin quantification9,17. The analysis methods of lignin are generally classified based on electromagnetic radiation, gravimetry, and solubility. The principle behind lignin estimation by&#160;electromagnetic radiation was based on the&#160;chemical property of lignin by which it absorbs light at specific wavelengths. These results were estimated based on the principle that lignin has a stronger UV absorbance than carbohydrates. In 1962, Bolker and Somerville used potassium chloride pellets to estimate lignin content in wood18. However, this method has drawbacks in the estimation of lignin content from herbaceous samples due to the presence of non-lignin phenolic compounds and the absence of an appropriate extinction coefficient. In 1970, Fergus and Goring found that the guaiacyl and syringyl compound absorption maxima were at 280 nm and 270 nm, which corrected the extinction coefficient issue of the Bolker and Somerville method19. Later, infrared spectroscopy, a highly sensitive technique for characterizing phenolics, was also used for lignin estimation with a small amount of plant biomass samples. One example of such technology&#160;was diffuse-reflectance Fourier transform spectrophotometry. This method, however, lacks a proper standard similar to the UV method20. Later, the lignin content was estimated by NIRS (near infrared spectroscopy) and NMR (nuclear magnetic resonance spectroscopy). Though, there are disadvantages in these methods, they do not alter the chemical structure of lignin, retaining its purity20.
The gravimetric Klason method is a direct and the most reliable analytical method for lignin estimation of woody stems. The basis for gravimetric lignin estimation is the hydrolysis/solubilization of non-lignin compounds and the collection of insoluble lignin for gravimetry21. In this method, the carbohydrates are removed by hydrolysis of the biomass with concentrated H2SO4 to extract lignin residue20,22. The lignin content estimated by this method is known as acid insoluble lignin or Klason lignin. Application of the Klason method depends on the plant species, the tissue type and the cell wall type. The presence of variable amounts of non-lignin components such as tannins, polysaccharides and proteins, results in proportional differences in the estimation of acid insoluble/soluble lignin contents. Hence, the Klason method is only recommended for lignin estimation of high-lignin content biomass such as woody stems17,23. Solubility methods such as acetyl bromide (AcBr), acid-insoluble lignin, and thioglycolic acid (TGA) are most commonly used methods for estimation of the lignin content from various plant biomass sources. Kim et al. established two methods for lignin extraction by solubilization. The first method extracts lignin as an insoluble residue by solubilizing cellulose and hemicellulose, while the second method separates lignin in the soluble fraction, leaving cellulose and hemicellulose as the insoluble residue24.
Similar methods employed in lignin estimation based on the solubility are thioglycolic acid (TGA) and acetyl bromide (AcBr) methods25. Both TGA and acetyl bromide methods estimate the lignin content by measuring the absorbance of the solubilized lignin at 280 nm; however, the AcBr method degrades xylans during the process of lignin solubilization and shows a false increase in the lignin content26. The thioglycolate (TGA) method is the more reliable method, as it depends on specific bonding with the thioether groups of benzyl alcohol groups of lignin with TGA. The TGA bound lignin is precipitated under acidic conditions using HCl, and the lignin content is estimated using its absorbance at 280 nm27. The TGA method has additional advantages of less structural modifications, a soluble form of lignin estimation, less interference from non-lignin components, and precise estimation of lignin due to specific bonding with TGA.
This TGA method is modified based on the kind of plant biomass sample used for lignin content estimation. Here, we modified and adapted the rapid TGA method of rice straws27 to cotton tissues to estimate the lignin content. Briefly, the dried powdered plant samples were subjected to protein solubilization buffer and methanol extraction to remove proteins and the alcohol soluble fraction. The alcohol insoluble residue was treated with TGA and precipitated lignin under acidic conditions. A lignin standard curve was generated using commercial bamboo lignin and a regression line (y = mx+c) was obtained. The &#34;x&#34; value uses average absorbance values of lignin at 280 nm, while &#34;m&#34; and &#34;c&#34; values were entered from the regression line to calculate unknown lignin concentration in cotton plant biomass samples. This method is divided into five phases: 1) preparation of plant samples; 2) washing the samples with water and methanol; 3) treatment of the pellet with TGA and acid to precipitate lignin; 4) precipitation of lignin; and 5) the standard curve preparation and lignin content estimation of the sample. The first two phases are primarily focused on the plant material preparation followed by water, PSB (protein solubilization buffer) and methanol extractions to obtain the alcohol insoluble material. Then, it was treated with TGA (thioglycolic acid) and HCl to form a complex with lignin in the third phase. At the end, HCl was used to precipitate lignin, which was dissolved in sodium hydroxide to measure its absorbance at 280 nm28.

