Standard Method For Biomass

Chemical Analysis and Testing
Laboratory Analytical Procedures
Table of Contents
(as of 09-23-98)
Issue Procedure
Date: Number:
11-01-94 Standard Method for Determination of Total Solids in Biomass ............................................... LAP-001
08-12-96 Determination of Carbohydrates in Biomass by High Performance

Liquid Chromatography ................................................................................................................. LAP-002
01-30-95 Determination of Acid-Insoluble Lignin in Biomass ................................................................... LAP-003
09-25-96 Determination of Acid-Soluble Lignin in Biomass ...................................................................... LAP-004
04-28-94 Standard Method for Ash in Biomass........................................................................................... LAP-005
08-12-96 Measurement of Cellulase Activities ............................................................................................ LAP-006
05-31-95 Preparation of Dilute-Acid Pretreated Biomass........................................................................... LAP-007
05-31-95 SSF Experimental Protocols: Lignocellulosic Biomass Hydrolysis

and Fermentation............................................................................................................................ LAP-008
08-26-96 Enzymatic Saccharification of Lignocellulosic Biomass ........................................................... LAP-009
04-22-94 Standard Method for the Determination of Extractives in Biomass.......................................... LAP-010
05-05-94 Determination of Ethanol Concentration in Biomass to Ethanol

Fermentation Supernatants by Gas Chromatography................................................................ LAP-011
07-05-94 Standard Test Method for Moisture, Total Solids, and Total Dissolved
Solids in Biomass Slurry and Liquid Process Samples ............................................................. LAP-012
08-26-96 HPLC Analysis of Liquid Fractions of Process Samples for Monomeric

Sugars and Cellobiose ................................................................................................................... LAP-013
08-12-96 Dilute Acid Hydrolysis Procedure for Determination of Total Sugars in

the Liquid Fraction of Process Samples ...................................................................................... LAP-014
09-25-96 HPLC Analysis of Liquid Fractions of Process Samples for Byproducts

and Degradation Products............................................................................................................. LAP-015
09-25-96 Determination of Starch in Biomass Samples by Chemical Solubilization

and Enzymatic Digestion ............................................................................................................... LAP-016
07-31-98 Determination of O-Acyl Groups in Biomass by High Performance

Liquid Chromatography ................................................................................................................. LAP-017
09-23-98 Determination of Insoluble Solids of Pretreated Biomass Material .......................................... LAP-018

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Ethanol Project
Chemical Analysis and Testing Task

Laboratory Analytical LAP-001
Procedure
Procedure Title: Standard Method for Determination
of Total Solids in Biomass
Author: Tina Ehrman Date:

10/28/94
ISSUE DATE: 11/1/94 SUPERSEDES: 8/17/92

Standard Method for Determination of Total Solids in Biomass
Laboratory Analytical Procedure #001
1. Introduction
1.1 Biomass samples are hygroscopic materials and can contain large and varying amounts
of moisture. To be meaningful, the results of chemical analyses of biomass are
typically reported on a dry weight basis. The following procedure describes the method
used to determine the amount of solids (or moisture) present in a solid biomass sample.
1.2 This procedure has been adopted by ASTM as an ASTM Standard Test Method.
2. Scope
2.1 This method is intended to determine the amount of total solids remaining after 105oC
drying of a solid sample.
2.2 All analyses shall be performed according to the guidelines established in the Ethanol
Project Quality Assurance Plan (QAP).
3. References
3.1 NREL CAT Task Laboratory Analytical Procedure #012, "Standard Test Method for
Moisture, Total Solids, and Total Dissolved Solids in Biomass Slurry and Liquid
Process Samples."
3.2 TAPPI Method T210 om-58. 1991. "Weighing, Sampling and Testing Pulp for
Moisture." Technical Association of the Pulp and Paper Industry Standard Methods.
3.3 Vinzant, T.B., L. Ponfick, N.J. Nagle, C.I. Ehrman, J.B. Reynolds, and M.E. Himmel.
1994. "SSF Comparison of Selected Woods From Southern Sawmills." Appl.
Biochem. Biotechnol. 45/46:611-626.
4. Significance and Use
4.1 The total solids content of a biomass sample is the amount of solids remaining after all
volatile matter has been removed by heating the sample at 105EC to constant weight.
Conversely, the moisture content is a measure of the amount of water (and other
components volatilized at 105oC) present in such a sample.
Procedure #001 Page 2 of 7

