ASTM E3181-20

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: E3181 − 20
Standard Practice for
Determination of the Converted Fraction of Starch and
Cellulosic Content From a Fuel Ethanol Production Facility
This standard is issued under the fixed designation E3181; 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.
1. Scope 2. Referenced Documents
1.1 This practice provides criteria for the sampling, testing, 2.1 ASTM Standards:
and calculation methodologies used for the quantification of E122 Practice for Calculating Sample Size to Estimate,With
the converted fraction of starch and cellulosic content. Specified Precision, the Average for a Characteristic of a
Furthermore, this practice covers procedures for the manage- Lot or Process
ment of the standard error associated with the sampling and E1755 Test Method for Ash in Biomass
testing of before conversion and after conversion samples from E1757 Practice for Preparation of Biomass for Composi-
a fuel ethanol production facility. tional Analysis
1.1.1 This practice can be used to determine the volume of E2586 Practice for Calculating and Using Basic Statistics
renewable fuel produced from the simultaneous conversion of
2.2 AOAC Standard:
starch and cellulosic material eligible for generating D3 RINs
AOAC 942.05 Ash of Animal Feed
under the United States (U.S.) Renewable Fuel Standard
2.3 Other Publications:
(RFS).
CFR 40 Code of Federal Regulations (CFR) Part 80 Subpart
1.2 This practice covers the collection and testing of het- M Renewable Fuel Standard
EPA-420-B-19-022 Guidance on Qualifying an Analytical
erogeneous material, including, but not limited to: corn,
Method for Determining the Cellulosic Converted Frac-
sorghum, wheat, mash, beer, whole stillage, dried distillers
tion of Corn Kernel Fiber Co-Processed with Starch
grains with solubles (DDGS), and dried distillers grains.
EPA-HQ-OAR-2012-0401; FRL-9910-40-OAR (July,
1.3 This practice is intended to be used in renewable fuel
2014) Additional Detail on the Calculation of the Cellu-
production facilities designed to produce renewable alcohols.
losic Converted Fraction, and Attribution of Batch RINs
Use of this practice in any other type of process has not been
for D-code Dependent Feedstocks
reviewed.
1.4 This practice can be utilized using either manual or
3. Terminology
automatic sampling techniques, so long as the criteria of this
3.1 Definitions:
practice are followed.
3.1.1 after conversion sample, n—a sample obtained from
1.5 This standard does not purport to address all of the
theprocessaftertheconversionprocessofstarchandcellulosic
safety concerns, if any, associated with its use. It is the
content is determined to be complete as defined by the
responsibility of the user of this standard to establish appro-
producer’s reaction standards.
priate safety, health, and environmental practices and deter-
3.1.2 amyloglucosidase, n—an enzyme that specifically
mine the applicability of regulatory limitations prior to use.
catalyzes the hydrolysis ofα-D-glucosidic bonds successively
1.6 This international standard was developed in accor-
from the non-reducing ends of oligo-and polysaccharides with
dance with internationally recognized principles on standard-
the release of β-D-glucose.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical 2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Barriers to Trade (TBT) Committee. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Available from AOAC International, 2275 Research Blvd., Suite 300,
This practice is under the jurisdiction of ASTM Committee E48 on Bioenergy Rockville, MD 20850-3250, http://www.aoac.org.
and Industrial Chemicals from Biomass and is the direct responsibility of Subcom- Available from the U.S. Government Printing Office, Superintendent of
mittee E48.05 on Biomass Conversion. Documents, 732 N. Capitol St. N.W. Mail Stop: SDE Washington, DC 20401.
Current edition approved Feb. 1, 2020. Published March 2020. DOI: 10.1520/ Available from the United States Environmental Protection Agency (EPA),
E3181-20. 1200 Pennsylvania Avenue, N.W., Washington, DE 20460, www.epa.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3181 − 20
3.1.3 ash, n—inorganic residue remaining after combustion, 3.1.18 lignin, n—a complex organic polymer with aromatic
determined by definite prescribed methods. components; non-carbohydrate found in cellulosic feedstock.