<table><tbody><tr><td>BioSpectrophotometer kinetic</td><td>Eppendorf kinetic</td><td>6136000010</td><td>For measuring absorbance at 280 nm</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td>For centrifuging&amp;nbsp; samples</td></tr><tr><td>Commercial bamboo lignin</td><td>Aldrich</td><td>1002171289</td><td>Used in the preparation of the standard curve</td></tr><tr><td>Distilled water</td><td>Fischer Scientific</td><td>16690382</td><td>Used in the protocol</td></tr><tr><td>Falcon tubes</td><td>VWR</td><td>734-0448</td><td>Containers for solutions</td></tr><tr><td>Freezer mill</td><td>Spex Sample Prep</td><td>68-701-15</td><td>For fine grinding of plant tissue samples</td></tr><tr><td>Heat block/ Thermal mixer</td><td>Eppendorf</td><td>13527550</td><td>For temperature controlled steps during lignin extraction</td></tr><tr><td>Hotplate stirrer</td><td>Walter</td><td>WP1007-HS</td><td>Used for preparation of solutions</td></tr><tr><td>Hydrochloric acid (HCL)</td><td>Sigma</td><td>221677</td><td>Used in the protocol</td></tr><tr><td>Incubator</td><td>Fisherbrand</td><td>150152633</td><td>For thorough drying of plant tissue samples</td></tr><tr><td>Measuring scale</td><td>Mettler toledo</td><td>30243386</td><td>For measuring plant tissue weight, standards and microfuge tubes</td></tr><tr><td>Methanol (100 %)</td><td>Fischer Scientific</td><td>67-56-1</td><td>Used in the protocol</td></tr><tr><td>Microfuge tubes (2 mL)</td><td>Microcentrifuge</td><td>Z628034-500EA</td><td>Containers for extraction of lignin</td></tr><tr><td>Plant biomass gerinder</td><td>Hanchen</td><td>Amazon</td><td>Used for crushing dried samples</td></tr><tr><td>pH meter</td><td>Fisher Scientific</td><td>AE150</td><td>Measuring pH for solutions prepared for lignin extraction</td></tr><tr><td>Temperature controlled incubator/oven</td><td>Fisher Scientific</td><td>15-015-2633</td><td>Used in the protocol</td></tr><tr><td>Thioglycolic acid (TGA)</td><td>Sigma Aldrich</td><td>68-11-1</td><td>Used in the protocol</td></tr><tr><td>Vacuum dryer</td><td>Eppendorf</td><td>22820001</td><td>Used for drying samples</td></tr><tr><td>Vortex mixer</td><td>Eppendorf</td><td>3340001</td><td>For proper mixing of samples</td></tr></tbody></table>

estimation of plant biomass lignin content using thioglycolic acid (tga)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Two different cotton experimental lines were compared for differences in their lignin contents in different tissues. The extracted lignin content of each sample was measured at 280 nm and recorded its respective absorbance values. The average absorbance values of each biological replicate were compared against the regression line of the lignin standard curve (Table 2, Figure 3C). The regression line, y = mx + c, is used to calculate the unknown lignin content of the extracte...

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Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

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Research

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Biochemistry

9.3K Views.  Texas Tech University. Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation. 