Issue Date: 11/1/94
Supersedes: 8/17/92
4.2 The results of the chemical analyses of biomass samples are typically reported on a
105oC dry weight basis. The total solids content of a sample is used to convert the
analytical results obtained on another basis to that of a dry weight basis.
5. Apparatus
5.1 Analytical balance, sensitive to 0.1 mg.
5.2 Automated infrared moisture analyzer (such as Denver Instrument Company IR-100
or equivalent) or a convection (drying) oven, with temperature control of 105 ± 3oC.
5.3 Desiccator.
5.4 Aluminum foil weighing dishes.
6. ES&H Considerations and Hazards
6.1 Follow all applicable NREL Laboratory Specific Hygiene Plan guidelines.
7. Sampling, Test Specimens and Test Units
7.1 Test specimens suitable for analysis by this procedure are "as received", air-dried,
milled, or extractive-free biomass solids and the solid fraction of samples generated
during the pretreatment, fractionation, or fermentation of biomass. If the total solids
(or moisture) content of the whole slurry or liquid fraction of these process samples are
to be determined, the CAT Task Laboratory Analytical Procedure #012, "Standard Test
Method for Moisture, Total Solids, and Total Dissolved Solids in Biomass Slurry and
Liquid Process Samples", must be used instead.
7.2 The test specimen shall consist of approximately 2 to 10 g of sample obtained in such
a manner as to ensure that it is representative of the entire lot of material being tested.
7.3 Materials containing a large amount of free sugars or proteins will caramelize or brown
under direct infrared heating elements. Total solids in these materials should be done
by the Convection Oven Procedure.
7.4 This procedure is not suitable for biomass samples that visibly change on heating, such
as unwashed acid-pretreated biomass still containing free acid.

Issue Date: 11/1/94
Supersedes: 8/17/92
8. Convection Oven Procedure
8.1 This method involves drying a sample at 105°C ± 3°C in a convection oven. Each
sample must be run in replicate (duplicates, at minimum).
8.2 Accurately weigh a predried aluminum foil weighing dish to the nearest 0.1 mg.
Record this weight.
8.3 Thoroughly mix the sample and then weigh out 1 to 5 grams, to the nearest 0.1 mg, into
the weighing dish. Record the weight of the sample plus weighing dish.
8.4 Place the sample into a convection oven at 105°C ± 3°C and dry to constant weight
(±0.1% change in the amount of moisture present upon one hour of reheating).
Typically overnight drying is required for very wet samples.
8.5 Remove the sample from the oven and place in a desiccator; cool to room temperature.
8.6 Weigh the dish containing the oven-dried sample to the nearest 0.1 mg and record this
weight.
9. Infrared Moisture Analyzer Procedure
9.1 This method employs an automated infrared moisture analyzer. Each sample should
be run in replicate (duplicates, at minimum).
9.2 Program the automated moisture analyzer for a standby temperature of 95oC, an
analysis temperature of 105oC, and a pre-determined end of analysis criteria of a rate
of weight change that does not exceed 0.05% in one minute.
9.3 Turn on the infrared heating elements. Once the analysis temperature of 105oC has
been reached, allow the instrument to equilibrate at that temperature for 30 minutes.
9.4 Place an aluminum foil weighing dish on the balance pan. For wetter samples, it may
be useful to place a quartz pad in the weighing dish to help disperse the moisture. Shut
the hood of the instrument and wait five minutes to insure that the dish and pad are
completely dry before taring the balance.
9.5 Quickly transfer 1 to 3 grams of the thoroughly mixed sample to the weighing dish.
Spread the sample as evenly as possible over the surface of the weighing dish.

Issue Date: 11/1/94
Supersedes: 8/17/92
9.6 As soon as the instrument balance stabilizes, shut the hood of the instrument and
proceed with the analysis, following the instructions in the instrument operation
manual.
9.7 Once the sample has been dried to constant weight, as determined by the programmed
analysis parameters, the analysis will be automatically terminated by the instrument.
10. Calculations
10.1 Calculate the percent total solids on a 105oC dry weight basis as follows (the automated
moisture analyzer will provide the calculated value as part of the instrument printout):
weight dried sample plus dish - weight dish

% Total solids = x 100
weight sample as received
10.2 If desired, the percent moisture can also be calculated:
(weight dried sample plus dish - weight dish)

% Moisture = [1 - ] x 100
11. Report
11.1 Report the result as percent total solids (or percent moisture) to two decimal places, and
cite the basis used in the calculations.
11.2 For replicate analyses of the same sample, report the average, standard deviation, and
%RPD.
12. Precision and Bias
12.1 Convection Oven Procedure: Data obtained by replicate testing of hybrid poplar in one
laboratory gave a standard deviation in moisture content of 0.19% and a CV% of
0.20%. Replicate testing of sodium tartrate gave a standard deviation in total solids of
0.21% and a CV% of 0.23%.

