Determining the lignin content and composition of plant cell walls starts with dried plant material, which is subjected to various extractions. The resulting material consists of cell walls only and then is split up to determine the lignin content. The wall material is subjected to hydrolysis by acetyl bromide and the solubilized mono als are quantified by UV spectrometry for the determination of lignin composition.
Wall material is hydrolyzed by cytolysis. The solubilized lignin components are then deriv to their volatile trimethyl saline derivatives, which can be separated and quantified using gas chromatography in conjunction with mass spectrometry. The determination of matrix polysaccharide composition and cellulose content is demonstrated in another video entitled Comprehensive Compositional Analysis of Plant Cell Walls, part Two Carbohydrates.
Hi, I'm Marcus Pauley, head of the Wall Analytical facility at the Great Lakes Bioenergy Research Center here at Michigan State University. I'm Cliff Foster manager of the analytical cell wall facility. We'll show you the procedure on how to determine content and composition of plant biomass samples.
Plants convert light energy from the sun into chemical energy such as biomass. Plant biomass consists mainly of cell walls, a complex network of a variety of polymers that encase all plant cells. Here we determine the composition of plant cell wall materials to assess the potential for transforming these lignin Cellose X, which are a highly abundant renewable resource into biofuels.
Today we'll focus on the polyphenol lignin in another video entitled Comprehensive Compositional Analysis of Plant Cell Walls, part two carbohydrates. We'll focus on the polysaccharide analysis. To begin this procedure, add three 5.5 millimeter stainless steel balls to a two milliliter sared screw cap tube and roughly 60 to 70 milligrams of air or freeze dried plant material.
The material can be ground to a fine powder by using eyewall, a grinding and dispensing robot. The eyewall takes the tube and shakes it for 30 seconds, where due to the metal balls, the material is ground to a fine powder, then the ground, but compact plant powder is loosened an alternative non robotic low throughput procedure. Facilitating a retro is presented in part two, add 1.5 M of 70%aqueous ethanol to the ground material vortex thoroughly and centrifuge at room temperature.
This treatment solubilize most proteins and cell organelles aspirate the supernatant and add a one-to-one volume to volume. Chloroform methanol solution to the alcohol insoluble pellet shake thoroughly to respin the pellet and centrifuge. The chloroform methanol extracts the hydrophobic compounds such as lipids after centrifugation, aspirate the supernatant and resuspend the pellet and 500 microliters of acetone.
To dry the sample evaporate the solvent with a stream of air at 35 degrees Celsius until dry, the dried samples can be stored at room temperature until further processing. To initiate the removal of starch from the sample reus, suspend the pellet and 1.5 mils of 0.1 molar sodium acetate pH five cap the tubes and heat them in a heating block to gelatinize the starch. After cooling the suspension on ice, add the starch degrading enzyme mix cap the tube and vortex thoroughly incubate the suspension overnight.
In the shaker at 37 degrees Celsius, a rotation shaker ensures proper mixing. After the overnight incubation, terminate the digestion by heating in a heating block at 100 degrees Celsius for 10 minutes. Then centrifuge and discard the supernatant, which contains a solubilized starch.
Wash the remaining pellet three times using 1.5 mils of water each time by vortexing, centrifuging, and decanting as demonstrated before. Finally, resuspend the pellet in 500 microliters of acetone and evaporate the solvent with a stream of air at 35 degrees Celsius until dry. The dried samples containing isolated cell wall or leg, no celluloses are ready for further analysis and can be stored at room temperature.
Start by weighing one to 1.5 milligrams of prepared cell wall material. The material can be weighed by hand or in an automated manner with the eye wall as demonstrated in part two. Rinse the tube walls with 250 microliters of acetone to collect the cell wall material on the bottom of the tube and evaporate the acetone very gently under airflow.
After the acetone has evaporated gently add 100 microliters of freshly made 25%volume to volume acetyl bromide in glacial acetic acid along the two walls. To prevent splashing a subtle bromide breaks down the lignin polymer into its acetylated monos. Then cap the volumetric flask and heat at 50 degrees Celsius for two hours.
Heat an additional hour with four texting every 15 minutes, and finally cool on ice At room temperature after cooling the flask add 400 microliters of two molar sodium hydroxide and 70 microliters of freshly prepared 0.5 molar hydroxyl lamine hydrochloride vortex, the volumetric flasks. Next, neutralize the solution by filling up the volumetric flask exactly to the two milliliter mark with glacial acetic acid cap and invert several times to mix pipette 200 microliters of the solution containing the hydrolyzed and solubilized mono lals into a UV specific 96 well plate and read the plate in a well plate reader at 280 nanometers. Read the plate at least three times and average the absorbance since particulates can cause a slight variation in absorbance values.
Determine the percentage of a subtle bromide soluble lignin or A BSL using the appropriate plant coefficient with the formula presented in the written protocol. Lignin is a polyphenol network that consists mainly of three subunits, P hydroxy phenol, gua ACell, and spring roll units due to determine the composition of lignin in the plant material. Start by transferring approximately two milligrams of cell wall material into a screw capped glass tube for cytolysis.
Next, get ready to prepare the 2.5%boron tri fluoride ethyl ate, and 10%ethane thiol solution in dioxane. Since dioxane is very hazardous, do not take any samples or equipment out of the hood. Remember to work very carefully and use a balloon filled with nitrogen gas to displace the lost volume in the dioxane bottle with nitrogen.
Prepare the solution by mixing the following volumes per sample 175 microliters dioxane 20 microliters ETSH five microliters BF three. After mixing, add 200 microliters of the solution to each sample. Purge the vial headspace with nitrogen gas and cap.
Immediately proceed to heat the samples at 100 degrees Celsius for four hours with gentle mixing every hour end. Theo ace lysis reaction by on ice for five minutes. Then add 150 microliters of 0.4 molar sodium bicarbonate to neutralize and vortex.
For the cleanup of the lignin breakdown products, add one mil of water and 0.5 mils of acetate vortex and let the phases separate. The ethyl acetate layer will be on top and water at the bottom. Transfer 150 microliters of the top Ethel acetate layer containing the lignin components into a two mil tube, making sure no water is transferred.
Following the transfer from the top Ethel acetate layer, evaporate the solvent using a concentrator with air. Add 200 microliters of acetone and evaporate. Repeat the treatment with acetone two times to remove excess water.
Next to deriv the lignin components to their volatile trimethyl Cy Lane derivatives. Add 500 microliters of ethyl acetate, 20 microliters of purine, and 100 microliters of NO biss trimethyl cyl acetamide to each tube. Incubate for two hours at 25 degrees Celsius.
At the end of the two hours, transfer 100 microliters of the reaction into a GC MS vial and add 100 microliters of acetone. Analyze the samples by GC equipped with a quadruple mass spectrometer or flame ionization detector. For column and details of the run, please consult the accompanying written protocol.
Here are some representative results of the lignin analysis using poplar wood as the plant material. According to this analysis, 22%of the poplar material consists of the polyphenol lignin. The composition of the lignin components was analyzed by GCMS.
Peaks are identified by relative retention times using tetra coane internal standard or by characteristic mass spectrum icons of 2 99 MZ 2 69 MZ and 2 39 MZ for the sg and h monomers respectively. The composition of the lignin components is quantified by setting the total peak area to 100%The compositional analysis shows that this particular sample contains mainly string or S units. However, Goya, aol, or G units are also abundant.
Obviously, the amount and mono lial unit composition can vary depending on plant tissue and species. We have just shown you how to determine the lignin content and composition of plant material for the analysis of polysaccharides. Turn into part two.
Thanks for watching and good luck with your experiments.
