Guidance on Qualifying an Analytical
Method for Determining the Cellulosic
Converted Fraction of Corn Kernel Fiber
Co-Processed with Starch
United States
Environmental Protection
tl	Agency

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Guidance on Qualifying an Analytical
Method for Determining the Cellulosic
Converted Fraction of Corn Kernel Fiber
Co-Processed with Starch
Compliance Division
Office of Transportation and Air Quality
U.S. Environmental Protection Agency
oEPA
United States	EPA-420-B-19-022
Environmental Protection
Agency	May 2019

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Guidance on Qualifying an Analytical Method for Determining the Cellulosic Converted
Fraction of Corn Kernel Fiber Co-Processed with Starch
Introduction
This document provides guidance on how to demonstrate that an analytical method for
determining the cellulosic converted fraction of corn kernel fiber co-processed with starch at a
traditional ethanol facility satisfies the applicable regulatory requirements. Public confidence in
the volumes of cellulosic ethanol produced under the Renewable Fuel Standard (RFS) program is
important to the integrity of the program. As such, affected stakeholders have sought
clarification regarding what methods should be used to determine the cellulosic converted
fraction. The intent of this guidance is to explain our interpretation of our regulatory
requirements and to articulate clear criteria for the type of analysis and demonstrations that EPA
believes would be an appropriate basis for registration under the program.
EPA evaluates registration applications on a case-by-case basis given the best available
information and science relating to analytical methods for determining the cellulosic converted
fraction of corn kernel fiber co-processed with corn starch. This guidance provides EPA's
current view on the manner in which facilities should demonstrate the accuracy of such
analytical methods and thus may satisfy the applicable registration requirements. However, any
decisions on individual facilities' registration applications will be made in the context of each of
those applications on the basis of the requirements of EPA's regulations. This guidance does not
create any new requirements and may not apply to a particular situation based on the
circumstances.
This guidance is organized into the following topics: (1) background and explanation of
the relevant regulations; (2) EPA's interpretation of the regulatory term "reasonable accuracy";
(3) establishing voluntary consensus standard body (VCSB) methods that are consistent with the
regulations; and (4) a summary of EPA's guidance for demonstrating that the results of an
analytical method for calculating the cellulosic converted fraction are reasonably accurate using
acceptable reference materials.
Background and Explanation of Regulations
In the 2014 Pathways II Final Rule,1 EPA added a pathway for the production of
cellulosic ethanol from corn kernel fiber2 and promulgated the regulations necessary to
implement this pathway. The Agency stated that given variations in individual conversion
processes, enzymes used, and other differences, the amount of finished fuel derived from the
cellulosic content of corn kernels (i.e., the cellulosic converted fraction) can vary. For example,
the process and enzymes used may be more effective in converting the sugars and starches in a
feedstock than the cellulose or hemicellulose. In such a case, the cellulosic content of the
feedstock may not be a good indicator of the amount of finished biofuel that is derived from
1	79 Federal Register 42128 (July 18, 2014).
2	See id. at 42147-48: 40 CFR 80.1426, Table 1 row K (production of cellulosic ethanol from crop residue,
including corn kernel fiber).
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cellulosic materials. Furthermore, depending on the conversion process used, the amount of
information needed to determine how much of the finished fuel is derived from the cellulosic
content will also vary.3 Therefore, the regulations include requirements for registration,
reporting, and recordkeeping related to calculating the cellulosic converted fraction to ensure a
high degree of confidence that cellulosic biofuel RINs are appropriately generated.
The regulations at 40 CFR 80.1450(b)(l)(xiii)(B)(3) require a producer of renewable fuel
seeking to generate cellulosic RINs who intends to produce a single type of fuel using two or
more feedstocks converted simultaneously (such as corn starch and corn kernel fiber) to provide
"chemical analysis data supporting the calculated cellulosic converted fraction (CF) and a
discussion of the possible variability that could be expected between reporting periods per
§80.145 l(b)(l)(ii)(U)(l). Data used to calculate the cellulosic CF must be representative and
obtained using an analytical method certified by a voluntary consensus standards body
[VCSB],or using a method that would produce reasonably accurate results as demonstrated
through peer reviewed references provided to the third-party engineer performing the
engineering review at registration."
