Name: estimation of crystalline cellulose content of plant biomass using the updegraff method
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Description: Watch this Scientific Journal Video about Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method at JoVE.com

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

1. Experimental preparation
<ol>
	<li>Grind dried plant material into a fine powder.</li>
	<li>Protein Solubilization Buffer (PSB): Prepare stock solutions of 1 M Tris (pH 8.8), 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 8.0) and autoclave them. Make fresh PSB buffer from these stock solutions with final concentrations of 50 mM Tris, 0.5 mM EDTA and 10% sodium dodecyl sulfate (SDS) in sterile water.</li>
	<li>Prepare 100 mL of 70% ethanol (v/v): 70 mL of 100% ethanol and 30 mL of sterile water.</li>
	<li>Prepare ...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semimicro+determination+of+cellulose+inbiological+materials.">Semimicro determination of cellulose inbiological materials.</a> Analytical Biochemistry. 32, 420-424 (1969).</li><li>Viles, F. J., Silverman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+starch+and+cellulose+with+anthrone.">Determination of starch and cellulose with anthrone.</a> Analytical Chemistry. 21, 950-953 (1949).</li><li>Scott, T. A., Melvin, E. 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Cotton fibers are natural fibers produced from the cottonseed. Cotton fiber is a single cell with ~95% cellulose content2 with high crystalline cellulose content with extensive applications in textile industry31. As, cotton fiber contains ~95% cellulose, we have used cotton root tissues for demonstration of the estimation of crystalline cellulose content. Cotton root tissues are moderately rich in crystalline cellulose content and represents a commonly available plant bioma...

