ASTM E3417-24

• Abstract:

  SIGNIFICANCE AND USE; 4.1 The procedure, in brief, consists of solubilization of biomass using cold caustic extraction, an enzymatic removal of starch and yeast glucan followed by precipitation of hemicellulosic material, and acid hydrolysis of the residual carbohydrate pellet before measurement of glucose and galactose derived from cellulose and hemicellulose. ; 4.2 The starch removal procedure is identical to that employed in the NREL assay2 which was itself adapted from the Megazyme-published starch analysis procedure: RTS-NaOH, 2019.5,6 ; 4.3 The cellulosic pellet hydrolysis and monosaccharide measurements are identical to those described in the NREL laboratory analytical procedure “Determination of Structural Carbohydrates and Lignin in Biomass,” NREL/TP-5100-42618 and “Determination of Cellulosic Glucan Content in Starch Containing Feedstocks,” NREL/TP-2800-76724.7 ; 4.4 This test method references Practices E1757 and E3181. ; 4.5 This test method is intended for use in the measurement of the fermentable portion of cellulosic content in pre- and post-fermentation corn biomass specifically for the apportionment of a percentage of the total ethanol produced in a fermentation as cellulosic.; SCOPE; 1.1 This procedure can be used to quantify the cellulose/hemicellulose-derived glucan and galactan (CHDGG) content in corn biomass samples that also contain varying levels of starch-derived and yeast-derived glucan. The method has been shown to provide accurate values for samples with cellulose content up to 40 % w/w.3 ; 1.2 Units—The values stated in SI units are to be regarded as the standard. No other units of 34 measurement are included in this standard. ; 1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. ; 1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

• Status: Published
• Publication Date: 31-Dec-2023
• Technical Committee: E48 - Bioenergy and Industrial Chemicals from Biomass
• Drafting Committee: E48.05 - Biomass Conversion

Overview

ASTM E3417-24 is an internationally recognized standard test method that provides a validated procedure for the determination of cellulose/hemicellulose-derived glucan and galactan content (CHDGG) in solid corn biomass samples. Developed by ASTM Committee E48 on Bioenergy and Industrial Chemicals from Biomass, this method is essential for accurately quantifying the fermentable cellulosic fraction of corn biomass, particularly in samples containing starch-derived and yeast-derived glucan. This standard addresses the critical analytical need for measuring the cellulosic content in corn biomass, a key factor in apportioning the percentage of ethanol produced from cellulosic sources in bioethanol production processes.

Key Topics

• Sample Preparation and Homogenization: Samples are prepared following ASTM Practice E1757 to ensure representativity and reliability.
• Removal of Non-Cellulosic Glucan: The method uses cold caustic extraction and enzymatic steps to remove starch and yeast-derived glucan, preventing overestimation of cellulosic content.
• Precipitation and Filtration: Hemicellulosic material is selectively precipitated and filtered, isolating the cellulosic pellet for further analysis.
• Acid Hydrolysis: The residual carbohydrate pellet, believed to contain mainly cellulose and hemicellulose, undergoes acid hydrolysis to release monosaccharides (glucose and galactose).
• Quantification by HPLC: Monosaccharide content is accurately measured using high-performance liquid chromatography, with calibration and verification standards to ensure data integrity.
• Correction and Calculation: Recovery standards are applied to correct for sugar loss during hydrolysis, and calculations provide cellulose and hemicellulose-derived sugar contents in the sample.

Applications

• Bioethanol Production Apportionment: The method allows for precise determination of the fermentable cellulosic portion in pre- and post-fermentation corn biomass. This is crucial in assigning the percentage of total ethanol produced as cellulosic, supporting compliance with regulatory frameworks such as the US Renewable Fuel Standard (RFS).
• Feedstock Characterization: Useful in research, pilot plants, or commercial biofuel facilities for characterizing solid corn biomass feedstocks and optimizing conversion processes.
• Quality Control and Facility Monitoring: The standard provides robust analytical support for quality assurance, process control, and documenting compliance in biofuel plants.
• Technical Reporting and Regulatory Submittals: Results obtained using ASTM E3417-24 can be included in environmental, sustainability, and regulatory reporting, supporting transparency and traceability in the biofuels value chain.

