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MEMORANDUM DATE: May 30, 2014 TO: Air and Radiation Docket EPA
- HQ
- OAR
- 2014
-0401 SUBJECT: Additional Detail on the Calculation of the Cellulosic Converted Fraction
This memorandum provides additional information about the implications for cellulosic fuel producers processing feedstock materials with an adjusted cellulosic content below EPA’s cellulosic content threshold of 75%
. All cellulosic biofuel processes using biomass feedstocks must employ a process with the demo nstrated capability of converting cellulosic material to biofuel. However, while processes using a feedstock with an adjusted cellulosic content greater than 75% will be allowed to generate cellulosic renewable identification numbers ( RINs ) for the entire fuel volume , processes using a feedstock with an adjusted cellulosic content below 75% will have to apportion different D
-codes to the RINs produced based on the amount of cellulosic fuel produced. In practice, such producers will treat the feedstock mater ial as two separate feedstocks
– one cellulosic feedstock and one non
- cellulosic feedstock
– and allocate RINs using existing regulations for generating fuels using multiple feedstocks . For most producers this apportio nment will be straight
-forward, but fo r producers simultaneously processing cellulosic and non
- cellulosic feedstocks in a single reactor using a biochemical process, the apportionment will be more complicated
– requiring additional record keeping and reporting requirements. The regulatory fram ework EPA established to verify that cellulosic RINs generated by such processes correspond to cellulosic biofuel involves verifying that the calculated converted fractions ( CF ) applied to each portion of the feedstock material (cellulosic or non - cellulosic) is accurate. Establishing the accuracy of these determined CF values requires obtaining reasonably accurate feedstock and process data to perform a mass balance accounting of the various feedstock materials allowing portions of the final fu el to be attributed either to the cellulosic or non
- cellulosic feedstock. This memorandum will explain: 1) why EPA has established separate requirements for various types of processes, 2 ) what types of data can be considered reasonably accurate to form th e basis for calculating cellulosic and non
cellulosic CF values for feedstocks, and 3) how accurate feedstock CF values and existing regulations can be used to apportion RINs for a single type of fuel produced from multiple feedstocks.2

I. Background a. T he cellulosic content threshold, and the implications for feedstocks with lower cellulosic contents EPA is finalizing a cellulosic content threshold for feedstocks to qualify for cellulosic RINs for the total volume of fuel produced. 1 If a feedstock has a n average adjusted cellulosic content of at least 75%, all of the fuel produced from the feedstock is eligible to generate cellulosic RINs, provided that the producer is using a method that can produce fuel from the cellulosic portion of the feedstock. “A djusted cellulosic content” is the percent of organic matter that is cellulosic material (cellulose, hemicellulose , and lignin)
. In other words, the percent cellulosic content is adjusted for the inorganic (ash) content of the feedstock. In cases where two or more feedstocks are processed simultaneously, producers are eligible to generate cellulosic RINs for a percentage of the fuel produced, based on the cellulosic converted fraction and the equations Ta ble 4 to § 80.1426 . The purpose of this Memorandum is to provide details on how the cellulosic converted fraction can be calculated , and examples of methods that could be used to generate the data needed to calculate the cellulosic converted fraction
. b. Registration, reporting and record keeping requirements for thermochemical and biochemical processes EPA is requiring all producers of cellulosic RINs converting cellulosic biomass to complete additional registration requirements. These additional requirements are designed to demonstrate that the process being registered is capable of performing cellulosic biomass conversion, and that the relevant unit process equipment to perform the described conversion to biofuel is both in place and operational. Process technology plays a key role in how much of the final fuel product is actually produced from cellulose, hemicellulose, or lignin. There are two basic processes for converting cellulosic feedstocks into fuel: thermochemical and biochemical. Thermochemical processes mainly consist of pyrolysis
– in which cellu losic biomass is decomposed with temperature to bio
- oils that can be further processed to produce a finished fuel
– and gasification
– in which cellulosic biomass is decomposed to synthesis gas (“syngas”) that with further catalytic processing can produce a finished fuel product. T hermochemical processes typically convert all of the organic components of the feedstock into finished fuel , and t he portion of the finished fuel derived from cellulosic material is proportional to the cellulosic content of the o rganic fraction of the feedstock. Therefore, EPA’s additional registration requirements for thermochemical producers are intended to demonstrate how much of the feedstock is cellulosic, and that the conversion equipment to be used in the process is both in place and functional at the time of registration. By contrast, t he biochemical process requires the release of sugars from biomass and the use of microorganisms to convert sugars into fuels. The amount of cellulosic material converted to fuel will depe nd on the details of the process, such as the types and combinations of enzymes used. Variations