Issue Date: 11/1/94
Supersedes: 8/17/92
12.2 Infrared Moisture Analyzer Procedure: Data obtained by replicate testing of hybrid
poplar in one laboratory gave a standard deviation in moisture content of 0.20% and
a CV% of 0.21%. Replicate testing of sodium tartrate gave a standard deviation in total
solids of 0.58% and a CV of 0.60%.
12.3 An inherent error in any moisture determination involving drying of the sample is that
volatile substances other than water are removed from the sample during drying.
13. Quality Control
13.1 Reported significant figures: Report the percent total solids (or percent moisture) to
two decimal places.
13.2 Replicates: At minimum, all samples and the method verification standard are to be
analyzed in duplicate.
13.3 Blank: This gravimetric analysis utilizes a balance blank with every batch of samples,
consisting of a weighing dish passed through all steps of the procedure. The difference
in weight must be less than the equivalent of a 0.5% error.
13.4 Relative percent difference criteria: For the infrared drying method the maximum
%RPD for duplicate analysis of a sample is 4.0%. For the convection oven method the
maximum %RPD is 1.1%. If the stated %RPD is exceeded, the sample must be rerun.
13.5 Method verification standard: A method verification standard must be run in duplicate
with every batch. Sodium tartrate is a suitable material for use as a method verification
standard, since the moisture content of this material is not greatly affected by its storage
conditions. The published "loss on drying" for sodium tartrate is 15.62% (84.38% total
solids).
13.6 Calibration verification standard: Not applicable.
13.7 Sample size: A minimum of two grams of sample are required for duplicate analyses.
If there is insufficient sample, the result will be flagged and the lack of precision will
be noted.
13.8 Sample storage: Samples shall be stored in an airtight container. Process samples and
high-moisture-content feedstock samples must be refrigerated. Every effort shall be
made to ensure that a representative aliquot is taken for analysis.
13.9 Standard storage: Not applicable.

Issue Date: 11/1/94
Supersedes: 8/17/92
13.10 Standard preparation: Not applicable.
13.11 Definition of a batch: Any number of samples which are analyzed and recorded
together. The maximum size of a batch would be limited by the equipment constraints.
A batch cannot be larger than what is practical with the equipment.
13.12 Control charts: The result of each replicate analysis of the method verification
standard is recorded along with the average, %RPD, and a laboratory book/page
reference. The average value obtained for each analysis of the method verification
standards is to be control charted.
13.13 Other: Biomass can rapidly gain or lose moisture when in contact with air. During the
weighing steps, minimize the amount of time the sample is exposed to the air.
Page 7 of 7
Procedure #001
Issue Date: 11/1/94
Supersedes: 8/17/92
Ethanol Project

Procedure
Determination of Carbohydrates in Biomass by High
Procedure Title: Performance Liquid Chromatography
Author: Raymond Ruiz and Tina Ehrman Date: 2-14-96

ISSUE DATE: 8-12-96 SUPERSEDES: 1-30-95
Determination of Carbohydrates in Biomass by High Performance Liquid Chromatography
1. Introduction
1.1 The carbohydrates making up a major portion of biomass samples are polysaccharides composed
primarily of glucose, xylose, arabinose, galactose, and mannose subunits. The polysaccharides
present in a biomass sample can be hydrolyzed to their component sugar monomers by sulfuric
acid in a two-stage hydrolysis process. The sample can then be quantified by ion-moderated
partition HPLC.
1.2 This procedure has been adopted by ASTM as the Standard Test Method for Determination of
Carbohydrates in Biomass by High Performance Liquid Chromatography, E1758-95.
2. Scope
2.1 This method covers the determination of carbohydrates, expressed as the percent of each sugar
present in a hydrolyzed biomass sample. The sample is taken through a primary 72% sulfuric
acid hydrolysis, followed by a secondary dilute-acid hydrolysis.
2.2 Sample material suitable for this procedure include hard and soft woods, herbaceous materials
(such as switchgrass and sericea), agricultural residues (such as corn stover, wheat straw, and
bagasse), waste-paper (such as office waste, boxboard, and newsprint), washed acid- and
alkaline-pretreated biomass, and the solid fraction of fermentation residues. All results are
reported relative to the 105°C oven-dried weight of the sample. In the case of extracted
materials, the results may also be reported on an extractives-free basis.
2.3 All analyses shall be performed according to the guidelines established in the Ethanol Project
Quality Assurance Plan (QAP).
3. References
3.1 Moore, W.E., and D.B. Johnson. 1967. Procedures for the Chemical Analysis of Wood and
Wood Products. Madison, WI: U.S. Forest Products Laboratory, U.S. Department of
Agriculture.
3.2 Ethanol Project Laboratory Analytical Procedure #001, "Standard Method for the Determination
of Total Solids in Biomass".
3.3 Ethanol Project Laboratory Analytical Procedure #003, "Determination of Acid-Insoluble Lignin
in Biomass".
3.4 NREL Ethanol Project Laboratory Analytical Procedure #004, "Determination of Acid-Soluble
Lignin in Biomass".