Related Standards

• ASTM E1757 - Practice for Preparation of Biomass for Compositional Analysis: Reference method for preparing representative biomass samples.
• ASTM E3181 - Practice for Determination of the Converted Fraction of Starch and Cellulosic Content From a Fuel Ethanol Production Facility: Analytical procedures for apportioning ethanol yield from different biomass fractions.
• NREL/TP-5100-42618 - Determination of Structural Carbohydrates and Lignin in Biomass: Referenced methodology for cellulosic pellet hydrolysis and monosaccharide measurement.
• NREL/TP-2800-76724 – Determination of Cellulosic Glucan Content in Starch Containing Feedstocks: Supporting method adapted for use in E3417-24.
• Megazyme RTS-NaOH, 2019 – Referenced starch analysis procedure forming the basis for starch removal steps.

Practical Value

ASTM E3417-24 fills a key analytical gap for the bioenergy sector, enabling accurate, repeatable quantification of cellulose and hemicellulose-derived sugars in corn biomass. By leveraging established chemical and enzymatic protocols, the standard supports operational efficiency, regulatory compliance, and the expansion of advanced biofuels production. Its use drives innovation in feedstock valorization, process optimization, and the accurate reporting of renewable fuel yields from cellulosic sources.

Keywords: ASTM E3417-24, cellulose, hemicellulose, glucan, galactan, corn biomass, bioethanol, biofuels, compositional analysis, regulatory compliance, feedstock characterization.