1 See Notice of Final Rulemaking placed in docket EPA
- HQ
- OAR
- 2012
- 0401.3

from process to process will establish different hydrolysis efficiencies for cellulosic and non
- cellulosic carbohydrates meaning that the cellulosic portion of the fuel will not necessarily be proportional to the cellulosic composition of the feedstock. For in situ processes, where cellulosic and non
- cellulosic material are simultaneously hydrolyzed to intermediate sugars or converted to fuel, the registration, reporting, and recordkeeping requirements, as well as the method for determining the cellulosic converted fraction, are different depending on whether the cellulosic biomass is converted by biochemical hydrolysis or by thermochemical treatment. EPA’s addi tional registration and record keeping requirements for in situ biochemical producers of cellulosic fuel are intended to establish and monitor the relative conversion efficiencies for both the cellulosic and non
- cellulosic portions of the feedstock in orde r to ensure that RIN assignments have been made appropriately.

II. Definition of the cellulosic Converted Fraction The EPA defines the converted fraction (CF) to be the average mass percent (typically a yearly average) representing that portion of the feedstock that is converted into renewable fuel . The CF is calculated by dividing the mass of the feedstock that is converted to renewable fuel by the mass of the feedstock that is capable of being converted to fuel
. The cellulosic converted fraction, therefore, is the average mass percent representing that portion of the cellulosic feedstock that is converted into renewable fuel. This calculation is straight forward for producers using a single feedstock that meets the 75 percent adjusted cellulosic content threshold , or mechanically separating the cellulosic and non
- cellulosic portions of a mixed feedstock and processing those portions separately
. H owever it is more complicated to calculate separate CF and cellulosic CF values among in situ biochemical process es because the mass of the resulting fuel corresponding to the non
- cellulosic and cellulosic portions cannot be directly measured
. However, existing EPA regulations detail how RINs can be allocated where multiple feedstocks are being simultaneously process ed. This memo provides more detail on how the formulas in 80.1426(f)(3)(vi) should be used to calculate RINs. III. Description of various process configurations for “in situ” processes Biochemical processes typically employ engineered yeast strains to ferment aqueous sugars into alcohols that can be separated and used as biofuels. Release of sugars from complex carbohydrates is facilitated by enzymatic treatment in a process called hydrolysis . The polymer ic linkages in these carbohydrates can be cellulosic or non - cellulosic, and this distinction determines which enzymatic treatment will result in hydrolyzing the chemical bonds linking the sugars together. In this case, enzymes can be viewed as keys that un
- lock the sugars, and only the proper key will fit a particular chemical bond ’s lock
. Released s ugars will be converted to biofuel in the same manner regardless of whether they were derived from cellulosic or non
- cellulosic carbohydrates . Therefore, hydrolysis is the critical step controlling the fraction of t he finished fuel that can be considered to be derived from cellulosic material. Determining the extent to which the cellulosic portion of a feedstock was hydrolyzed can be quantified in a number of ways, but this validation is more difficult for processes that perform in situ4

hydrolysis of both the cellulosic and non
- cellulosic feedstock portions . This is due to the fact that the fraction of sugars or fuel derived from either type of carbohydrate cannot be measured directly, but rather requires a mass balance accounting to identify the source of the sugars or fuel
. Figure 1 shows a simplified example of a biochemical process performing in situ biochemical hydrolysis, and Figure 2 shows a separated hydrolysis scenario. **Figure 1 –**  **Simplified sche matic of a**  **biochemical biofuel process with in situ**  **hydrolysis**

