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

Summary

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.

Abstract

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.

Introduction

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 cellu....

Protocol

1. Experimental preparation

1. Grind dried plant material into a fine powder.
2. 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.
3. Prepare 100 mL of 70% ethanol (v/v): 70 mL of 100% ethanol and 30 mL of sterile water.
4. Prepare 100 mL of methanol: chloroform in a 1:1 ratio (50 mL methanol and 50 mL chloroform).
5. Prepare 82.5 mL of Updegraff reagent. Add 75 mL of 80% ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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