Determining the matrix polysaccharide composition and crystalline cellulose content 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 matrix polysaccharide composition. The wall material is treated with a weak acid to hydrolyze, non crystalline polysaccharides, which are mainly the hemo celluloses into their monosaccharides.
The monosaccharides are then deriv into their corresponding volatile aldol acetates, which are then analyzed and quantified by gas chromatography coupled to a mass spectrometer for the determination of crystalline cellulose. Content walls are treated with the graph reagent, a mixture of acids that strip away non crystalline wall components. The remaining crystalline cellulose is degraded into its monomer glucose, which can be assayed, chole metrically.
The determination of lignin content and composition is demonstrated in another J video publication entitled Comprehensive Compositional Analysis of Plant Cell Walls, part One Lignin. 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 Cell Wall Analytical facility.
Today we'll show you how to determine the composition of the polysaccharides present in the plant cell wall material that is cellulose in various Homo celluloses. Here at the Great Lakes Bioenergy Research Center, we develop technology to convert these sugars into biofuels. In order to identify an optimal plant feedstock, we first need to assess the kind and abundance of sugars that is present in this biomass.
In another video entitled Comprehensive Compositional Analysis of Plant Cell Walls, part one Lignin, we'll focus on the polyphenol analysis. So let's get started. Begin this procedure by isolating cell walls.
Grind roughly 60 to 70 milligrams of dried plant material with 5.5 millimeter stainless steel balls in a two mil SAR TED screw cap tube. Using a REM mill as a high throughput alternative, use a grinding and dispensing robot termed eyewall as described in part one. When grinding is complete, remove the steel balls.
Continue to isolate cell walls and remove residual starch as shown in part one and described in the accompanying written protocol. Weigh out two milligrams of DS starch cell wall material into a two mill sared tube. A high throughput way of performing this task is the eyewall robot.
The robot takes a tube containing ground plant material and pokes a hole in the bottom of the tube. The pierced tube is then placed on a VI feeder. An empty tube is placed on a balance and shifted under the VI feeder through vibration material percolates through the hole in the loaded top tube into the empty bottom tube until the desired amount of material is accumulated.
Add 100 micrograms of acetol as an internal standard. Rinse the tube walls with acetone to collect the cell wall material on the bottom of the tube and evaporate the acetone very gently under airflow. Next, perform weak acid hydrolysis.
Carefully add 250 microliters of two molar trichloroacetic acid or TFA to each sample, making sure no material is splashed up onto the tube walls. Cap the tubes tightly and incubate at 121 degrees Celsius in a heating block for 90 minutes. Then cool the heating blocks and samples on ice and centrifuge the tubes at 10, 000 RPM for 10 minutes.
Transfer 100 microliters of the acidic supernatant containing the solubilized sugars of the matrix polysaccharide to screw cap vials, making sure not to disturb the pellet material. The pellet can be used for the crystalline cellulose assay. Evaporate the TFA in a glass tube under a gentle stream of air in an evaporation device.
Add 300 microliters of two propanol to remove excess acid vortex and evaporate at 25 degrees Celsius. Repeat for a total of three times. In order to enable identification of the monosaccharides by gas chromatography, perform an aldol acetate derivitization.
First, reduce the monosaccharides to their corresponding open ring aldols, which are then acetylated. Consult the accompanying written protocol for detailed instructions. Each step of the procedure follows the previously shown protocol, addition of reagent incubation evaporation under a stream of air, followed by removal of excess reagent by various washing steps.
In the final steps, the aldol acetates are extracted. First, add 500 microliters of ethyl acetate and swirl lightly. Then add two mils of water, cap the tubes and vortex, centrifuge the tubes at 2000 RPM for five minutes to obtain clear separate layers with ethyl acetate on top containing the Al Dettol acetates and water on bottom pipette 50 microliters of ethyl acetate layer into GCMS vials with inserts dilute by adding 100 microliters of acetone to the GC vial and the GC vial can be stored at four degrees Celsius.
If the GCMS analysis does not immediately proceed, inject the samples into a gas chroma photographer that is equipped with a quadruple mass spectrometer. For column and details of the run, please consult the accompanying written protocol. The starting material for determining the crystalline cellulose content can be isolated cell wall material or wall material already treated with the weak acid TFA, either immediately after acid treatment or in storable dried form.
After removing excess TFA with iso propanol as shown previously, add to the TFA pellet in the two M sars to tube one M of up degra reagent made up of acetic acid, nitric acid and water, add an eight to one to two volume to volume ratio. Cap the tube tightly vortex and heat on a heating block at 100 degrees Celsius for 30 minutes. As a result of this treatment, only crystalline cellulose remains in the pellet.
All other polysaccharides are hydrolyzed cool samples in the block on ice to room temperature or cooler. Then centrifuge the samples at 10, 000 RPM for 15 minutes after centrifugation, discard the supernatant ensuring that the pellet is not disturbed and no material from the pellet is removed. For this purpose, leave approximately 150 microliters of the supernatant in the tube, wash the pellet several times with water.
Next, perform a same and hydrolysis with the concentrated sulfuric acid in order to completely hydrolyze the crystal and cellulose pellet into glucose. Start by adding 175 microliters of 72%sulfuric acid to the sarta tube and incubate for 30 minutes at room temperature. After the initial incubation, vortex the sample and incubate for another 15 minutes.
Then add 825 microliters of water and vortex and centrifuge the samples at 10, 000 RPM for five minutes. There might be some brown and soluble material, mostly lignin remaining in the tube while the glucose is in the supernatant. The glucose content of the supernatant is assay using the Colormetric anthone assay.
This assay is performed in a 96 well polystyrene microtiter plate. Repair a glucose standard curve in duplicate following the instructions in the accompanying written protocol. Prepare the samples by combining 10 microliters of each sample, supernat and 90 microliters of water into separate wells of the same microtiter plate as the standard finish by adding 200 microliters of freshly prepared and thrown reagent.
Next, heat the plate for 30 minutes at 80 degrees Celsius in an oven with an aluminum heat spreader. Observe that the glucose containing samples turn from yellow to blue green when heating is complete. Let the plate cool to room temperature and shake thoroughly.
Read the absorption at 625 nanometers using a microtiter plate reader. The content of glucose, the building block of cellulose is calculated based on the absorbance compared to the standard curve established on the same plate. Here are some representative results of the polysaccharide analysis of a piece of poplar wood.
The gas chromatogram peaks are identified by mass profiles and or retention times of standards. The monosaccharides are quantified based on standard curves. The sugar analysis of the matrix polysaccharides shows a high abundance of xylose representing hemi cellulose xam, but other sugars are also present.
According to the analysis, 40%of the poplar material consists of crystal and cellulose. Please note that these contents and compositions can vary from plant to plant or between different plant anatomies. We have just shown you how to determine the polysaccharide content and compositional analysis of lignano cellulosic biomass for the analysis of polyphenol lignin.
Tune into part one. Thanks for watching and good luck with your experiments.