**Figure 2 –**  **Simplified schematic of a biochemical biofuel process with separated hydrolysis**5

To determine the relative amounts of cellulosic and non
- cellulosic feedstock hydrolyzed , a ma ss balance accounting of the converted feedstock and resulting products and co
- products is required
. There are a number of ways to perform a mass balance accounting for the converted portion of the cellulosic feedstock. I t will be easier for producers using separated hydrolysis because the cellulosic conversion can be quantified directly from the cellulosic hydrolysis unit as indicated by the dashed boundary line in Figure 2 . In this case, complex analysis of the inputs a nd outputs is not required because the aqueous sugars in the hydrolysis unit’s effluent are assumed to be derived from cellulosic carbohydrates. Similarly, if the cellulosic feedstock and resulting intermediates were kept separate from the downstream ferm entation, separations, and refining, then all of the resulting fuel wo uld be assumed cellulosic as well. A direct accounting for the converted cellulosic fraction will allow RINs to be easily attributed. By contrast, in situ processes require a more compl ex mass balance accounting to confirm the portions of the biofuel resulting from cellulosic hydrolysis relative to non
- cellulosic hydrolysis. Assessment methods can be designed in a number of ways, but would typically account for cellulosic conversion by e ither quantifying overall biofuel increases relative to non
- cellulosic treatment, or by directly analyzing the structural make
- up of the process’ feedstock and residual materials, as well as the overall fuel yield, to determine relative feedstock conversio n. EPA is allowing producers to design analytical protocols specific to their process’ configuration as long as the mass balance accounting calculation method is verified by an independent engineer, these calculations can independently identify both a cel lulosic and non
- cellulosic CF, the data used to perform the calculations is regularly reported along with the calculated CF values, and the data itself is collected and reported in scientifically reasonable manner.
IV. Expectations for data collection and analysis

a. General requirements regarding data used to calculate C F EPA is providing clarifying information on the type of methods that can be used to generate data to calculate the cellulosic converted fraction. Data used to calculate the cellulosic CF must be obtained using an analytical method certified by a voluntary consensus standards body (VCSB) , or using a method that would produce reasonabl y accurate results. If a VCSB
- approved method is not used to generate the data required to calculate the cellul osic CF for a given process, then the producer will need to show that the method used is an adequate means of generating the data by providing peer reviewed references to the third party engineer performing the engineering review at registration. An examp le of an appropriate Voluntary Consensus Standards Body test method certified by a voluntary consensus standards body is ASTM E1758, “Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography.” Since this ASTM test method has been adjudicated through the voluntary consensus standards body process we are confident in its use in practice. An example of another type of method would be a non
-voluntary consensus standards body test method such as the National Renewable Energy Laboratory (NREL) Laboratory Analytical Procedure to measure structural6

carbohydrates 2 and is an example of such a methodology that is commonly presented in peer
- reviewed literature. 3 Since this NREL method has not been adjudicated through the voluntary consensus standards body process, the user of these types of non
-VCSB test methods should have the independent third party engineering review certify that the non
-VCSB test method would provide reasonabl y accurate results. These examples of test method analyses quantify the amount of cellulose, hemicellulose, and lignin in biomass. Data from these analyses can be used in a mass balance equation to determine the cellulosic CF. In t he next sections we describe some of the types of methods that can be used to determine the cellulosic fraction of a feedstock, which can be used to calculate the cellulosic CF of the finished fuel. b. Structural Carbohydrate Analysis One method of determining the cellulose and hemicellulose content of biomass is using a High Performance Liquid Chromatography (HPLC) structural carbohydrate analysis, also known as a dietary fiber analysis
. This type of structural carbohydrate analysis has been commonly used by researchers who are interested i n the composition of a feedstock that will be used for biofuel
. 4 For this type of test, acid hydrolysis is used to break down both cellulose and hemicellulose into monomeric sugars ( i.e., cellulose is converted to glucose; hemicellulose is converted to xy lose, galactose, arabinose, and mannose). The concentration s of these sugars are measured using HPLC
. The glucose concentration can be used as a measure of the cellulose concentration in the plant, and the sum of the xylose, galactose, arabinose, and man nose concentrations can be used as a measure of the hemicellulose concentration. The sample can also be analyzed for acetyl content, which is part of hemicellulose. Examples of this type of procedure are the National Renewable Energy Lab (NREL) Laboratory Analytical Procedure , “Determination of Structural Carbohydrates and Lignin in Biomass” and ASTM E1758, “Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography.” Although this type of method has been successfully used to mea sure cellulose and hemicellulose for many feedstocks, an important caveat is that the results obtained are highly dependent on the exact methods used, and small changes to the procedures can have a large effect on the results. 5 In addition, the NREL