Issue Date: 8/12/96
Supersedes: 1/30/95
3.5 NREL Ethanol Project Laboratory Analytical Procedure #010, "Standard Method for the
Determination of Extractives in Biomass".
3.6 TAPPI Test Method T264 om-88, "Preparation of Wood For Chemical Analysis." In Tappi Test
Methods. Atlanta, GA: Technical Association of the Pulp and Paper Industry.
3.7 Vinzant, T.B., L. Ponfick, N.J. Nagle, C.I. Ehrman, J.B. Reynolds, and M.E. Himmel. 1994.
"SSF Comparison of Selected Woods From Southern Sawmills." Appl. Biochem. Biotechnol.,
45/46:611-626.
4. Terminology
4.1 Prepared Biomass - Biomass that has been prepared by lyophilization, oven drying, air drying,
and in some instances by extraction, to reduce the moisture content of the sample so it is
suitable for carbohydrate analysis.
4.2 Oven-Dried Weight - The moisture-free weight of a biomass sample as determined by LAP-001,
"Standard Method for Determination of Total Solids in Biomass".
5.1 The percent sugar content is used in conjunction with other assays to determine the total
composition of biomass samples.
6. Interferences
6.1 Samples with high protein content may result in percent sugar values biased low, as a
consequence of protein binding with some of the monosaccharides.
6.2 Test specimens not suitable for analysis by this procedure include acid- and alkaline-pretreated
biomass samples that have not been washed. Unwashed pretreated biomass samples containing
free acid or alkali may change visibly on heating.
7. Apparatus
7.1 Hewlett Packard Model 1090 HPLC, or equivalent, with refractive index detector.
7.2 HPLC columns, BioRad Aminex7 HPX-87C and/or Aminex7 HPX-87P (or equivalent).
7.3 Guard columns, cartridges appropriate for the column used.
Note: Deashing guard column cartridges from BioRad, of the ionic form H+/CO3%,
are an option when using an HPX-87P column. These cartridges have been found
to be effective in eliminating baseline ramping.
7.4 Analytical balance readable to 0.1 mg.

Issue Date: 8/12/96
Supersedes: 1/30/95
7.5 Convection ovens with temperature control to 45 ± 3°C and 105 ± 3°C.
7.6 Autoclave capable of maintaining 121 ± 3°C.
7.7 Water bath set at 30 ± 3°C.
7.8 Desiccator containing anhydrous calcium sulfate.
8. Reagents and Materials
8.1 Reagents
8.1.1 High purity sugars for standards (98%+) - two sets of glucose, xylose, galactose,
arabinose,and mannose from different lots or manufacturers.
8.1.2 72% w/w H2SO4 (12.00 ± 0.02 M or specific gravity 1.6389 at 15.6 °C /15.6°C).
8.1.3 Calcium carbonate, ACS reagent grade.
8.1.4 Water, 18 megohm deionized.
8.2 Materials
8.2.1 Glass test tubes, 16x100 mm.
8.2.2 125 mL glass serum bottles, crimp top style, with rubber stoppers and aluminum seals
to fit.
8.2.3 pH paper, suitable to cover the pH range of 4 to 7.
8.2.4 Disposable nylon syringe filters, 0.2 µm.
8.2.5 Disposable syringes, 3 mL.
8.2.6 Autosampler vials, with crimp top seals to fit.
8.2.7 Erlenmeyer flasks, 50 mL.
9.2 72% H2SO4 is very corrosive and must be handled carefully.
9.3 Use caution when handling hot glass bottles after the autoclave step, as they may have become
pressurized.