Video: Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Environment

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The analysis does not usually require extensive sample preparation; structural and chemical information can be obtained without labeling. However, each Raman image contains thousands of spectra; this raises difficulties when extracting hidden information, especially for components with similar chemical structures. This work introduces a multivariate analysis to address this issue. The protocol establishes a general method to visualize the main components, including lignin, cellulose, and hemicellulose within the plant cell wall. In this protocol, procedures for sample preparation, spectral acquisition, and data processing are described. It is highly dependent upon operator skill at sample preparation and data analysis. By using this approach, a Raman investigation can be performed by a non-specialist user to acquire high-quality data and meaningful results for plant cell wall analysis.

This protocol aims to present a general method to visualize lignin, cellulose, and hemicellulose in plant cell walls using Raman imaging and multivariate analysis.

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Bioengineering

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Chemistry

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

Biology

Fractionation of Lignocellulosic Biomass using the OrganoCat Process

The shift from a petroleum-based to a more sustainable and bio-based economy requires the development of new refinery concepts to maintain the supply of raw materials and energy. For these novel and sustainable biorefinery concepts, it is important to use catalysts and solvents that are aligned with the principles of Green Chemistry. Therefore, the implementation of biogenic alternatives can be a promising solution. The lignocellulose pretreatment and fractionation process presented herein-OrganoCat-is an integrated fractionation of lignocellulose into its main components using biogenic acids such as 2,5-furandicarboxylic acid as catalyst. Hemicelluloses and other non-cellulosic polysaccharides are selectively depolymerized by the diluted acid and dissolved, while the crystalline cellulose remains in the solid pulp. In the presence of a second organic phase consisting of biogenic 2-methyltetrahydrofuran, disentangled lignin is extracted in situ. The process allows for the efficient fractionation of the three main components-lignin, cellulose, and non-cellulosic sugars. This helps to focus on the quality of the lignin, the improvement of enzymatic hydrolysis of the cellulose-enriched pulp, and the mild non-cellulosic sugar extraction with low degradation.

OrganoCat is a method for the pretreatment and fractionation of lignocellulose under mild conditions into lignin, fermentable sugars, and cellulose pulp. In a biogenic, biphasic solvent system of water and 2-methyltetrahydrofuran with 2,5-furancarboxylic acid as catalyst, the OrganoCat products are separated in situ for straightforward product recovery.

The shift from a petroleum-based to a more sustainable and bio-based economy requires the development of new refinery concepts to maintain the supply of ...

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements as well as power and heat consumption and by using environment-friendly solvents. Deep eutectic solvents (DESs) are key, green, and low-cost solvents in sustainable biorefineries. They are transparent mixtures characterized by low freezing points resulting from at least one hydrogen bond donor and one hydrogen bond acceptor. Although DESs are promising solvents, it is necessary to combine them with an economic heating technology, such as microwave irradiation, for competitive profitability. Microwave irradiation is a promising strategy to shorten the heating time and boost fractionation because it can rapidly attain the appropriate temperature. The aim of this study was to develop a one-step, rapid method for biomass fractionation and lignin extraction using a low-cost and biodegradable solvent. 
In this study, a microwave-assisted DES pretreatment was conducted for 60 s at 800 W, using three kinds of DESs. The DES mixtures were facilely prepared from choline chloride (ChCl) and three hydrogen-bond donors (HBDs): a monocarboxylic acid (lactic acid), a dicarboxylic acid (oxalic acid), and urea. This pretreatment was used for biomass fractionation and lignin recovery from marine residues (Posidonia leaves and aegagropile), agri-food byproducts (almond shells and olive pomace), forest residues (pinecones), and perennial lignocellulosic grasses (Stipa tenacissima). Further analyses were conducted to determine the yield, purity, and molecular weight distribution of the recovered lignin. In addition, the effect of DESs on the chemical functional groups in the extracted lignin was determined by Fourier-transform infrared (FTIR) spectroscopy. The results indicate that the ChCl-oxalic acid mixture affords the highest lignin purity and the lowest yield. The present study demonstrates that the DES-microwave process is an ultrafast, efficient, and cost-competitive technology for lignocellulosic biomass fractionation.

Deep eutectic solvent-based, microwave-assisted pretreatment is a green, fast, and efficient process for lignocellulosic fractionation and high-purity lignin recovery.

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements ...