2 “Determination of Structural Carbohydrates and Lignin in Biomass,” National Renewable Energy Laboratory. Available at: http://www.nrel.gov/biomass/analytical_procedures.html 3 See e.g . Sluiter, J.B. et al. (2010 ) Compositional Analysis of Lignocellulosic Feedstocks. 1. Review and Description of Methods. Journal of Agricultural and Food Chemistry, 58, 9043
- 9053. Templeton, D.W. et al. (2010 ) Compositional Analysis of Lignocellulosic Fe edstocks. 2. Method Uncertainties. Journal of Agricultural and Food Chemistry, 58, 9054
-9062. 4 E .g. Guo, G. et al., 2009. “Characterization of enzymatic saccharification for acid
- pretreated lignocellulosic materials with different lignin composition.” E nzyme and Microbial Technology, 45:80
- 87; Dahlberg, J. et al., 2011. “Compositional and agronomic evaluation of sorghum biomass as a potential feedstock for renewable fuels.” Journal of Biobased Materials and Bioenergy, 5:1
- 7; Fedenko, J.R. et al. 2013. “B iomass production and composition of perennial grasses grown for bioenergy in a subtropical climate across Florida, USA.” Bioenergy Research, 6:1082
- 1093. 5 For example, using a sample particle size that is smaller than recommended can cause a low bias in measured carbohydrate content. For more details about how procedure changes can affect the results, see the Laboratory Analytical Procedures, “Summative Mass Closure” and “Determination of Structural Carbohydrates and Lignin in Biomass,” National Renewable Energy Laboratory. Available at: http://www.nrel.gov/biomass/analytical_procedures.html7

and ASTM procedures warn that biases may be introduced if the sample has a high protein content, or if extractives have not been removed. Because the HPLC structural carbohydrate analysis only measures cellulose and hemicellulose, a separate method is needed to measure the lignin concentration. A procedure for measuring the lignin concentration is detailed in the NREL method cited above, as well as in ASTM E1721 “Determination of Acid
- Insoluble Residue in Biomass.” Briefly, the acid hydrolysis step th at breaks down the cellulose and hemicellulose also separates lignin into an “acid soluble” portion and an “acid insoluble” portion. The concentration of acid soluble lignin can be measured using a UV
-Visible spectrophotometer. The concentration of acid insoluble lignin can be measured gravimetrically. In addition to the HPLC structural carbohydrate method , near infrared spectroscopy (NIRS) can be used to determine the structural carbohydrate composition , as well as the presence of other components
. 6 N IRS measures the chemical bonds in a biomass sample , which can be used to determine its composition. This method is rapid and non
- destructive . In order for measurements to be made using NIRS, the spectra must be calibrated using other methods, such as a n HPLC compositional analysis.
c. Detergent Fiber Analysis For a determination of the total fiber content of biomass, a neutral detergent fiber (NDF) test can be used. The NDF method dissolves non -fiber components such as proteins, lipids, and sugars. The leftover material is the total fiber, which can be measured gravimetrically. This method is detailed in AOAC 2002.04 Amylase
-Treated Neutral Detergent Fiber in Feeds
. 7 This method is commonly used for determining the forage potential of biomass. Th e NDF m ethod can be used along with an acid detergent fiber (ADF) test and an acid detergent lignin (ADL) test to determine the amount of cellulose, hemicellulose and lignin, rather than just the total fiber content . The ADF method breaks down hemicellulose, so that cellulose and lignin remain. Th e measurement of ADF can be subtracted from the measurement of NDF to determine the concentration of hemicellulose in the sample . The ADL test measures the lignin concentration by breaking down both cellulose and hemicellulose using sulfuric acid and ignition
. The ADL measurement can be subtracted from the ADF measurement to determine cellulose concentration
. The detergent fiber analyses are much simpler than the HPLC structural carbohydrate analysis . However, these two types of methods do not always give the same results. For example, one study show ed that the detergent fiber methods overestimate c ellulose and hemicellulose and underestimate lignin relative to the structural