Issue Date: 8/12/96
Supersedes: 1/30/95
10.1 Test specimens suitable for analysis by this procedure are as follows:
- biomass feedstocks, dried and reduced in particle size, if necessary.
- pretreated biomass, washed free of any residual acid or alkali.
- the solids fraction of fermentation residues.
10.2 The sample must not contain particles larger than 1 mm in diameter. If milling is required to
reduce the particle size of the test specimen, a laboratory mill equipped with a 40 mesh (or
smaller) screen should be used.
10.3 The total solids content of the "as received" test specimen (prior to any drying or extraction
steps) must be determined by LAP-001 in parallel with the carbohydrate analysis. Record this
value as %Tas received.
10.4 Material with a total solids content less than 85%, on a 105°C dry weight basis, will require
drying by lyophilization, oven drying, or air drying prior to milling or analysis. The amount of
moisture lost as a result of the preparation procedure must be determined. This moisture content
is used to calculate the total solids content of the sample based on its preparation and is recorded
as %Tprep. This value is used to correct the weight of the prepped material used in the
carbohydrate analysis, as described in the calculations section. The prepared sample should be
stored in a manner to ensure its moisture content does not change prior to analysis.
Note: Preparing samples for analysis by oven drying can produce hard chunks of
material. This material must then be milled to reduce the size of the large pieces to
less then 1 mm in diameter. The sample is then redried prior to testing.
10.5 Some samples may require extraction prior to analysis, to remove components that may interfere
with the analysis. LAP-010, "Standard Method for the Determination of Extractives in
Biomass", is used to prepare an extractives-free sample with a moisture content suitable for
carbohydrate analysis. As part of this procedure, the percent extractives in the prepared sample,
on a 105°C dry weight basis, is determined. This value, recorded as % extractives, can be used
to convert the % sugar reported on a extractives-free basis to an as received (whole sample)
basis.
10.6 The test specimen shall consist of approximately 0.3 g of sample. The test specimen shall be
obtained in such a manner to ensure that it is representative of the entire lot of material being
tested.
11. Procedure
11.1 This procedure is suitable for air-dried, lyophilized, and extracted biomass samples, as well as
for samples that have been oven dried at a temperature of 45°C or less. It is not suitable for
samples that have been dried at a temperature exceeding 45°C.

Issue Date: 8/12/96
Supersedes: 1/30/95
Note: The total solids content of the original sample, %Tas received, must be determined
using LAP-001, prior to any preparatory steps. The total solids content of the sample
based on its preparation, %Tprep , must also be known.
11.2 Determine the total solids content of the prepared or extractives-free biomass sample by LAP-
001 and record this value as %Tfinal .
Note: Samples for total solids determination (LAP-001) must be weighed out at the
same time as the samples for the carbohydrate determination. If this is done later, it
can introduce an error in the calculation because ground biomass can rapidly gain or
lose moisture when exposed to the atmosphere.
11.3 Weigh 0.3 ± 0.01 g of the prepared or extractives-free sample to the nearest 0.1 mg and place
in a 16x100 mm test tube. Record as W1, the initial sample weight in grams. Each sample must
be run in duplicate, at minimum.
11.4 Add 3.00 ± 0.01 mL (4.92 ± 0.01 g) of 72% H2SO4 and use a glass stirring rod to mix for 1
minute, or until the sample is thoroughly wetted.
11.5 Place the test tube in the water bath set at 30 ± 1°C and hydrolyze for 2 hours.
11.6 Stir the sample every 15 minutes to assure complete mixing and wetting.
11.7 Weigh out 0.3 ± 0.01 g of each high purity sugar (predried at 45°C) to the nearest 0.1 mg, and
place each in its own 16x100 mm glass test tube. Add acid, hydrolyze, and stir these sugars as
described in the previous three steps. These sugar recovery standards (SRS) will be taken
through the remaining steps in the procedure in parallel with the samples. The calculated
recovery of the SRSs will be used to correct for losses due to the destruction of sugars during
the hydrolysis process. It may be useful to run selected SRSs in duplicate, particularly if
specific sugars are deemed critical.
11.8 Prepare a method verification standard (MVS) by weighing out 0.3 ± 0.01 g of a well
characterized standard material suitable for analysis. Add acid, hydrolyze, and stir the MVS as
was done with the samples and SRSs (see 11.4-11.6 above). This MVS will be taken through
the remaining steps in the procedure in parallel with the samples and the SRSs, and is used to
test the reproducibility of the method as a whole.
Note: A suitable method verification standard, Populus deltoides, may be obtained

from NIST (research material #8492).
11.9 Upon completion of the two hour hydrolysis step, transfer each hydrolyzate to its own serum
bottle and dilute to a 4% acid concentration by adding 84.00 ± 0.04 mL deionized water. Be
careful to transfer all residual solids along with the hydrolysis liquor. The total weight added
to the tared bottle is 89.22 g (0.3 g sample, 4.92 g 72% H2SO4, and 84.00 g deionized water).
Since the specific gravity of the 4% acid solution is 1.0250 g/mL, the total volume of solution,
VF , is 87.0 mL.
11.10 Stopper each of the bottles and crimp aluminum seals into place.