Further, EPA anticipated that the converted fraction may vary over time, and therefore
established reporting requirements at 40 CFR 80.145 l(b)(l)(ii)(U) that require recalculation and
recertification to EPA of the cellulosic converted fraction at specified intervals (annually for low
volume producers and every 500,000 gallons of cellulosic RINs generated for larger volume
producers). The initial cellulosic converted fraction is based on the data submitted at registration
and this upfront cellulosic converted fraction determination applies to RINs generated until a
new cellulosic converted fraction allocation is available and reported. Given the natural variation
in cellulosic content and conversion efficiencies, EPA recognized some variation would exist in
the amount of biofuel that is derived from the cellulosic components of a feedstock. The
regulations require that if the cellulosic converted fraction deviates from the previously
calculated cellulosic converted fraction by 10 percent or more, a producer is required to alert
EPA to this change in addition to updating the formula used to calculate RIN allocations.4 This
regulatory requirement reflects EPA's recognition that variation within 10 percent of previously
calculated numbers may result under normal operating conditions, but that larger variations raise
significant concerns that the process or feedstock has significantly changed compared to what
was approved at registration. As a practical matter, if EPA observes a high variability (i.e., over
10 percent) in the recertified cellulosic converted fraction, this may signal the need for additional
inquiry with the producer to understand the cause of that variability.
Interpretation of the "Reasonable Accuracy" Requirement
This section provides a detailed explanation of how a producer of renewable fuel should
demonstrate that an analytical method will produce reasonable accurate results. As there is
currently no VCSB method for demonstrating how much of the cellulosic material is being
converted into biofuel, ethanol producers have sought to use the second option in
§ 80.1450(b)(l)(xiii)(B)(3): calculating the cellulosic converted fraction using a non-VCSB
method that would produce reasonably accurate results as demonstrated through peer reviewed
3	Id. at 42134.
4	See id at 42135.
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references. Some stakeholders have requested that EPA provide our interpretation of the peer
reviewed reference requirement. EPA interprets this provision as requiring that peer reviewed
references not only evaluate the potential performance of a non-VCSB analytical method but
also demonstrate the accuracy of the results of that method. That is, the references must
demonstrate to EPA that not only is the analytical method theoretically capable of producing
accurate results, but that its application has, in fact, yielded a calculation of the cellulosic
converted fraction that is reasonably accurate. Both criteria must be satisfied in order to
demonstrate that the method used to calculate the cellulosic converted fraction would produce
reasonably accurate results. This is consistent with the purpose of the additional registration and
reporting requirements for fuels produced from co-processing two or more different
feedstocks—to ensure the accurate assignment of RINs to the cellulosic versus non-cellulosic
components of the finished fuel.5 That is, to uphold the integrity of RIN assignment and thus of
the program, it is critical that the analytical method has been demonstrated to produce reasonably
accurate results.
As explained further below, the wide degree of variability in the data EPA has reviewed
alerted us to the fact that it is not possible, as a technical matter, to assess whether a method is
accurately measuring how much of a cellulosic feedstock is converted into fuel without
comparing the performance of the method to a known, representative reference material.6 EPA's
concern about the lack of a benchmark against which to assess the performance of non-VCSB
analytical methods led us to approach the National Institute of Standards and Technology (NIST)
in August 2017 to pursue the development of a reference material containing both starch and
cellulose.7 Such reference material would establish the "true value" of cellulosic content against
which to evaluate the results of analytical methods and will allow peer reviewers and the Agency
to determine whether those methods can produce reasonably accurate results for the cellulosic
converted fraction.8 Accurate calculations of cellulosic conversion are necessary to apportion
RINs correctly per the co-processing requirements under 40 CFR 80.1426(f)(3)(vi) and thus
provide industry with a path forward on RFS registrations. A work group composed of NIST, the
National Renewable Energy Laboratory (NREL), EPA, and industry was established to develop
5	40 CFR 80.1426(f)(3)(vi) contains the requirements for assigning RINs to renewable fuels produced using two or
more feedstocks processed simultaneously, including situations in which only one of the feedstocks is cellulosic.