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3417 − 24
Standard Test Method for
Determination of Cellulose/Hemicellulose-Derived Glucan
and Galactan Content in Solid Corn Biomass Samples
This standard is issued under the fixed designation E3417; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
The US biofuel industry generates ~15 billion gal of bioethanol annually. Most of this bioethanol
is produced using processes that convert the starch content of corn biomass to glucose through
enzymatic degradation and subsequently to ethanol via yeast fermentation. Starch [a branched
polymer of α-(1-4)-D-glucose units] is the source of the majority of the glucose content present in
corn. A tiny proportion of bioethanol is generated using processes that convert cellulosic feedstock to
glucose before yeast fermentation. The emergence of in-situ corn kernel fiber (CKF) conversion
processes that allow the simultaneous conversion of starch and cellulosic content in biomass presents
an excellent opportunity for the sector. While starch measurement methodology has been well
described in literature, the analytical methodology required to accurately measure cellulosic content
in corn biomass has not been published to date despite the urgent requirement for the same to be used
in the assignment of a small but valuable percentage of total ethanol produced during such a
fermentation as cellulosic.
The procedure outlined herein seeks to address this need by modifying the assay described in the
seminal 2021 work of Sluiter et al.
1. Scope 1.4 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This procedure can be used to quantify the cellulose/
ization established in the Decision on Principles for the
hemicellulose-derived glucan and galactan (CHDGG) content
Development of International Standards, Guides and Recom-
in corn biomass samples that also contain varying levels of
mendations issued by the World Trade Organization Technical
starch-derived and yeast-derived glucan. The method has been
Barriers to Trade (TBT) Committee.
shown to provide accurate values for samples with cellulose
content up to 40 % w/w.
2. Referenced Documents
1.2 Units—The values stated in SI units are to be regarded
2.1 ASTM Standards:
as the standard. No other units of 34 measurement are included
E1757 Practice for Preparation of Biomass for Composi-
in this standard.
tional Analysis
E3181 Practice for Determination of the Converted Fraction
1.3 This standard does not purport to address all of the
of Starch and Cellulosic Content From a Fuel Ethanol
safety concerns, if any, associated with its use. It is the
Production Facility
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
3. Terminology
mine the applicability of regulatory limitations prior to use.
3.1 Definitions:
3.1.1 biomass, n—substance wholly comprised of living or
recently living (non-fossil) material.
This test method is under the jurisdiction of ASTM Committee E48 on
3.1.1.1 Discussion—This method is selective for corn bio-
Bioenergy and Industrial Chemicals from Biomass and is the direct responsibility of
Subcommittee E48.05 on Biomass Conversion.
mass which is defined as the biomass from large kernels set in
Current edition approved Jan. 1, 2024. Published January 2024. DOI: 10.1520/
rows on a cob from a cereal plant.
E3417-24.
Sluiter, J. B. et al., “Direct Determination of Cellulosic Glucan Content in
Starch-Containing Samples,” Cellulose, 2021, https://doi.org/10.1007/s10570-020- For referenced ASTM standards, visit the ASTM website, www.astm.org, or
03652-2. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
See Supporting data document - Determination of cellulosic carbohydrate Standards volume information, refer to the standard’s Document Summary page on
content in corn biomass samples the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3417 − 24
3.1.2 calibration verification standard, CVS, n—standards 3.2.4 SRS—Sugar Recovery Standard
used in determining the quality of the calibration curve as well
3.2.5 YDC—Yeast Degrading Cocktail
as the quality of the standard reagents used in preparing the
calibration standards.
4. Significance and Use
3.1.2.1 Discussion—The concentration of the CVS should
4.1 The procedure, in brief, consists of solubilization of
be set in the middle of the chosen calibration range and is used
biomass using cold caustic extraction, an enzymatic removal of
to ensure the calibration remains accurate throughout the
starch and yeast glucan followed by precipitation of hemicel-
entirety of the sample run. Additional CVS concentrations at
lulosic material, and acid hydrolysis of the residual carbohy-
the lower or higher end of the calibration curve can also be run
drate pellet before measurement of glucose and galactose
to ensure accuracy of a sample’s respective concentrations.
derived from cellulose and hemicellulose.
3.1.3 cellulose, n—a crystalline, straight chain glucan with
β-(1→4) linkages.
4.2 The starch removal procedure is identical to that em-
ployed in the NREL assay which was itself adapted from the
3.1.4 cellulose/hemicellulose-derived glucan and galactan,
Megazyme-published starch analysis procedure: RTS-NaOH,
n—carbohydrate content present in a sample comprising glucan
5,6
2019.
and galactan, but specifically does not include glucan from
starch or yeast.