Issue Date: 8/12/96
Supersedes: 1/30/95
11.11 Set the autoclave to a liquid cycle to prevent loss of sample from the bottle in the event of a
loose crimp seal. Autoclave the samples in their sealed bottles for 1 hour at 121 ± 3°C.
11.12 After completion of the autoclave cycle, allow the samples to cool for about 20 minutes at room
temperature before removing the seals and stoppers.
11.13 These autoclaved solutions may also be used for the determination of acid-insoluble residue
and/or acid-soluble lignin, in parallel with this carbohydrate determination.
Note: If acid-insoluble lignin and/or acid-soluble lignin determinations are to be

conducted on a sample, the residual solids must be collected by filtering the
hydrolyzate through an ashed and weighed filtering crucible prior to proceeding with
the carbohydrate determination. Refer to LAP-003, "Determination of Acid-
Insoluble Lignin in Biomass", for details. If an acid-soluble lignin determination is
to be conducted, a portion of the filtrate must be reserved for analysis. Acid-soluble
lignin should be analyzed within 24 hours, preferably within 6 hours of hydrolysis.
Refer to the procedure "Determination of Acid-Soluble Lignin in Biomass" (LAP-
004) for details.
11.14 Transfer 20 mL aliquots of each hydrolyzate, or filtrate, to 50 mL Erlenmeyer flasks.
11.15 Neutralize with calcium carbonate to a pH between 5 and 6. Do not over-neutralize. Add the
calcium carbonate slowly with frequent swirling to avoid problems with foaming. Monitor the
pH of the solution with pH paper to avoid over-neutralization.
11.16 Filter the neutralized hydrolyzate using a 3 mL syringe with a 0.2 µm filter attached. One
portion of the hydrolyzate should be filtered directly into a sealable test tube for storage. A
second portion should be filtered directly into an autosampler vial if the hydrolyzate is to be
analyzed without dilution. If the concentration of any of the analytes is expected to exceed the
validated linear range of the analysis, dilute the hydrolyzate as required and filter into an
autosampler vial for analysis.
Note: It is advisable to determine the initial glucose concentration of the sample

using an alternative technique, such as a YSI glucose analyzer, in order to predict
whether or not the glucose in the sample will fall within the linear range of the
analysis.
11.17 The portion of the neutralized hydrolyzate filtered into the test tube should be securely sealed,
labeled, placed in the refrigerator, and reserved in case a repeat analysis is required. The sample
should be stored for no longer than two weeks.
11.18 Prepare a series of sugar calibration standards in deionized water at concentrations appropriate

Issue Date: 8/12/96
Supersedes: 1/30/95
for creating a calibration curve for each sugar of interest. A suggested scheme for the HPX-87C
column is to prepare a set of multi-component standards containing glucose, xylose, and
arabinose in the range of 0.2 -12.0 mg/mL. For the HPX-87P column, galactose, and mannose
should be included as additional components in the standards. Extending the range of the
calibration curves beyond 12.0 mg/mL will require validation.
11.19 Prepare an independent calibration verification standard (CVS) for each set of calibration
standards, using sugars obtained from a source other than that used in preparing the calibration
standards. The CVS must contain precisely known amounts of each sugar contained in the
calibration standards, at a concentration that falls in the middle of the validated range of the
calibration curve. The CVS is to be analyzed after each calibration curve and at regular
intervals in the HPLC sequence, bracketing groups of samples. The CVS is used to verify the
quality of the calibration curve(s) throughout the HPLC run.
11.20 Analyze the calibration standards, the CVS, the samples, the SRSs, and the MVS by HPLC
using a Biorad Aminex7 HPX-87C or HPX-87P column for glucose, xylose, and arabinose. If
mannose and galactose are also to be determined, a Biorad Aminex7 HPX-87P column must
be used instead. For many analyses, it is useful to run the same samples on both columns and
compare the results. The following instrumental conditions are used for both the HPX-87C and
the HPX-87P columns:
Sample volume: 50 µL.

