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...

Watch this Scientific Journal Video about Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method at JoVE.com

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 ...

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