The formula in this provision for assigning RINs to the different components of the finished fuel incorporates the
value for the cellulosic converted fraction. Thus, accurate assignment of RINs depends in part on accurate
calculation of the cellulosic converted fraction.
6	A representative reference material per the National Institute of Standards and Technology (NIST) (see
https://www.nist.gov/srm/srm-definitions) is a material, sufficiently homogeneous and stable with respect to one or
more specified properties, which has been established to be fit for its intended use in a measurement process. Uses
may include calibrating a measurement system, assessing a measurement procedure, assigning values to other
materials, and quality control. In this instance, EPA expects to evaluate the performance of non-VCSB methods
based on their ability to return results consistent with the known cellulosic value for the reference material.
7	NIST, with stakeholder support, is developing representative reference materials using samples taken from a corn
ethanol facility. The candidate reference materials were prepared from dried, ground and blended corn grain biomass
intermediates before and after conversion to ethanol. Representative reference materials will allow the peer
reviewers and the Agency to directly evaluate whether a non-VCSB method is able to achieve accurate results rather
than relying on an opinion that the analytical steps in a method should be capable of doing so.
8	Accuracy describes how closely the measured value approximates its true value. A representative reference
material that has been validated by an independent body is needed to provide a "true value" against which to
evaluate the ability of particular analytical methodology to determine results that approach the true value.
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candidate materials and advance this process. NIST recently distributed the candidate materials
for intra-laboratory analysis, an important step forward in the process.
While NIST is seeking input on both starch and cellulosic values of the candidate
materials,9 there is an expectation within the workgroup that the first round of laboratory
samples will focus predominantly on starch10 and that the cellulosic analysis will follow once an
acceptable value has been determined for starch.11 EPA believes it is not possible for an
analytical method that is designed to focus on starch or some other non-cellulosic component(s),
where the accuracy is determined for various components and not for cellulose directly by
comparison to a representative reference material, to yield reasonably accurate calculations for
cellulose. Peer reviewers and other outside parties have expressed concerns that resistant and
retrograde starch present in samples due to feedstock handling or processing could impact
laboratory results and cellulosic calculations. That is, measurements of starch may not
themselves be accurate enough to be used to derive reasonably accurate estimates of the
cellulosic converted fraction. This is especially true when cellulose is determined by mass
balance via subtraction of a starch measurement because the variability of the starch
measurements is likely to overwhelm the percent mass of cellulose that is converted. For a
starch-based method in which cellulosic conversion is not measured directly, uncertainty in the
starch measurement is propagated to the cellulose calculation. On average, the incoming corn
feedstock is approximately 70 percent starch and 6 percent cellulosic fiber. Uncertainty of +/- 2
percent in the starch measurement translates to uncertainty of +/- 1.4 percent of total feedstock
mass. Where industry expects approximately 2 percent total cellulosic corn mass conversion,
carrying the +/- 1.4 percent uncertainty from the starch mass over to the 2 percent cellulosic
mass yields an uncertainty in the calculation of cellulosic mass of +/- 70 percent.12 In general,
EPA does not believe that a result with 70 percent uncertainty can be considered "reasonably
accurate." Furthermore, the results of a recent EPA statistical analysis13 indicate that it is
possible, given the potential range of pre- and post-fermentation starch and cellulose values, that
starch-based calculations could yield clearly nonsensical results for the cellulosic converted
fraction, e.g., negative values and values in which over 100 percent of cellulosic mass is
converted. These results again demonstrate that calculations based on starch reference values
alone cannot ensure that resulting estimates of cellulosic conversion are reasonably accurate.
9	Candidate materials are samples sent to laboratories for independent testing to help develop reference materials.
For example, NIST has sent out candidate materials for testing at several laboratories and has requested reported
values for starch and cellulose. Based on the quality of the data, statistical assessment, stability of the samples and
other factors, NIST could proceed to develop "reference materials" from these "candidate materials."