Cellulose is the primary load-bearing component of cell walls, which is present in both primary and secondary cell walls. The cell wall is an extracellular matrix that surrounds plant cells and is primarily composed of cellulose, lignin, hemicellulose, pectin, and matrix proteins. Approximately one third of plants biomass is cellulose1 and it plays significant roles in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. Cotton fiber contains 95% cellulose2 content, while trees contain 40% to 50% of cellulose depending on the plant species and organ types3. The cellulose is composed of repeating units of cellobiose, a disaccharide of glucose residues connected by &#946;-1,4 glycosidic bonds4. Cellulosic ethanol is produced from the glucose derived from the cellulose present in the plant cell walls5. Cellulosic fiber is made up of several micro fibrils in which each micro fibril acts as core unit with 500-15000 glucose monomers1,6. The cellulose homopolymer is synthesized by plasma membrane embedded cellulose synthase complexes (CSC&#39;s)1,7. Individual cellulose synthase A (CESA) proteins synthesize glucan chains and the adjacent glucan chains are connected by hydrogen bonds to form crystalline cellulose1,8. Cellulose exists in several crystalline forms with two predominant forms, cellulose I&#945; and cellulose I&#946; as native forms9. In higher plants, cellulose exists in cellulose I&#946; form while lower plant cellulose exists in I&#945; form10,11. Overall, the cellulose plays a significant role in imparting strength and rigidity to the plant cell walls.
First generation biofuels are primarily produced from corn starch, cane sugars, and beet sugars, which are food sources, while second-generation biofuels are focusing on the biofuel production from non-food plant biomass cell wall material12. Accurate estimation of crystalline cellulose content is not only important for fundamental research on cellulose biosynthesis and cell wall dynamics but also for applied biofuel and bio products research. Various methods have been developed and optimized for estimation of cellulose in the plant biomass, and the Updegraff method is the most widely used method for cellulose estimation. The first reported method for cellulose estimation was by Cross and Bevan in 190813. The method was based on the principle of alternate chlorination and extraction by sodium sulphate. However, the cellulose obtained by the original as well as modified protocols of Cross and Bevan method showed contamination of small fractions of lignin in addition to a substantial amount of xylans and mannans14. Despite several modifications to remove lignin and hemicelluloses from the cellulose fraction, the Cross-Bevan method retained a considerable amount of mannans along with cellulose. Later, Kurschner&#39;s method was developed by employing nitric acid and ethanol to extract cellulose15. This method stated that total lignin and 75% of pentosans were removed but the true cellulose results were the same as those estimated by chlorination method of Cross and Bevan. Another method (Norman and Jenkins) was developed by employing methanol-benzene, sodium sulphate, and sodium hypochlorite to extract cellulose16. This method also retained some fraction of lignin (3%) and significant amounts of pentosans leading to in accurate estimation of cellulose. Later, Kiesel and Semiganowsky used a different approach to hydrolyze cellulose using 80% concentrated sulfuric acid, and the hydrolyzed reduced sugars were estimated by Bertrand&#39;s method17. The two methods, Waksman&#39;s and Stevens18 and Salo14,19 which were developed based on Kiesel and Semiganowsky&#39;s method, also yielded 4-5% less cellulose content compared to earlier methods20.
The Updegraff method is the most widely used method for the estimation of crystalline cellulose content. This method was first described by Updegraff for the measurement of cellulose in 196921. The Updegraff method integrates the Kurschner method (use of nitric acid), Kiesel and Seminowsky methods (hydrolysis of cellulose into glucose monomers using sulfuric acid) with some modifications, and the anthrone assay of Viles and Silverman for simple colorimetric estimation of glucose and crystalline cellulose content22. The principle of this method is the use of acetic acid and nitric acid (Updegraff reagent) to eliminate hemicellulose and lignin from the homogenized plant tissues, which leaves acetic/nitric acid resistant cellulose for further processing and estimation15. The acetic/nitric acid resistant cellulose is treated with 67% sulfuric acid to break the cellulose into glucose monomers and the released glucose monomers are estimated by anthrone assay21,23. Several