6 E.g . Sanderson, M.A. et al., 1996. “Compositional analysis of biomass feedstocks by near infrared reflectance spectroscopy.” Biomass and Bioenergy, 11(5):365
- 370; Vogel, K.P. et al., 2011. “Quantifying actual and theoretical ethanol yields for switchgrass strains using NIRS analyses.” Bioenergy Research, 4:96
- 110. 7 See also, Mertens, D.R., 2002. “Gravimetric determination of amylase
-treated neutral detergent fiber i n feeds with refluxing in beakers or crucibles: Collaborative study.” Journal of AOAC International, 85(6):1217
- 1240.8

carbohydrate analysis. 8 Another st udy has shown that NDF and ADF cannot be used to accurately determine glucan and xylan content on an extractives
-free basis. 9 d. Quantification of Starch Content Measurement of the starch content can be used to indirectly determine the cellulosic converted fractio n. 10 To measure the starch content, enzymes are used to convert the starch to maltodextrin and then to glucose. A subsequent reaction forms a dye in proportion to the amount of glucose present. The absorbance of this dye is measured with a spectrophotometer . Standards are used to calibrate the spectrophotometer measurements and determine the starch content of the initial sample
. V. Allocating RINs using the 1426 equations

a. Description of how EPA views cellulosic and non
- cellulo sic components of a feedstock as 2 separate feedstocks for RIN allocation For simplicity, EPA selected to specify that if a single plant’s average adjusted cellulosic content is less than 75%, then the feedstock is considered to be two feedstocks
– specifi cally a cellulosic and non
cellulosic feedstock. This designation allows the equations in Table 4 to 80.1426 to be used to allocate both cellulosic and non
- cellulosic RINs for the resulting mixed batch of fuel. This will require a separate converted fracti on (CF) to be calculated for both the cellulosic and non
- cellulosic fractions. b. Calculation of the cellulosic CF and identification of the volume of D3
- RINs The converted fraction (CF) is a unitless average mass percent representing that portion of the feedstock that is converted int o renewable fuel. This is calculated by dividing the mass of the feedstock that is converted to renewable fuel by the mass of the feedstock that is capable of being converted to fuel . For in situ biofuel producers, this calcul ation will require some type of material balance accounting because the fuel will be a mixture of both cellulosic and non
- cellulosic fuel. T here are several possible combinations of system boundaries and analytical methods that could be used to perform a m ass balance accounting of cellulosic conversion , which would offer the data needed to calculate the corresponding cellulosic biofuel production via an in situ process. Thus, there are different ways to obtain the data to be used to calculate the CF values need to perform RIN allocation
. W e will present a n example of how to use data characterizing feedstock composition , cellulosic conversion and certain process parameters to allocate RINs . In this example we assum e that an in situ process is simultan eously processing corn starch and cellulosic polysaccharides associated with corn kernel fiber to produce ethanol.

8 Dien, B.S., et al., 2006. “Chemical composition and response to dilute
- acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass.” Biomass and Bioenergy, 30:880
-891. 9 Wolfrum, E.J., et al., 2009. “Correlating detergent fiber analysis and dietary fiber analysis data for corn stover collected by NIRS.” Cellulose, 16:577
- 585. 10 e.g. Megazyme Total Starch Assay (Amylglucosidase/alpha
-Amylase Method); AOAC Official Method 996.11, “Starch (Total) in Cereal Products, Amylglucosidase
- alpha
-Amylase Method”; AACC Method 76.13, “Total Starch Assay Procedure (Megazyme Amyloglucosidase/alpha