Eluant: 0.2 µm filtered and degassed, deionized water.
Flow rate: 0.6 mL/min.
Column temperature: 85°C.
Detector: refractive index.
Run time: 20 minutes data collection plus a 15 minute post-run.
12. Calculations
12.1 Create a calibration curve by linear regression analysis for each sugar to be quantified. From
these curves, determine the concentration in mg/mL of the sugars present in each solution
analyzed by HPLC.
12.2 Calculate the amount of sugar recovered from each SRS taken through the two-stage hydrolysis.
This will give an estimate of the amount of each individual sugar destroyed during the
hydrolysis procedure.
% R srs = C 2 x 100%
C1

Issue Date: 8/12/96
Supersedes: 1/30/95
Where: %R srs = % recovery of sugar recovery standard
C1 = known concentration of sugar recovery standard before hydrolysis, in
mg/mL
C2 = concentration of sugar recovery standard detected by HPLC after
hydrolysis, in mg/mL
12.3 Use the percent recovery of the appropriate sugar recovery standard to correct sugar
concentration values (in mg/mL) obtained from HPLC for each sugar detected in the hydrolyzed
sample.
% R srs
C corr = C spl ÷
100%
Where:
Ccorr = concentration of sugar in hydrolyzed sample corrected for hydrolysis,
in mg/mL
Cspl = concentration of sugar detected in the hydrolyzed sample by HPLC,
in mg/mL
%Rsrs = % recovery of sugar recovery of standard, as determined in the
previous step
12.4 For lyophilized, air dried, or oven dried samples, or for samples requiring no preparation,
calculate the percentage of each sugar present in the sample on an as received 105°C dry weight
basis as follows:
1g 1g
C corr x xV F C corr x xV F
1000 mg 1000 mg
% Sugar = x 100% = x 100%
% T as received % T final
W1 x W1 x
% T prep 100%
Where: W1 = initial weight of sample, in grams

VF = volume of filtrate, 87.0 mL
Ccorr = concentration of sugar in hydrolyzed sample corrected for loss on hydrolysis,
is determined in previous step, in mg/mL
%Tas received = % total solids content of the original sample (prior to any preparation
steps) on a 105°C dry weight basis, as determined by the LAP-001
%Tprep = % total solids content of the sample as determined during the preparation
of the sample for analysis (by lyophilization, oven-drying, or air drying)
%Tfinal = % total solids content of the prepared sample used in this carbohydrate
analysis, on a 105°C dry weight basis, as determined by the LAP-001
Note: If the sample used in the analysis required no preparation (analyzed as

received), then %Tprep = 100% and %Tfinal = %Tas received.

Issue Date: 8/12/96
Supersedes: 1/30/95
12.5 If the biomass was prepared according to the "Standard Method for the Determination of
Ethanol Extractives in Biomass" (LAP-010), first calculate the percentage of each sugar present
on an extractives-free 105EC dry weight basis and then correct this value to an as received
(whole sample) 105°C dry weight basis.
12.5.1
Calculate the percentage of each
1 g sugar on an extractives-free basis as follows:
C corr x x VF
1000 mg
% Sugar extractives - free = x 100%
% T final
W1 x
100%
Where: Ccorr = concentration of sugar in hydrolyzed sample corrected for loss on

hydrolysis, as determined above, in mg/mL
W1 = initial weight of extracted sample, in grams
%Tfinal = % total solids content of the prepared sample used in this
carbohydrate analysis (in this case, extracted sample), on a 105°C dry
weight basis, as determined by the LAP-001
12.5.2 Correct the % sugar value on an extractives-free basis, calculated above, to an as

received (whole sample) 105°C dry weight basis as follows:
(100% - % extractives)
% Sugar whole sample = % Sugar extractives - free x
100%
Where:
% Sugarextractives-free = % sugar on an extractives-free 105°C dry weight basis,
as determined in the previous step
% extractives = % extractives in the extracted sample as described in the
Standard Method for the Determination of Extractives in Biomass
13. Report
13.1 Report the percent sugar present in the sample, to two decimal places, on a whole sample 105°C
dry weight basis or on an extractives-free basis. Cite the basis used in the report.
13.2 For replicate analyses of the same sample, report the average, standard deviation, and relative
percent difference (RPD).