10	For the purposes of this letter, we refer to a starch or "starch only" reference material or method as one that
reports values for any component(s) other than cellulose, which may include starch, fats, proteins, and/or ash. Some
proposed methods directly measure the conversion of starch and other, non-cellulosic components, and then subtract
the starch values from the total to derive the cellulosic component of the finished fuel.
11	It is our understanding that NIST also requested cellulosic values from labs participating in evaluation of the
candidate reference material, but it is yet unclear whether participation and data quality will be sufficient to establish
a reference value for cellulose from this "phase 1" effort.
12	If starch is 70 percent of the mass and the coefficient of variation is +/- 2 percent, then the uncertainty surrounding
the starch measurement is +/- 1.4 percent total mass. Industry has indicated that they expect 2 percent of the total
mass which is fiber to be converted to cellulosic ethanol. Therefore, given just the variability on the starch
measurement that is part of the analytical process to determine cellulose, there is a variability of 2 percent mass +/-
1.4 percent mass or +/-70 percent.
13	See Appendix.
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Based on our work with NIST, on feedback received from labs such as NREL, the
National Corn to Ethanol Research Center, and other technical experts in the ethanol industry,
and on EPA's own modeling, we believe that reasonable accuracy should be demonstrated by
validating that the results of a non-VCSB analytical method for calculating the cellulosic
converted fraction are within 20 percent of the reported cellulosic value of a representative
reference material.
First, the reference material and reported values should be representative of the mixture
of starch and cellulose present in the feedstock. As explained above, based on work to date, EPA
does not believe that the use of a reference material that only reports known values for starch (or
for starch and other non-cellulosic components) can provide a reasonably accurate estimate of
cellulose conversion when starch and cellulose are processed simultaneously. In our technical
judgment, a reference material that is representative of the feedstocks actually being processed is
necessary to accurately determine the cellulosic converted fraction, which is in turn needed to
accurately apportion RINs. Second, while EPA evaluates the accuracy of results reported in
registration applications on a case-by-case basis, as a general matter we currently would consider
results within 20 percent of the known value for the cellulosic component of the representative
reference material to be reasonably accurate. Such a benchmark is a corollary to assessing
accuracy relative to reference material; once a "true value" has been established, a benchmark
defines the universe of results around that value that should be deemed "reasonably accurate."
Additionally, our experience reviewing results to date has indicated that a benchmark will be a
useful tool and provide clarity to facilities aiming to achieve reasonably accurate results. In this
instance, EPA's reporting regulations provide that deviations of up to 10 percent from a
previously calculated cellulosic converted fraction are acceptable, i.e., that the additional
reporting requirement is not triggered if deviation from the previous converted fraction is less
than 10 percent.14 While this benchmark may be a reasonable value to adopt in the context of
calculating the cellulosic converted fraction, it is also reasonable to allow for some operational
flexibility and/or natural variability that may be outside the control of the party. Therefore, given
our current understanding we believe that reasonable accuracy is generally achieved for the
purposes of 40 CFR 80.1450(b)(l)(xiii)(B)(3) when values are within 20 percent of the reported
cellulosic component of representative reference material.
In summary, EPA believes that a representative reference material that contains and
reports values for both starch and cellulose should be used to ensure that a non-VCSB test
method is producing reasonably accurate results under the particular circumstances presented by
co-processing starch and cellulose in the presence of resistant and retrograde starch. If the peer
reviewed references demonstrate that a party using a non-VCSB analytical method has satisfied
both criteria as outlined above, the application should be acceptable for registration under the
pathway in the RFS program, assuming all other registration requirements are met.
14 See 40 CFR 80.145l(b)(l)(ii)(U).
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VCSB Methods for Determining the Cellulosic Converted Fraction
The preceding discussion addresses the use of a representative reference material to
validate non-VCSB analytical methods under the second option in 40 CFR
80.1450(b)(l)(xiii)(B)(3). The same considerations regarding the use of a non-VCSB method
under this second option are also relevant for purposes of the first option—obtaining the data
used to calculate the cellulosic converted fraction using a VCSB-certified analytical method.