-Amylase Method) ”9

Data characterizing the feedstock corn composition (Table 1) and process specific data to elucidate the relative starch and cellulosic conve rted fraction (CF) will be required to appropriately allocate RINs. For this example, t he validation protocol for obtaining the cellulosic CF requires performing an analytic process that involves 1) remov ing starch, 2 ) quantifying a n initial l evel of cellu losic polysaccharides prior to hydrolysis, 3) quantify ing a final level of cellulosic polysaccharides following hydrolysis, and 4) identifying the cellulosic ethanol by calculating the difference between the initial and final levels of cellulosic polysacch arides. In this example, the CF is determined to be the identified cellulosic ethanol (Step 4) divided by the theoretical ethanol yield for the total initial amount of cellulosic polysaccharides. Once the amount of ethanol resulting from the cellulosic polysaccharides has been identified, data characterizing the combined feedstock composition can be used to identify the cellulosic portion of the feedstock. An analysis of a representative sample of the feedstock would be used to characterize the corn comp osition (Table 1). The data from these various analytical methods can be used to allocate RINs for the example batch (Table 2)
. **Table 1 –**  **Relevant feedstock characteristics and composition** Corn Composition Kernel Mass (M) , lb wet 61.2 lb/ bu Corn Moisture (m) , % wet 15% Kernel Mass, lb dry 52.0 lb/b u Starch, % of kernel dry 75.0% Cellulosic polysaccharides, % of kernel dry 4.0%
RIN allocation for the cellulosic and non
- cellulosic portions of the biofuel can be performed by applying the RIN volume ( V RIN ) equation.

= ∙ ∙ + Where EV is the equivalence value for the renewable fuel (in this case it is 1 since the fuel is ethanol) , V S is the standardized volume of the renewable fuel produced , and FE is the feedstock energy value
. Note that this equation will be applied separately to calculate the RIN volume for each D
- code. The feedstock energy ( FE) is characterized by the FE equation .

= ∙ ( 1
− ) ∙ ∙ Where M is the mass of the feedstock, m is the feedstock moisture con tent, CF is the converted fraction and E is the energy content of the components of the feedstock converted to fuel (Table 2).10

**Table 2 –**  **Example: assumed**  **inputs , intermediate values, and calculations**  Example in situ RIN Allocation Calculation Assumed feedstock for example (M), lb wet Total standardized volume of fuel (VS ), gal 1,000,000 64,158 Note: 77,000 Btu/gal ethanol Starch Cellulose Comments Feedstock, thousand lb dry / batch 637 .5 34 Account for moisture content and composition (Table 1). Energy Content (E), Btu/lb dry 8,000 8,000 Theoretical feedstock energy assuming complete conversion 11

. Converted Fraction (CF) 0.95 0.35 Calculated using method approved at registration 12

. Feedstock Energy (FE) , thousand Btu 4, 845 , 000 95 , 200 Obtained by multiplying the dry feedstock, E, and CF. RIN Volume (V RIN ), gallons 62 , 922 1, 236 Number of D6 and D3 RINs that would be assigned respectively for this example batch. For the example presented , 1,000,000 pound s of wet corn feedstock was processe d to produce 64,158 gallons of ethanol. B ased on established fe edstock characteristics, the wet feedstock was composed of 637,500 pounds of starch and 34,000 pounds of cellulose on a dry mass basis. By establishing and using the dry mass of the se components for subsequent calculation , the moisture content term in the FE equation simplifies to 1. Multiplying the dry mass of each feedstock by its corre sponding converted fraction ( CF) and energy content ( E) yields the feedstock energy ( FE) . Using the resulting FE values for starch and cellulose respectively, and applying the V RIN equation for allocating RIN volumes to batches with t hese two feedstocks yields the RIN volume for each type of D
- code. Note that since the fuel produced is ethanol, the energy equivalence value ( EV ) is 1. This example shows how information about the wet feedstock mass, total fuel output, feedstock characteristics, and calculated process parameters can be used to allocate separate RINs for an in situ process converting both starch and cellulose to ethanol.

11 This theoretical value will depend on the process’ fermentation and relevant reaction chemistry as well as the processes ability to convert C5 as well as C6 carbohydrates. 12 CF values are typically an annual mass percent average, but for in situ processes ge nerating D3 RINs, a new CF will have to be calculated for every 500,000 cellulosic RINs generated or every year
– whichever comes first. The same CF is used for all RIN allocation between each required update.