Quantitative 31P NMR Analysis of Lignins and Tannins

The development of sustainable biorefinery products is confronted, among others, with the challenge of lignin and tannin valorization. These abundant, renewable aromatic biopolymers have not been widely exploited due to their inherent structural complexity and high degrees of variability and species diversity. The lack of a defined primary structure for these polyphenols is further compounded with complex chemical alterations induced during processing, eventually imparting a large variety of structural features of extreme significance for any further utilization efforts.
Consequently, a protocol for the rapid, simple, and unequivocal identification and quantification of the various functional groups present in natural polyphenols, is a fundamental prerequisite for understanding and accordingly tailor their reactivity and eventual utility.
Quantitative 31P NMR offers the opportunity to rapidly and reliably identify unsubstituted, o-mono substituted, and o-disubstituted phenols, aliphatic OHs, and carboxylic acid moieties in lignins and tannins with broad application potential.
The methodology consists of an in situ quantitative lignin or tannin labeling procedure using a suitable 31P containing probe, followed by the acquisition of a quantitative 31P NMR spectrum in the presence of an internal standard. The high natural abundance of the 31P nucleus allows for small amounts of the sample (~30 mg) and short NMR acquisition times (~30-120 min) with well-resolved 31P signals that are highly dependent on the surrounding chemical environment of the labeled OH groups.

Quantitative 31P NMR Analysis of Lignins and Tannins

31P NMR is a powerful tool for the structural elucidation of polyphenols. This fast, easy, precise, quantitative, and highly reproducible analytical procedure, that allows for the quantification and differentiation of the different types of hydroxy, phenolic, and carboxylic groups in lignins and tannins has now become a routine analytical tool.

The development of sustainable biorefinery products is confronted, among others, with the challenge of lignin and tannin valorization. These abundant, ...

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Lignin Micro-/Submicron Particle Synthesis for Bioflavonoid Release

Green Synthesis, Characterization, Encapsulation, and Measurement of the Release Potential of Novel Alkali Lignin Micro-/Submicron Particles

The applicability of biopolymer micro-/nano- technology in human, veterinary medicine, pharmaceutical, and food technology is rapidly growing due to the great potential of biopolymer-based particles as effective carrier systems. The use of lignin as a basic heteropolymer biomatrix for the design of innovative micro-/submicron formulations allows the achievement of increased biocompatibility and offers various active functional groups presenting opportunities for customization of the physicochemical properties and bioactivities of the formulations for diverse applications. The aim of the present study was to develop a simple and ecofriendly methodology for the synthesis of lignin particles with micro- and submicron size; to evaluate their physicochemical, spectral, and structural characteristics; and to examine their capacity for encapsulation of biologically active molecules and potential for in vitro release of bioflavonoids in simulated gastrointestinal media. The presented methodologies apply cheap and green solvents; easy, straightforward, quick, and sensitive processes requiring little equipment, non-toxic substances, and simple methods for their characterization, the determination of encapsulation capacity towards the poorly water-soluble bioactive compounds morin and quercetin, and the in vitro release potential of the lignin matrices.

Author Spotlight: Development and Characterization of Eco-Friendly Lignin-Based Microparticles for Enhanced Delivery of Bioflavonoids

We describe novel, simple methodologies of synthesis and characterization of biocompatible lignin micro- and submicron particles. These formulations provide a facile approach for the utilization of the heteropolymer, as well as an alternative for the rational design of multifunctional carrier matrices with potential applicability in biomedicine, pharmaceutical technology, and the food industry.

The applicability of biopolymer micro-/nano- technology in human, veterinary medicine, pharmaceutical, and food technology is rapidly growing due to the ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

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Chapters in this video

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

Extraction of Lignin with High β-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

Fractionation of Lignocellulosic Biomass using the OrganoCat Process

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Quantitative ³¹P NMR Analysis of Lignins and Tannins

Label-free in situ Imaging of Lignification in Plant Cell Walls

Green Synthesis, Characterization, Encapsulation, and Measurement of the Release Potential of Novel Alkali Lignin Micro-/Submicron Particles