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.

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Foster, C. E., Martin, T. M., Pauly, M. Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin. J. Vis. Exp. (37), e1745, doi:10.3791/1745 (2010).


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.


1. Cell Wall Isolation

  1. Grind roughly 60-70mg of air- or freeze-dried plant material with 5.5 mm stainless steel balls in a 2 ml Sarstedt screw cap tube using an iWall, a grinding and dispensing robot (30 s). An alternative non-robotic low-throughput procedure using a ball-mill (retschmill) is presented in Part II2.
  2. Add 1.5 ml of 70% aqueous ethanol to the dispensed ground material, and vortex thoroughly.
  3. Centrifuge at 10,000 rpm for 10 min to pellet the alcohol insoluble residue.
  4. Aspirate or decant the supernatant.
  5. Add 1.5 ml of chloroform/methanol (1:1 v/v) solution to the residue and shake tube thoroughly to resuspend the pellet.
  6. Centrifuge at 10,000 rpm for 10 min and aspirate or decant the supernatant.
  7. Resuspend pellet in 500 μl of acetone.
  8. Evaporate the solvent with a stream of air at 35°C until dry.
    If needed dried samples can be stored at room-temperature until further processing.
  9. To initiate the removal of starch from the sample resuspend the pellet in 1.5 ml of a 0.1 M sodium acetate buffer pH 5.0.
  10. Cap the Sarstedt tubes and heat for 20 min. at 80°C in a heating block.
  11. Cool the suspension on ice.
  12. Add the following agents to the pellet: 35 μl of 0.01% sodium azide (NaN3), 35 μl amylase (50 μg/mL H2O; from Bacillus species, Sigma); 17 μl pullulanase (17.8 units from Bacillus acidopullulyticus; Sigma). Cap the tube and vortex thoroughly.
  13. The suspension is incubated over night at 37°C in the shaker. Orienting the tubes horizontally aides improved mixing.
  14. Heat suspension at 100°C for 10 min in a heating block to terminate digestion.
  15. Centrifuge (10,000 rpm, 10 min) and discard supernatant containing solubilized starch.
  16. Wash the remaining pellet three times by adding 1.5 ml water, vortexing, centrifuging, and decanting of the washing water.
  17. Resuspend pellet in 500 μl of acetone.
  18. Evaporate the solvent with a stream of air at 35°C until dry. It may be necessary also to break up the material in the tube with a spatula for better drying.
    The dried material presents isolated cell wall (lignocellulosics). If needed dried samples can be stored at room-temperature until further processing.

2. Lignin Content

This method is based on a reported method by Fukushima and Hatfield 3.

  1. Weigh 1 - 1.5 mg of prepared cell wall material (see 1) into 2 ml volumetric flask leaving one tube empty for a blank.
  2. rinse tube walls with 250 μl of acetone to collect the cell wall material on the bottom of the tube, and evaporate the acetone very gentle under airflow.
  3. Gently add 100 μl of freshly made acetyl bromide solution (25% v/v acetyl bromide in glacial acetic acid) along the tube walls to prevent splashing.
  4. Cap volumetric flask and heat at 50°C for 2hrs
  5. Heat for an additional hour with vortexing every 15 minutes.
  6. Cool on ice to room temperature.
  7. Add 400 μl of 2M sodium hydroxide and 70 μl of freshly prepared 0.5 M hydroxylamine hydrochloride. Vortex volumetric flasks.
  8. Fill up volumetric flask exactly to the 2.0 ml mark with glacial acetic acid, cap and invert several times to mix.
  9. Pipette 200 μl of the solution into a UV specific 96 well plate and read in an ELISA reader at 280nm.
  10. Determine the percentage of acetyl bromide soluble lignin (%ABSL) using an appropriate coefficient (Poplar = 18.21; Grasses = 17.75; Arabidopsis = 15.69) with the following formula:
    % ABSL Calc:Figure 1

    Multiplication of %ABSL with 10 results in the ug/mg cell wall unit

    It helps to do at least 3 plate reads to average the absorbance (abs) since particulates can cause a slight variation in absorbance values. Note: 0.539 cm represents the pathlength, but depending on the plate this might need to be determined.

3. Lignin Composition

This method is adopted from a recent method published by Robinson and Mansfield5.