3.1.3.1 Discussion—Test Method E1755 andAOAC 942.05
3.1.19 makeup water, n—any liquid stream added to the
are acceptable prescribed methods.
process to prepare the mash slurry in a renewable fuel
production facility.
3.1.4 backset, n—a recycled liquid stream that also contains
some level of dissolved and suspended solids used as makeup
3.1.20 mash, n—a mixture or slurry of water (including
water in a renewable fuel production facility.
recycled water streams) and ground grain.
3.1.5 beer, n—the mash after it has undergone fermentation
3.1.21 monosaccharide, n—any of the class of sugars (for
and has been deemed complete by virtue of the carbohydrates
example, glucose) that cannot be hydrolyzed to give a simpler
being converted into renewable fuel by enzymes and ferment-
sugar.
ing organisms.
3.1.22 oligosaccharide,n—acarbohydratewhosemolecules
3.1.6 before conversion sample, n—a sample obtained from
are composed of a relatively small number of monosaccharide
the process before any starch or cellulosic content conversion
units.
has taken place.
3.1.23 polysaccharide, n—a carbohydrate (for example,
3.1.7 cellulose, n—a crystalline, straight chain glucan with starch, cellulose, or glycogen) whose molecules consist of a
β(1→4) linkages. long chain of sugar molecules bonded together.
3.1.24 predominantly cellulosic, adj—feedstock that has an
3.1.8 cellulosic content, n—as defined by EPA RFS docu-
average adjusted cellulosic content of 75 %, measured on a dry
mentation: the sum of cellulose, hemicellulose, and lignin in
mass basis; furthermore, this ‘‘adjusted cellulosic content’’ is
cellulosic feedstock.
the percent of organic (non-ash) material that is cellulose,
3.1.9 cellulosic feedstock, n—any feedstock composed pre-
hemicellulose, or lignin (CFR 40, Part 80 and EPA-HQ-OAR-
dominately of cellulose, hemicellulose, or lignin.
2012-0401; FRL-9910-40-OAR).
3.1.9.1 Discussion—The EPA allows renewable fuel pro-
3.1.25 Renewable Fuel Standard (RFS), n—program autho-
duced from cellulosic feedstocks with an average adjusted
rized under the Energy PolicyAct of 2005 and expanded under
cellulosic content of 75 % (on a dry mass basis) to be eligible
the Energy Independence and SecurityAct of 2007; it requires
for the generation of the D3 RIN code.
an increasing amount of renewable fuel to be blended into the
3.1.9.2 Discussion—For example, in a facility producing
U.S. liquid fuel supply each year through the year 2022.
ethanolfromcorn,theEPAconsidersthecornkernelfibertobe
3.1.26 resistant starch, n—starch that cannot be readily
a cellulosic feedstock.
monomerized by commercially available alpha amylase and
3.1.10 certification run, n—a group of converted fraction
glucoamylase; this type of starch can be caused by several
determinations to establish the converted fraction of starch and
different factors: physical inaccessibility, molecular structure
cellulosic content in a process that involves the simultaneous
configuration, cross linkage, and lipid binding.
conversion of feedstocks that are predominantly cellulosic and
3.1.26.1 Discussion—When resistant starch is suspected to
feedstocks that are not predominantly cellulosic.
be present, it is important that the starch measurement tech-
3.1.11 converted fraction (CF), n—a unitless average mass
nique used be designed to access resistant starch. Many starch
fraction representing the portion of the feedstock converted to
procedures have options that include, for example, dimethyl
either cellulosic or non-cellulosic fuel.
sulfoxide (DMSO) or potassium hydroxide (KOH) to ensure
complete access and hydrolysis of any resistant starch present.
3.1.12 DDG, n—the dried whole stillage; typically sold as
an animal feed component.
3.1.27 RINs, n—Renewable identification numbers (RINs)
are unique numbers generated to represent a volume of
3.1.13 DDGS, n—the dried whole stillage with solubles;
renewable fuel used for compliance to the RFS program.
typically sold as an animal feed component.