modifications of the original Updegraff method were used to simplify the procedure and cellulose estimation by anthrone assay24. Broadly, this method can be divided into five phases. In the first phase, the plant material is prepared. In the second phase, the crude cell wall is separated from the total biomass, as cellulose is the key component of plant cell walls. Later, in the third phase, the cellulose is separated from the non-cellulosic cell wall components by treating with Updegraff reagent. In the fourth phase, the acetic/nitric acid resistant cellulose is broken into glucose monomers by sulfuric acid treatment. Sulfuric acid treatment of cellulose results in formation of 5-hydroxymethylfurfural compounds from the reaction of glucose monomers with sulfuric acid. Finally, in the last phase, the anthrone generates a greenish blue complex by boiling with the furfural compound generated in the previous phase25. This anthrone based colorimetric method was first used in 1942 by Dreywood. Anthrone is a dye that identifies furfural compounds of pentose and hexose dehydrated products such as 5-hydroxymethylfurfural, under acidic conditions. Reaction with hexose produces an intense color and better response compared to pentoses25. The amount of bound glucose is measured by spectrophotometer absorbance at 620 nm and the intensity of the greenish blue complex is directly proportional to the amount of sugar in the sample. The measured absorbance values were compared with a glucose standard curve regression line to calculate the glucose concentration of the sample. The measured glucose content was used to estimate the cellulose content of the plant biomass.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Fisher Chemical</td>
			<td>A18-500</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Anthrone</td>
			<td>Sigma Aldrich</td>
			<td>90-44-8</td>
			<td>For colorimetric assay</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td>For centrifugation</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Mallinckrodt</td>
			<td>67-66-3</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>6381-92-6</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Millipore Sigma</td>
			<td>EM-EX0276-4S</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1004-090</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma</td>
			<td>SKU A6283</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Heat block/ ThermoMixer F1.5</td>
			<td>Eppendorf</td>
			<td>13527550</td>
			<td>For controlled temperatures</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Fisherbrand</td>
			<td>150152633</td>
			<td>Used for drying plant sample</td>
		</tr>
		<tr>
			<td>Measuring Scale</td>
			<td>Mettler Toledo</td>
			<td>30243386</td>
			<td>For specific quantities</td>
		</tr>
		<tr>
			<td>Methanol 100 %</td>
			<td>Fisher Chemical</td>
			<td>A412-500</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Microplate (96 well)</td>
			<td>Evergreen Scientific</td>
			<td>222-8030-01F</td>
			<td>For anthrone assay</td>
		</tr>
		<tr>
			<td>Nitric acid</td>
			<td>Sigma Aldrich</td>
			<td>695041</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Polypropylene Microvials (2 mL) / screw capped tubes</td>
			<td>BioSpec Products</td>
			<td>10831</td>
			<td>For high temperatures</td>
		</tr>
		<tr>
			<td>Spectrophotometer(Multimode Detector)</td>
			<td>Beckmancoulter DTX880</td>
			<td>1000814</td>
			<td>For measuring absorbances</td>
		</tr>
		<tr>
			<td>Spex SamplePrep 6870 Freezer / Mill</td>
			<td>Spex Sample Prep</td>
			<td>68-701-15</td>
			<td>For grinding plant tissues into fine powder</td>
		</tr>
		<tr>
			<td>Sulphuric acid</td>
			<td>J.T.Baker</td>
			<td>02-004-382</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma Aldrich</td>
			<td>151-21-3</td>
			<td>Used in the PSB buffer</td>
		</tr>
		<tr>
			<td>Tubes (2 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-408-138</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Tris Hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>&#160;1185-53-1</td>
			<td>Used in the PSB buffer</td>
		</tr>
		<tr>
			<td>Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Vacuum dryer (vacufuge plus)</td>
			<td>Eppendorf</td>
			<td>22820001</td>
			<td>For drying samples</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisherbrand</td>
			<td>14-955-151</td>
			<td>For mixing</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Thermoscientific</td>
			<td>TSGP02PM05</td>
			<td>For temperature controlled conditions at specific steps</td>
		</tr>
		<tr>
			<td>Weighing Paper</td>
			<td>Fisher Scientific</td>
			<td>09-898-12A</td>
			<td>Used in the protocol</td>
		</tr>
	</tbody>
</table>