This section addresses EPA's assessment of the current15 ASTM effort to establish a VCSB
method.
For the reasons laid out above—the need to accurately apportion RINs to the cellulosic
and non-cellulosic components of the finished fuel consistent with RIN generation requirements
and to uphold public trust in the integrity of the program—any VCSB analytical method should
yield accurate calculations of the cellulosic converted fraction. In general, EPA believes that the
collaborative development process and adoption of a VCSB-certified analytical method signifies
that the method will produce results that are agreed to be sufficiently accurate to achieve the
intended regulatory purpose.16 However, it is EPA's understanding that the method currently
being considered under the ASTM process—development of performance standards for
validating starch conversion—is intended to serve as a VCSB method for calculating cellulosic
conversion. Given our concerns as articulated in the previous section, EPA does not believe that
such a method, based on direct measurements of non-cellulosic components only, can produce
accurate results for cellulose, even if it has been deemed a "VCSB" method. Further, EPA may
have concerns if the method was to provide that results within two standard deviations of the
value reported for the reference material are acceptable and the NIST process yielded a product
with a high variability (e.g., a reference material with a relative standard deviation of 30
percent), as there is the possibility of an absurd result being deemed an acceptable estimate of
cellulosic conversion. EPA would not consider such an outcome to be sufficiently accurate to
inform RIN allocation and thus does not believe that use of a method such as the one currently
under consideration in the ASTM process would be consistent with the regulatory scheme.
Therefore, given EPA's current understanding of the potential accuracy of starch-based
methods for calculating cellulosic conversion, if the current ASTM effort or another VCSB
process results in certification of a starch-based/starch-only method intended for use under
40 CFR 80.1450(b)(l)(xiii)(B)(3), we recommend that parties relying on that method also
include in their registration applications a demonstration that it produces reasonably accurate
results according to the criteria in the previous section. EPA will continue to monitor the state of
the evolving science in this field and will revisit this guidance as needed and update stakeholders
as appropriate.
15	As of April 2019
16	EPA interprets the two options in 40 CFR 80.1450(b)(l)(xiii)(B)(3) as functional equivalents—two different ways
of ensuring results are of sufficient accuracy to inform RIN allocation. Therefore, in the context of this regulatory
provision, EPA believes a VCSB method should provide a reasonable degree of confidence in the results.
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Summary of EPA's Guidance for Demonstrating Reasonable Accuracy
Peer reviews submitted with registration requests for adding the cellulosic pathway for
co-processing corn kernel fiber and starch that rely on a non-VCSB analytical method to
determine the cellulosic converted fraction must not only assess whether it is theoretically
possible for the chosen method to measure cellulose accurately, but must also review the data to
ensure that the method has actually provided reasonably accurate results. EPA recommends that
peer reviews make this demonstration by documenting that the method returns cellulosic values
for a representative reference material within 20 percent of the mean value reported for that
material by an independent body such as NIST. If the references demonstrate that a party using a
non-VCSB analytical method has satisfied the criteria as outlined above in this guidance, the
registration application should be approvable, assuming all other registration requirements are
met.
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Appendix: EPA's Monte Carlo Evaluation of Starch-only Reference Materials for
Cellulosic Ethanol from Corn Kernel Fiber
Executive Summary:
EPA conducted a Monte Carlo simulation to evaluate whether it would be reasonable for
labs to use starch only values from representative reference materials currently under
development by NIST to evaluate method accuracy for determining the cellulosic converted
fraction. The Monte Carlo simulations showed that the error associated with starch
measurements is large enough to produce expected cellulosic conversion results that could not be
deemed to be reasonably accurate. Hence, EPA believes that demonstrating reasonable accuracy
of cellulosic measurements will likely require a representative reference standard with a
cellulosic value.