3.1.27.1 D3 RINs, n— the RIN code assigned to gallons of
3.1.14 feedstock, n—any raw material used as a carbohy-
renewable fuel produced from any cellulose, hemicellulose, or
drate source for fermentation.
lignin that has lifecycle greenhouse gas emissions that are at
3.1.15 GHG, n—greenhouse gas; greenhouse gases include:
least 60 % less than the baseline lifecycle greenhouse gas
carbon dioxide, methane, nitrous oxide, and fluorinated gases.
emissions.
3.1.16 glucan, v—refers to any polymer of glucose, regard-
3.1.27.2 D6 RINs, n—the RIN code assigned to gallons of
less of structure.
renewable fuel produced from renewable biomass and has
lifecycle greenhouse gas emissions that are at least 20 % less
3.1.17 hemicellulose, n—a polymeric, branched carbohy-
than baseline lifecycle greenhouse gas emissions, unless the
drate with mixed C5 and C6 monomeric sugars, typically with
fuel is exempt from this requirement.
a xylose or mannose backbone.
3.1.17.1 Discussion—For the purpose of this practice, C5 3.1.28 starch, n—a polysaccharide consisting of glucose
sugars are five carbon sugars derived from hemicellulose. C6 monomers joined inα 1,4 linkages; the simplest form of starch
sugars are six carbon sugars derived from either starch, is the linear polymer amylose, while amylopectin is the
hemicellulose, or cellulose. branched form.
E3181 − 20
3.1.28.1 Discussion—For the purposes of this practice, for cellulosic content to a commercially available reference
starch shall include the soluble C6 glucan mono- and oligosac- material with established and documented concentrations of
charides. Since the EPA definition for “cellulosic content” is cellulosic content. When the commercially available reference
specific to the “sum of cellulose, hemicellulose, and lignin in material is analyzed, the test method shall achieve a mean test
cellulosic feedstock,” the soluble C6 glucan mono- and oli- value that falls within 20 % of the reported cellulosic content
gosaccharides shall be included with the starch total (CFR 40, value (EPA-420-B-19-022) as documented with the commer-
Part80).Sincetheonlydifferencebetweenstarchandcellulose cially available reference material. Test values that fall within
is the orientation of the 1, 4 linkage, to determine the starch 20 % of the reported cellulosic content value are deemed
content of a sample that contains both starch and cellulose, an reasonably accurate.
assay that utilizes amyloglucosidase is necessary. 5.1.1.1 Discussion—Producers generating RINs with a D
code of 3 or a D code of 7 using two or more different
3.1.29 total solids, n—the amount of total suspended and
feedstocks (at least one of which does not have at least 75 %
dissolved matter contained in a sample.
average adjusted cellulosic content) which are processed si-
3.1.30 whole stillage, n—the beer once the renewable fuel
multaneously through an in situ biochemical hydrolysis treat-
has been removed.
mentwillsimilarlyhaveadditionalregistrationrequirementsto
helpensurethatcellulosicRINsarebeinggeneratedaccurately.
4. Significance and Use
At the time of registration, such a producer must submit: (1)
4.1 In 2014, the U.S. EPA published the final rules adding
the overall fuel yield, including supporting data demonstrating
renewable fuel pathways under the RFS Program. The rules
this yield and a discussion of the possible variability in overall
qualified kernel fiber as a cellulosic feedstock meeting the
fuelyieldthatcouldbeexpectedbetweenreportingperiods; (2)
60 % greenhouse gas (GHG) reduction and qualifies for the
the cellulosic converted fraction that will be used for generat-
generation of D3 RINs. These rules allow for two approaches
ing RINs under § 80.1426(f)(3)(vi); including chemical analy-
for kernel fiber conversion (CFR 40, Part 80 and EPA-HQ-
sis data (described in more detail below) supporting the
OAR-2012-0401; FRL-9910-40-OAR):
calculated cellulosic converted fraction and a discussion of the
4.1.1 Producers of cellulosic fuels derived from conversion
possible variability that could be expected between reporting
of feedstocks that are predominantly cellulosic, where “pre-
periods; and (3) a description of how the cellulosic converted
dominantly cellulosic” is defined as feedstock that has an
fraction is determined and calculations showing how the data
average adjusted cellulosic content of 75 %, measured on a dry
were used to determine the cellulosic converted fraction. Data
mass basis; furthermore, this ‘‘adjusted cellulosic content’’ is
used to calculate the cellulosic converted fraction by producers
the percent of organic (non-ash) material that is cellulose,
using in situ biochemical hydrolysis treatment who seek to
hemicellulose, or lignin (CFR 40, Part 80 and EPA-HQ-OAR-
generate cellulosic RINs must be representative and obtained
2012-0401; FRL-9910-40-OAR).