  1. Transfer approximately 2 mg of cell wall material (see 1.) into a screw capped glass tube for thioacidolysis.
  2. carefully prepare the 2.5% boron trifluoride diethyl etherate (BF3), 10% ethanethiol (EtSH) solution. You must use a balloon filled with nitrogen gas to displace the lost volume in the dioxane bottle with nitrogen. Dioxane is very hazardous, do not take samples or equipment out of the hood. Volumes needed for the preparation of the solution per sample: 175 μl dioxane; 20 μl EtSH; 5 μl BF3.
  3. Add 200 μl of EtSH, BF3, dioxane solution to each sample.
  4. Purge vial headspace with nitrogen gas and cap immediately.
  5. Heat at 100°C for 4 hours with gentle mixing every hour.
  6. End reaction by cooling on ice for 5 minutes.
  7. Add 150 μl of 0.4M sodium bicarbonate, vortex
  8. For the clean-up add 1 ml of water and 0.5 ml of ethyl acetate, vortex and let phases separate (ethyl acetate on top, water on bottom).
  9. Transfer 150 μl of the ethyl acetate layer into a 2 ml Sarstedt tube. Make sure no water is transferred.
  10. Evaporate solvent by a concentrator with air.
  11. Add 200 μl acetone and evaporate (repeat for a total of two times remove excess water).
  12. For the TMS derivatization add 500 μl of ethyl acetate, 20 μl of pyridine, and 100 μl of N,O-bis(trimethylsilyl) acetamide to each tube.
  13. incubate for 2 hours at 25°C.
  14. Transfer 100 μl of the reaction into a GC/MS vial and add 100 μl of acetone.
  15. Analyze the samples by GC equipped with a quadrupole mass-spectrometer or flame ionization detector. An Agilent HP-5MS column is installed (30 mm X 0.25 mm X 0.25 μm film thickness). The following temperature gradient is used with a 30 min solvent delay and a 1.1 ml/ min flow rate: Initial hold at 130 °C for 3 min; a 3 °C/ min ramp to a 250 °C and hold for 1 min; allow equilibration to the initial temperature of 130 °C.
  16. Peaks are identified by relative retention times using tetracosane internal standard (optional) or by characteristic mass spectrum ions of 299 m/z, 269 m/z, and 239 m/z for S, G, and H monomers, respectively (see Fig. 2). The composition of the lignin components is quantified by setting the total peak area to 100%

4. Representative Results

An example of a wall analysis is presented in Figure 2. In this case poplar stem (wood) was analyzed by the various procedures outlined in the protocol section. An example chromatogram of the separation of lignin-components after thioacidolysis and TMS-derivatization is shown. Clearly, the relative abundance of syringyl- (S), guaiacyl- (G), and p-hydroxyphenol- (H) units can be determined. The content of acetyl bromide soluble lignin is self-explanatory, one can expect values of between 20-50% of the wall dry weight. One should note that acetyl bromide does not solubilize all of the lignin present in the wall, and that the degree of solubilization can vary depending on the material. However, this method is relatively easy to carry out and rapid and gives an excellent approximation of the lignin content in a lignocellulosic material.

Figure 1
Figure 1: Overview of lignocellulosic analysis. Cell walls (lignocellulosics) are isolated from crude dried plant material. The wall material is then weighted into aliquots and subdivided for the various assays. Wall material is treated with acetyl bromide and the solubilized lignin quantified by UV-spectroscopy. For the determination of the lignin composition, wall material is subjected to thioacidolysis. The solubilized phenolics undergo TMS derivatization and can then be separated and quantified by GC-MS analysis. The matrix polysaccharide composition and crystalline cellulose content protocol is discussed in Part II2.

Figure 2
Figure 2: Comprehensive lignocellulosic analysis of poplar wood. Wood chips from poplar (Populus tremoloides) were subjected to the described protocols.
Ligin composition; H p-hydroxyphenyl ;G guaiacyl; S syringyl units.


The described methods enable a rapid quantitative assessment of the lignin content and composition of lignocellulosic plant biomass. Using the iWall robot approximately 350 samples can be ground and dispensed per day. The throughput of the various analytical methods per person varies. Using the protocols described here, 30 samples can be processed for lignin content, and 15 for lignin composition per day. Due to the quantitative nature of the data optimal feedstock crops, variety or genotypes can be assessed in terms of their suitability for biofuel production.


We are grateful to Matthew Robert Weatherhead for excellent technical service and John Ralph, University of Wisconsin for valuable advice, discussions, and the poplar wood sample. This work was funded by the US Department of Energy (DOE) Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494) and by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (award no. DE-FG02-91ER20021).


Name Company Catalog Number Comments
Hydroxylamine Hydrochloride Sigma-Aldrich 255580
Acetyl Bromide Aldrich 135968
Ethanethiol Sigma-Aldrich E3708
Borontrifluoride diethyl etherate Fluka 15719
N,O,-Bis(trimethylsilyl) acetimide Fluka 15241
Dioxane Sigma-Aldrich 296309
Spectromax Plus 384 Molecular Devices Plus384
GC-MS Agilent Technologies 6890 GC/5975B MSD (lignin composition)
5.5mm Stainless Steel Balls Salem Ball Company (N/A)
96 well plate heat spreader Biocision Coolsink 96F
Heating block Techne Dri-block DB-3D
Sample concentrator Techne FSC400D