Background:
In 2014, EPA finalized regulations that added corn kernel fiber as an approved RFS
pathway for producing cellulosic biofuel, provided that parties developed testing methods that
could estimate the portion of the fuel derived from corn kernel fiber with "reasonable
accuracy."17 However, over the last several years EPA has observed data showing very high
variability in results reported for various facilities for the cellulosic converted fraction, where
those results were obtained using non-VCSB analytical methods. EPA was also approached by
technical experts in the space regarding concerns with starch assays, which are the basis of these
non-VCSB methods. Based upon these observations, EPA approached NIST in August of 2017
to encourage NIST to develop representative reference materials to provide both starch and
cellulosic values and which EPA could rely on to evaluate non-VCSB analytical methods. It is
EPA's understanding that NIST has sent out materials for the first phase of reference material
development - interlaboratory study and requested laboratories to report both starch and
cellulosic values. However, it is also EPA's understanding that industry has expressed a desire to
move forward based on the starch values first under "phase 1" and then cellulosic values for
"phase 2."
In order to inform an assessment of whether current efforts underway with NIST, NREL
and the ASTM workgroup can achieve reasonably accurate calculations of the cellulosic
converted fraction, EPA applied a Monte Carlo simulation based on some preliminary data and
feedback from industry. This analysis has enabled us to model simulations that present
uncertainty and play them out in replication for validation. The remainder of this document
discusses EPA's Monte Carlo assumptions, results and our preliminary conclusions from this
analysis.
17 40 CFR 80.1450(b)(l)(xiii)(B)(3) provides that data used to calculate the cellulosic converted fraction must be
obtained using either a method certified by a voluntary consensus standards body (VCSB), or a method that would
produce reasonably accurate results as demonstrated through peer reviewed references. As there is not currently a
VCSB-certified method, efforts to date have focused on the second option.
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EPA's Monte Carlo Simulations
Monte Carlo simulations look at the probability of an outcome described by a normal
distribution curve using a random number generator based on the mean and standard deviation of
that distribution. The simulations let us evaluate all the possible outcomes for cellulosic
conversion based on the normal distribution of measurements required as inputs to the
calculation. Being able to evaluate a number of simulations allows for better decision making in
evaluating whether analytical methods are robust enough for measuring cellulosic conversion.
A simplistic model of cellulose conversion that can be evaluated via Monte Carlo
simulation is thus: Cellulose Converted = Cellulose Feedstock pre-fermentation - Cellulose in
DDG post fermentation. We also need to make a few assumptions for our Monte Carlo scenarios
as there is no direct measurement for cellulose. Cellulose, pre and post-fermentation, is derived
from two or more measurements of other components in the corn kernel fiber. The first
assumption for the Monte Carlo simulations is that the cellulosic determination is simplified to
two measurements, one being the starch assay evaluated by the candidate reference material.
Thus, the cellulosic component of the feedstock pre-fermentation is defined as some larger
"measurement one" (Ml) minus the initial starch content of the feedstock pre-fermentation
(Mstarch pre). The cellulosic content of the feedstock post-fermentation is similarly simplified as
"measurement three" (M3) minus the residual starch content of the feedstock post-fermentation
(Mstarch post). Thus, our overall equation being modeled is CCF=(Mi-Mstarchpre) - (M3-Mstarch post).
The second set of assumptions needed to perform the assessment of whether starch
measurements are capable of accurately capturing the cellulosic converted fraction involves the
level of starch contained in the pre and post-fermented corn and the variability associated with
these starch measurements. EPA is basing this part of the analysis on preliminary data captured
from calls with stakeholders working on the NIST reference materials. The Monte Carlo
simulations thus assume an initial starch measurement of 70% starch by mass with a relative
standard deviation of two percent. The post fermentation starch measurement used in these
simulations was 9% by mass with a ten percent relative standard deviation. Lastly, we need to
make a final assumption with regard to the cellulosic converted fraction to complete our analysis.
Most parties that have approached the agency regarding this technology have claimed that corn
naturally contains around 6% by mass of cellulose and that they are able to achieve a cellulosic
converted fraction of one to three percent mass percent. In this analysis, EPA is assuming that
there is 6% by mass fiber in the feedstock and that 2% by mass of the cellulosic material is
converted into ethanol along with the starch that is fermented. With the assumptions outlined
above, we can simplify our equation to CCF=((Spre+ 6) -SPre))-((SPost+4) - Spost) where "S"
represents starch measurements pre and post-fermentation. Using the measurement data denoted
above, this equation representing a theoretical 2% cellulosic converted fraction is thus; CCF=
(76% +/- 2% - 70% +/- 2%) - (13% +/- 10% - 9% +/- 10%).
