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

Summary

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.

Abstract

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.

Protocol

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. 
    
    
    
    
    

Discussion

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.

Materials

List of materials used in this article

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