estimation of crystalline cellulose content of plant biomass using the updegraff method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

Cotton plants grown in the green house were selected for this study. Two different experimental lines of cotton were selected for comparative analysis of cellulose content. For each experimental line, the root tissue was collected from three biological replicates. A total of 500 mg of tissue was homogenized and 20 mg of it was used for crude cell wall extraction. Later, 5 mg of crude cell wall extract was used for Updegraff reagent treatment to remove hemicellulose and lignin from cellulose. The purified cellulose was hy...

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Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...

Here, we demonstrate Updegraff method, the world's most widely used method for the estimation of crystalline cellulose content. Cellulose is basically made up of glucose monomers linked by beta-1, 4 glycosidic bonds. We divided this protocol into five phases.In the first phase, we prepare the plant biomass material. In the second phase, we extract the crude cell wall. In the third phase, we use Updegraff treatment in which Updegraff is made up of acetic acid and nitric acid that helps in the removal of hemicellulose and lignin from our samples.In the fourth phase, we subject it to acid hydrolysis with the help of sulfuric acid that produces the glucose monomers. Finally, in the fifth phase, we use anthrone assay to estimate the crystalline cellulose content. Collect the plant material from the greenhouse, wash them thoroughly with water, air dry for two days, separate the tissue and transfer the tissue to the individually labeled containers and incubate in the incubator at 49 degrees Centigrade for 10 days.Then finally, cut into pieces using scissors or blade. Freeze the tissue and load into mortar and pestle for fine grinding into uniform powder or alternatively use Freezer/Mill. Here, we demonstrate using Freezer/Mill, so we load our tissue samples into these grinding vials and place them in the Freezer/Mill and run the 10 CP speed for three cycles.The tissue powder is collected in the Falcon tube as shown here. Weigh 20 mg of the tissue powder and transfer into a pre-weighed two mL tube. Add one mL of protein solubilization buffer to remove proteins.Vortex.Centrifuge at 15, 000 RPM for five minutes. Discard the supernatant. Repeat this step two more times.Add one mL of sterile water to the retained pellet.Vortex. Centrifuge at 15, 000 RPM for five minutes. Repeat this step two more times.Add one ML of 70%ethanol to the retained pellet.Vortex. Transfer to a preheated 70 degrees Centigrade block with simultaneous shaking for one hour. After one-hour incubation, centrifuge at 15, 000 RPM for five minutes.Repeat this step one more time. To the saved pellet, add one mL of 100%methylone.Vortex. Centrifuge at 15, 000 RPM for five minutes.Add one mL of chloroform methanol.Vortex. Centrifuge at 15, 000 RPM for five minutes. Discard the supernatant and add one mL of acetone.Vortex.Incubate at room temperature for five minutes. Centrifuge at 15, 000 RPM for five minutes. Re-discard the supernatant and air dry them at 37 degrees Centigrade overnight or continue by vacuum drying.Load your samples into the vacuum dryer and air dry for two to three hours or until the pellet is completely dry. Weigh five mg of the cell wall extract obtained from the previous step. Transfer into a two mL screw cap tube.Also include positive control at this point. Use two mg of Whatman filter paper for that purpose. Transfer to a two mL screw cap tube.Add 1.5 mL of Updegraff reagent to each weighed cell wall extract. Vortex and incubate at 100 degrees Centigrade in a preheated block for 30 minutes. After 30 minutes incubation, transfer to a rack.Allow it to cool on bench for 10 minutes. Centrifuge at 15, 000 RPM for 10 minutes. Discard the supernatant without disturbing the pellet.Add one mL of sterile water.Vortex. Centrifuge at 15, 000 RPM for 10 minutes. Discard 500 microliters of the supernatant.Add one mL of acetone.Vortex. Centrifuge at 15, 000 RPM for five minutes. Discard one mL of the supernatant.Add one mL of acetone.Vortex. Centrifuge at 15, 000 RPM for five minutes. Discard the supernatant completely.Add one mL of acetone.Vortex. Centrifuge at 15, 000 RPM for five minutes. Discard the supernatant.Dry at 37 degrees Centigrade overnight. After a one-night incubation, transfer to a rack. Add one mL of 67%sulfuric acid.Vortex.Incubate at 25 degrees Centigrade for one hour with simultaneous shaking. After one-hour incubation, make sure the pellet is completely dissolved. Transfer to a rack.This is the final step of the cellulose extraction. Now the samples are ready for sample dilution. For sample dilution, use 10 microliters of each sample from the previous step and add 490 microliters of sterile water.Repeat this for all the experimental samples. For the glucose standard curve preparation, prepare a fresh stock of one mg per mL glucose and prepare zero micrograms to 200 micrograms concentrations by adding one mg per mL glucose in 20 microliter increments and make up the final volume to one mL with sterile water. Take 500 microliter of each prepared standard in a screw cap tube and add one mL 0.2%anthrone.And now the samples are ready for anthrone assay. Add one mL of freshly prepared anthrone. Mix immediately by vortexing and transfer to ice.Repeat for all the standards and samples in the same way. Then transfer to a preheated 100 degrees Centigrade block for 10 minutes. After 10 minutes incubation, transfer to ice and incubate for five minutes on ice.After incubation, take 200 microliters of each sample in three replicates. Repeat for both standards and samples in the same way. And a loaded 96-well plate was loaded into a Multimode Detector.Using Smart Max Pro program, we set the filter setting to 620 nanometers. Plate type to 96-well type. Select all the read area.Click OK.Read. Read plate. You obtain the absorbance readings of both standards and samples, which will be collected in the Excel sheet for further analysis and estimation of cellulose content.Glucose standard curve is prepared by using the glucose concentrations in micrograms per mL on x-axis and the normalized absorbance values of the respective concentration at 620 nanometers on the y-axis. We get the regression line with m and c values in the plotted scatter graph we use to estimate the sample crystalline cellulose content. In the first step, we averaged the OD values at 620 nanometers.And then we normalized the data by subtracting the blank value. We calculate the glucose concentration using the formula x is equal to OD minus c/m where we plug in c and m values from the previous step of the regression line in the standard curve. Since the total volume that was used for anthrone assay is 500 microliters, we divide it by dilution factor two.Since the sample volume that was used was only 10 microliters, we'll further divide by 10 to get micrograms per microliter concentration of glucose which is equal to mg per mL concentration. And then we used the moisture factor 1.11 which is the moisture gain in the process. And finally, we calculate the percent crystalline cellulose content by dividing it with the cell wall weight, which is close to five mg, and multiply by 100 to get the percent.Then we averaged the crystalline cellulose content of three biological replicates to get the value for individual samples, which will be compared against another experimental line average of three biological replicates. And t-test can be used to test their level of significance. Here in table A, we show the table values of the concentration and the respective OD at 620 nanometers, which was further used to plot a scatter graph and a regression line with the m and c values that were used in the C for obtaining the cellulose content differences between the two experimental lines.In conclusion, both samples show cellulose differences. Thanks for watching.

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