The scenario outlined above is an optimistic analysis that does not consider compounded
variability from sampling and handling heterogenous material from large vessels at the facility
nor more than two analytical steps required to derive the cellulosic converted fraction.
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Nonetheless, we want to examine whether the variability in the starch measurements would
preclude an accurate calculation of the cellulosic converted fraction when the only comparison
available is to a known starch value. The Monte Carlo simulations will take a random value for
each of the four measurements required to determine cellulose based on the starch performance
data. We chose to run these Monte Carlo simulations in Microsoft Excel and the formula for the
scenario presented above is: CCF = =((NORMINV(RAND(),76,1.52)-
NORMINV(RAND(),70,1.4))-(NORMINV(RAND(),13,1.3)-NORMINV(RAND(),9,0.9))).
The normal distribution for pre and post-fermented starch measurements can be depicted
graphically using the mean and standard deviations provided above. Using the expected value at
three standard deviations allows one to predict a maximum and minimum cellulosic value for
both pre and post fermentation.
Post Fermentation
^ 05
	Max Cellulose 11%	
;	6 7 8 9 10 11 12 13 14 15 16 17 IS 19
Percent Starch
Starch	Starch--Fiber
Cellulose = (starch + fiber) - starch
Cellulosic conversion = Cellulose Pre Fermentation - Cellulose Post Fermentation
From the figure above, there are expected values for cellulose that would both exceed the
theoretical value of cellulose in the feedstock and which are negative. These values indicate that
fiber was not consumed in the process, but created; these results are nonsensical but theoretically
possible due to the variability of the measurements. The figure above represents the assumptions
that were input into the Monte Carlo simulations.
The results of the Monte Carlo simulations can be presented as a histogram of the
expected cellulosic converted fraction values that result from the equation presented above. The
following histogram represents individual results from 1000 simulations.
Pre Fermentation
Max Cellulose 16%
65 66 67 68 69 70 71 72 73 74 7S 76 77 78 79 80 81
Percent SUrch
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Histogram of Individual Predicted Values
160	,
-6	-4	-2	0	2	4	6	8	10
% Cellulose Converted
As can be viewed from the histogram results of the compiled Monte carlo simulations, a
large proportion of absurd results (i.e. both negative and very high cellulosic conversions) are
expected. The key findings from the Monte Carlo simulations include a high frequency (25-30%)
of absurd results and a large number of negative cellulosic conversions (-20%). These expected
outcomes are due to determining a small difference (i.e. the cellulose converted) from a large
value (i.e., measurement of starch mass) with associated measurement error.
One option for dealing with measurement variability would be to employ composite
sampling to produce a result closer to the population mean. Therefore, EPA expanded the Monte
Carlo simulations to look at various numbers of samples composited into ten discrete sampling
events.
Sampling Events
# samples
1
2
3
4
10
2.26
1.65
3.35
2.37
20
1.87
2.40
2.96
2.30
50
1.55
1.74
2.15
2.10
100
1.63
1.88
2.43
1.78
5
6
7
8
9
3.03
3.04
2.41
1.83
2.09
2.62
3.13
3.09
1.60
1.68
2.31
2.65
2.30
2.33
2.35
2.35
2.43
2.10
2.26
2.31
Range
10 mean stdev min	max
1.25
2.33
0.67
1.25
3.35
1.49
2.31
0.63
1.49
3.13
2.24
2.17
0.32
1.55
2.65
1.97
2.11
0.29
1.63
2.43
However, the number of samples that are likely to be required to achieve reasonable
accuracy (within +/- 20% of the accepted value) appears to be unrealistic for industry to execute
(e.g. between 50 and 100 samples per event). Therefore, EPA believes that to show reasonable
accuracy of cellulosic measurements, the path forward with corn kernel fiber will likely require a
representative reference standard with a cellulosic value.
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