4.3 The cellulosic pellet hydrolysis and monosaccharide
3.1.4.1 Discussion—For the purpose of this test method,
measurements are identical to those described in the NREL
cellulose/hemicellulose-derived glucan and galactan is abbre-
laboratory analytical procedure “Determination of Structural
viated as CHDGG.
Carbohydrates and Lignin in Biomass,” NREL/TP-5100-42618
3.1.5 cellulosic content, n—as defined by EPA RFS
and “Determination of Cellulosic Glucan Content in Starch
documentation, the sum of cellulose, hemicellulose, and lignin
Containing Feedstocks,” NREL/TP-2800-76724.
in cellulosic feedstock.
4.4 This test method references Practices E1757 and E3181.
3.1.6 cellulosic pellet, n—solid fraction of sample remain-
4.5 This test method is intended for use in the measurement
ing on quartz filter after precipitation and filtration following
of the fermentable portion of cellulosic content in pre- and
starch and yeast glucan removal.
post-fermentation corn biomass specifically for the apportion-
3.1.7 galactose, n—six-carbon monosaccharide common in
ment of a percentage of the total ethanol produced in a
biomass.
fermentation as cellulosic.
3.1.8 glucose, n—six-carbon monosaccharide common in
biomass.
5. Apparatus
3.1.9 glycogen, n—high molecular weight branched ho-
5.1 Analytical balance with precision to 0.1 mg.
mopolymer found in yeast consisting of linear α-(1,4)-glucosyl
chains with α-(1,6)-branch points. 5.2 Water bath capable of maintaining 50 °C 6 2 °C, 40 °C
6 2 °C, and 30 °C 6 2 °C.
3.1.10 hemicellulose, n—a polymeric, branched carbohy-
drate with mixed C5 and C6 monomeric sugars, typically with
5.3 HPLC system equipped with a refractive index detector,
a xylose or mannose backbone.
column oven capable of reaching 85 °C, and the following
3.1.11 starch, n—a polysaccharide consisting of glucose column: Concise Separations CarboSep CHO782, Lead Form
+ -
column (or equivalent) with ionic form H /CO deashing
monomers joined in α 1,4 linkages; the simplest form of starch
is the linear polymer amylose, while amylopectin is the guard column.
branched form.
NOTE 1—The deashing guard column should be replaced every ~50
3.1.12 yeast degrading cocktail, n—an enzymatic cocktail
injections to protect the analytical column.
for degradation of yeast glucan.
3.1.12.1 Discussion—The reagent should contain the neces-
6. Reagents and Materials
sary suite of hydrolytic activities to effect yeast glucan removal
6.1 Purity of Reagents—Reagent grade chemicals shall be
under the conditions specified in this standard, but should not
used in all tests. Unless otherwise indicated, it is intended that
exhibit contaminating activities that would degrade cellulosic
all reagents conform to the specifications of the Committee on
or hemicellulosic derived glucan and galactan.
Analytical Reagents of the American Chemical Society where
3.1.13 yeast glucan, n—any glucan from a yeast source.
3.1.13.1 Discussion—Includes compounds such as yeast
β-glucan; a branched polymer of 1,3/1,6-β-D-glucose units,
McCleary, B. V., Charmier, L. M. J., and McKie, V. A., Megazyme, “Measure-
glycogen, and trehalose; an α-1,1-linked glucosyl disaccharide.
ment of Starch: Critical Evaluation of Current Methodology,” Starch, 1800146, No.
71, 2018, pp. 1-13, DOI: 10.1002/star.201800146.
3.2 Abbreviations:
The Megazyme starch assay procedure referenced in this test method can be
3.2.1 CF—Converted Fraction
found at:
https://www.megazyme.com/documents/Booklet/K-TSTA-100A_DATA.pdf.
3.2.2 CHDGG—Cellulose/Hemicellulose-Derived Glucan
All NREL biomass compositional analysis laboratory procedures referenced in
and Galactan
this test method can be found at: https://www.nrel.gov/bioenergy/biomass-
3.2.3 CVS—Calibration Verification Standard compositional-analysis.html.
E3417 − 24
such specifications are available. Other grades may be used, 6.2.1 Positive displacement automatic pipettors capable of
provided it is first ascertained that the reagent is of sufficiently multi-dispensing with corresponding compatible tips.
high purity to permit its use without lessening the accuracy of 6.2.2 Pressure tubes, 120 mL capacity, glass, with screw on
the determination. polytetrafluoroethylene (PTFE) caps and O- ring seals or glass
6.1.1 D-(+)-glucose, ≥99.5 % (GC). vessels (that is, 250 mL Erlenmeyer flask, beaker, or serum
6.1.2 An aliquot of high purity D-(+)-glucose, ≥99.5 % bottle–a glass vessel matching close to or slightly larger than a
(GC) from a different source (manufacturer or lot), to be used 47 mm diameter bottom), autoclave safe. Pressure vessels shall
to prepare CVS. be pressure rated for at least 10.5 kg ⁄cm at 120 to prevent
6.1.3 D-(+)-galactose, ≥99.5 % (GC). potential rupture during the autoclave step.
6.1.4 An aliquot of high purity D-(+)-galactose, ≥99.5 % 6.2.3 PTFE or glass stir rods, in appropriate size for dilute
(GC) from a different source (manufacturer or lot) to be used to acid hydrolysis vessel.
prepare CVS. 6.2.4 Magnetic stir bars, 10 mm × 6.4 mm.
6.1.5 Sodium hydroxide pellets (for solution preparation) or 6.2.5 Nylon or polypropylene sample racks to accommodate
solution, ACS grade. tubes with outer diameters (OD) of 30 mm and 16 mm.
6.1.6 Water, HPLC grade, 0.2 μm filtered (or deionized 6.2.6 Metal sample racks to accommodate tubes with OD
18 MΩ ⁄cm filtered water). 38.1 mm (if using pressure tubes for acid hydrolysis).