using an analytical method certified by a voluntary consensus
4.1.2 Producers of cellulosic fuels derived from the simul-
standards body (VCSB) or using a non-VCSB method that
taneous conversion of feedstocks that are predominantly cel-
would produce reasonably accurate results. If using a non-
lulosic and feedstocks that are not predominantly cellulosic
VCSB approved method to generate the data required to
(CFR 40, Part 80 and EPA-HQ-OAR-2012-0401; FRL-9910-
calculate the cellulosic converted fraction for a given fuel, then
40-OAR).
the producer will need to show that the method used is an
4.2 Producers that wish to gain approval to the pathway that
adequate means of providing reasonably accurate results by
claims simultaneous conversion of feedstocks that are pre-
providing peer reviewed references to the third-party engineer
dominantly cellulosic and feedstocks that are not predomi-
performing the engineering review at registration. A full
nantly cellulosic are required to quantify the amount of
description of the formulas in § 80.1426(f)(3) used to calculate
renewable fuel that is derived specifically from cellulosic
RINs for renewable fuel described by two or more pathways,
content and from starch. To accomplish this, the producer
including methods used to calculate the converted fraction, can
needs to quantify the amount of cellulosic content and starch
be found in the associated memo to the docket (CFR 40, Part
present before the conversion process begins and after the
80).
conversion process is complete. These measurements of cellu-
5.1.2 It has been demonstrated that using sample prepara-
losic content and starch content before and after conversion are
tion drying temperatures above 65 °C when drying and prepar-
used to calculate a converted fraction of each, which is then
ing samples for analysis can cause the formation of resistant
used to ratio the renewable fuel produced accordingly and
starch which can lead to the generation of erroneous results.
assign those respective gallons the D6 or D3 RIN code (CFR
For this reason, samples that need to be dried prior to analysis
40, Part 80 and EPA-HQ-OAR-2012-0401; FRL-9910-40-
shall follow the guidance of Practice E1757.
OAR).
5.1.3 It is critical that any starch method used for the
determination of the converted fraction of starch for the
5. Test Method Requirements
purposes of this practice be designed to access resistant starch.
5.1 Anytestmethodusedforthequantificationofstarchand
Manystarchprocedureshaveoptionsthatinclude,forexample,
cellulosic content and the related converted fraction of each
shall be evaluated for both accuracy and precision.
5.1.1 Agreement of the test method for cellulosic content
A possible source of reference materials is NIST (National Institute of
may be demonstrated by showing agreement of the test method Standards and Technology).
E3181 − 20
dimethyl sulfoxide (DMSO) or potassium hydroxide (KOH) to where:
ensure complete access and hydrolysis of all starch present.
σ = the standard deviation of the sample results, and
5.1.4 Precision of the test methods shall be established and
n = the number of sample results.
documented by quantifying the reproducibility of the test
7.1.1.1 The standard error goes down by a factor of the
methods for starch and cellulosic content. To determine the
square root of the increase in the number of sample analysis
reproducibility of the test methods, six replicates of one
replicates.The variation in the results, therefore, goes down by
prepared sample of representative substrate shall be analyzed.
50 % for every fourfold increase in the number of sample
The standard deviation of the test method shall be calculated
analysis replicates performed. See Appendix X2 for an ex-
and documented (this is the uncertainty of the testing method).
ample of the reduction in standard error experienced in
proportion to the number of sample replications performed.
6. Sample Collection Requirements
7.1.2 The number of replications on each sample may be
6.1 The number of subsamples needed to achieve a repre-
increased to the extent necessary to lower the standard error of
sentative composite sample shall be determined by using
the result reported.
Pra
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