  1. Carroll, A., Somerville, C. Cellulosic Biofuels. Annual Review of Plant Biology. 60, 165-165 (2009).
  2. Foster, C. E., Martin, T., Pauly, M. Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part II: Carbohydrates. J Vis Exp. (2010).
  3. Fukushima, R. S., Hatfield, R. D. Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method. J. Agric. Food Chem. 49, (7), 3133-3133 (2001).
  4. Pauly, M., Keegstra, K. Cell-wall carbohydrates and their modification as a resource for biofuels. Plant J. 54, (4), 559-559 (2008).
  5. Robinson, A. R., Mansfield, S. D. Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling. Plant J. 58, (4), 706-706 (2009).
  6. Somerville, C. Toward a systems approach to understanding plant-cell walls. Science. 306, (5705), 2206-2206 (2004).
  7. Teeri, T. T., Brumer, H. Discovery, characterization and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endo-transglycosylases. Biocatalysis and Biotransformation. 21, 173-173 (2003).



  1. Erratum: Please note that acetyl-bromide (Lignin content; 3) under the conditions described solubilizes lignin as a polymer with very little degradation to monolignols. This is contrary to what is said in the video.

    Posted by: Anonymous
    March 13, 2010 - 8:06 PM
  2. Congratulations by the clear description of a high troughput method. It will be very usefull to our work in lignocellulose characterization in sugarcane. I still have some doubts about how good is the method compared to pyrolysis-GC-MS method we are running, were we can quantitate about ²0 different molignols. We have purified lignin sample that we previously analyse the pyr-GC-MS. If you are interested, I will be glad to provide you

    Posted by: Marcelo Ehlers L.
    July 21, 2010 - 7:59 PM
  3. Hello,
    I work in University of Poitiers (France). Thank you for your good work. I would like solubilise the Lignin for analyse it with MALDI-TOF. I have a question. If i use Acetyl-Bromide, i will break the molecule of Lignin or no ? Thanks for your answer.
    Best regards.

    Posted by: Anonymous
    January 23, 2012 - 5:48 AM
  4. Hello, thanks for the comments. The acetyl bromide soluble lignin assay is said to solubilize the lignin rather than break it down. There is a paper from Hatfield and Fukushima (²68/1/IND43704044.pdf) that briefly discuss the structure of the lignin after the acetyl bromide treatment. Best of luck!


    Posted by: Cliff F.
    January 23, 2012 - 10:42 AM
  5. Hello;
    I'm a master student working at the university of Copenhagen. I've a question about the cell wall isolation in the part one of the protocol: I tried to grind my samples ( barley straw ) following literally your protocol ( so straw in plastic vials, grinding balls inside ecc). I didn't used any robot just a grinding machine. At the end I obtained some samples in a very fine podwer but in others the powder was just bulked on the bottom of the vial in a quite hard layer. I broke it up for the lignin quantification. I was wondering if that can bring some trouble during the analysis. I really don't know how that's happen.

    Posted by: gianluca b.
    January 24, 2014 - 7:04 AM
  6. Hi. Just to clarify, did this happen before or after the cell wall extractions? When you dry the biomass at the end of the starch removal process, you might get a recalcitrant pellet but that depends on the tissue type. We mainly see this occur with leaf tissue that is low in quantity (maybe around 10 mg or less) so to avoid this, we make sure to start with 60-70 mg of dry tissue.

    Posted by: Cliff F.
    January 24, 2014 - 10:20 AM
  7. For the Lignin monomers (S,G, and H), what chemicals would I use for making standards?? I've ran a few of my TMS derivitized samples through GC/MS and am not getting any peaks. Before I determine if I need to re-do the experiment I would like to run some standards at different concentrations. Please let me know!


    Posted by: Emily G.
    September 3, 2014 - 1:04 PM
  8. Hi Emily. The lignin monomer standards are not commercially available so they need to be synthesized if you want to use them. John Ralph had a recent publication outlining the synthesis (J. Agric. Food Chem., 2012, 60 (4), pp 922–928) and use of the thioacidolysis monomers. They included internal standard response factors for GC-MS and GC-FID so if synthesis is out of the question, you could make due with their measurements.

    One important note is that since the publication, we have found that the biomass to thioacidolysis reaction mixture volume can impact the monomer yield. Now that we have the synthetic standards, we can track this phenomenon a lot better and have made a small adjustment to the initial reaction volume. We now use 1ml of reaction mixture with 2mg of biomass. This biomass to reaction mixture volume seems to maximize the monomer yields and we don't see the blackening of the biomass anymore (which sometimes would happen). Hopefully this will help! If you want to discuss the method in any more detail feel free to send me an email (

    Posted by: Cliff F.
    September 4, 2014 - 3:49 PM
  9. Hi there,
    Thank you very much for posting the video.
    The pullulanase from from Bacillus acidopullulyticus from sigma appeared to be discontinued, and there are several option for the amylase. Could you recomment an specific enzyme (preferably with a CAS number?).

    Another question, do you think that the particle size of the grinded material affect the results?

    Again thank you. The video is really helpful.


    Posted by: Yohanna C.
    June 16, 2015 - 4:17 AM

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