6.1.7 Sulfuric acid, 72 % w/w (specific gravity 1.6338 at 6.2.7 Conical polypropylene centrifuge tube, 50 mL, with a
20 °C). tightly fitting screw cap.
6.1.8 Glacial acetic acid, ACS grade. 6.2.8 Quartz fiber filters, 47 mm, efficiency rating: >99 %
6.1.9 Thermostable α-amylase. (3000 U ⁄mL; one Unit of retention dispersed oil particulate (DOP) at 3 μm.
α-amylase activity is defined as the amount of enzyme required 6.2.9 Autosampler vials and caps, 2 mL fill volume.
to release one μmole of 4-nitrophenol from blocked 6.2.10 pH paper (range 2-9).
4-nitrophenyl-maltoheptaoside per minute (in the presence of 6.2.11 Aluminum sample pans or porcelain crucible equiva-
excess α-glucosidase) at pH 6.5 and 40 °C). lent.
6.2.12 Disposable syringes, 3 mL, fitted with 0.2 μm nylon
NOTE 2—Note that both starch degrading enzymes must be essentially
syringe filters.
devoid of cellulose or hemicellulose degrading activities. E-BSTAA and
6.2.13 Disposable culture tubes, 16 mm × 100 mm (if using
E-AMGDF available from Neogen, 620 Lesher Place, Lansing, MI 48912,
have been shown to be suitable for this application. Equivalent enzymes
pressure tubes for acid hydrolysis).
from an alternative supplier with the same or better purity specification
6.2.14 Duran bottles, 250 mL capacity.
can also be used.
7. Hazards
6.1.10 Amyloglucosidase—(3260 U ⁄mL; one unit of amylo-
glucosidase activity is defined as the amount of enzyme
7.1 Adhered to the general safety measures that apply to all
required to release one μmole of D-glucose reducing-sugar chemical substances. For more information regarding the safe
equivalents per minute from soluble starch at pH 4.5 and
usage and handling of the reagents and components, please
40 °C).
refer to the associated safety data sheets (SDS).
6.1.11 Yeast-degrading enzyme cocktail (YDC,
7.2 This test method should not be carried out by anyone
>800 U ⁄mL; one unit of Yeast Degradation activity is defined
other than a trained and experienced laboratory analyst.
as the amount of enzyme required to release one μmole of
D-glucose reducing-sugar equivalents per minute from soluble 8. Reagent Preparation
yeast beta glucan (USP 1048288) at pH 5 and 40 °C. Product#
8.1 Sodium hydroxide solution (1.7M) for starch solubili-
E-YDC, Neogen, 620 Lesher Place, Lansing, MI 48912,
zation can be purchased or prepared as follows. Add 68 g of
www.neogen.com).
sodium hydroxide pellets to 900 mL of deionized filtered water
6.1.12 Calcium carbonate, ACS reagent grade.
in a 1 L volumetric flask. Dissolve by stirring, taking care as
6.1.13 Calcium chloride dihydrate, ACS reagent grade.
this solution will become hot. Adjust to a final volume of 1 L,
6.1.14 Ethanol, 99 % v/v.
allow to cool to room temperature. Store in the refrigerator.
6.1.15 Ethanol, 95 % v/v.
8.2 Sodium hydroxide solution (4M) for pH adjustment can
6.1.16 Ethanol, 85 % v/v.
be purchased or prepared as follows. Add 80 g of sodium
6.2 Materials:
hydroxide pellets to 400 mL of filtered deionized water in a
500 mL volumetric flask. Dissolve by stirring, taking care as
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
this solution will become hot. Adjust to a final volume of
Standard-Grade Reference Materials, American Chemical Society, Washington,
500 mL.
DC. For suggestions on the testing of reagents not listed by the American Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
8.3 Sodium acetate buffer (600 mM, pH 3.8) can be pur-
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
chased or prepared as follows. Add 69.6 mL of glacial acetic
copeial Convention, Inc. (USPC), Rockville, MD.
acid (1.06 g ⁄mL) to 1600 mL of deionized filtered water and
The sole source of supply of the YDC reagent known to the committee at this
time is Neogen, 620 Lesher Place, Lansing, MI 48912. If you are aware of
adjust to pH 3.8 using a 4M sodium hydroxide solution. Add
alternative suppliers, please provide this information to ASTM International
1.48 g of calcium chloride dihydrate and dissolve by stirring.
Headquarters. Your comments will receive careful consideration at a meeting of the
Quantitatively transfer volume to a 2 L volumetric flask and
responsible technical committee, which you may attend. Appendix X.1 provides
additional information on this reagent. adjust the final volume to 2 L with deionized filtered water.
E3417 − 24
9. Sample Preparation 10.3.2 Allow to stand at 4 °C for 120 min. Alternatively,
this solution can be left to precipitate at 4 °C overnight
9.1 Before analysis, test material should be prepared ac-
(approximately 16 h).
cording to Practice E1757.
10.3.3 Vacuum filter each reaction mixture through a sepa-
9.2 Care shall be taken to ensure a representative sample is
rate quartz fiber filter.
used for analysis. The material should be at room temperature
10.3.4 Rinse all particulates from tube and the sides of the
before sampling.
filtration apparatus using a minimum of 30 mL of 85 % v/v
10. Procedure ethanol; ensure a complete transfer of material to filter.
10.3.5 Place pellet and quartz filter onto an aluminum pan or
10.1 α-Glucan Removal Procedure (Starch and Glycogen):
porcelain crucible and place in a freeze dryer overnight to dry.
10.1.1 Prepare a reagent blank in duplicate with each set of
Drying conditions: ambient temperature, <1 mBar, condenser
determinations. This shall contain no sample
...