Issue Date: 8/12/96
Supersedes: 1/30/95
14.1 Data obtained by replicate testing of a hybrid poplar in one laboratory, using a HPX-87P
column, gave a standard deviation in glucose content of 1.90% and a CV of 3.95%.
14.2 Data obtained by replicate testing of an extractives-free hybrid poplar sample in five different
laboratories gave a standard deviation of 1.90% and a CV of 4.0%.
15. Quality Control
15.1 Reported significant figures: Report the percentage of each sugar present in the hydrolyzed
sample to two decimal places, on a whole sample 105°C dry weight basis or on an extractives-
free basis. Cite the basis used in the calculation.
15.2 Replicates: At minimum, all samples and the method verification standard are to be analyzed
in duplicate.
15.3 Blank: The only requirement is a reagent blank, which starts out as an empty 16x100 mm test
tube (ie, no sample) which is taken through all the procedural steps.
15.4 Relative percent difference criteria: The RPD for glucose must be less than 6.1%. If the RPD
is too large, the sample must rerun.
15.5 Method verification standard: A method verification standard must be run in duplicate with
every batch. This method utilizes a well characterized standard material suitable for analysis.
For example, NIST 8492 (Populus deltoides) is used as the MVS in carbohydrate analysis of
hardwoods.
15.6 Calibration verification standard: Calibration verification standards shall be independently

prepared and analyzed as described in section 11.19 of this procedure.
15.7 Sample size: A minimum of 0.6 grams of sample (on a dry weight basis) are required for
duplicate analyses. If there is insufficient sample, the result will be flagged and the lack of
precision will be noted.
15.8 Sample storage: Samples shall be stored in an airtight container and refrigerated.
15.9 Standard storage: Standards should be kept frozen in airtight vials or test tubes. Vortex the
standards vigorously upon thawing to ensure thorough mixing.
15.10 Standard preparation: Standards are prepared according to section 11.18 of this procedure.
15.11 Definition of a batch: Any number of samples which are analyzed and recorded together. The
maximum size of a batch would be limited by the equipment constraints. A batch cannot be
larger than what is practical with the equipment.
15.12 Control charts: The result of each replicate analysis of the method verification standard is
recorded along with the average, RPD, and a laboratory book/page reference. The average
value obtained for each analysis of the method verification standards is to be control charted.

Issue Date: 8/12/96
Supersedes: 1/30/95
Ethanol Project

Procedure
Determination of Acid-Insoluble Lignin in Biomass
Procedure Title:
Author: David Templeton and Tina Ehrman Date: 1-19-95
ISSUE DATE:1-30-95 SUPERSEDES: 8/19/92
Determination of Acid-Insoluble Lignin in Biomass
1. Introduction
1.1 Biomass is composed largely of cellulose, a polymer of glucose; hemicellulose, a

complex polymer of which the main chain consists primarily of xylans or
glucomannans; and lignin, a complex phenolic polymer. Unlike the other cell wall
components of biomass, the lignin is mostly insoluble in mineral acids. For this
reason, lignin can be analyzed gravimetrically after hydrolyzing the cellulose and
hemicellulose fractions with sulfuric acid.
1.2 This method contains two different procedures for determining acid-insoluble lignin.
Both approaches have been shown to give equivalent results. Procedure A presents
an approach where the acid-insoluble lignin procedure also generates the solutions
required for total carbohydrate and acid-soluble lignin determinations, thereby making
possible the "summative" analysis of the same sample. Procedure B is a modification
of the classic "Klason lignin" determination. Although the filtrate generated from this
procedure can be used to determine acid-soluble lignin, total carbohydrates should be
determined on a completely separate sample.
1.3 This procedure has been adopted by ASTM as an ASTM Standard Test Method.
2. Scope
2.1 This test method covers the determination of acid-insoluble lignin of hard and soft
woods, herbaceous materials (such as switchgrass and sericea), agricultural residues
(such as corn stover, wheat straw, and bagasse), wastepaper (such as office waste,
boxboard, and newsprint), acid and alkaline pretreated biomass, and the solid fraction
of fermentation residues. All results are reported relative to the 105oC oven-dried
weight of the sample. In the case of extracted materials, the results may also be
reported on an extractives-free basis.
2.2 The residue collected contains the acid-insoluble lignin and any condensed proteins
from the original sample. An independent nitrogen analysis would be required to
determine the acid-insoluble lignin content
separate from the condensed protein fraction and is outside the scope of this procedure.
3. References

Issue Date: 1/19/95
Supersedes: 8/19/92
3.1 Ehrman, C.I., and M.E. Himmel. 1994. "Simultaneous Saccharification and
Fermentation of Pretreated Biomass: Improving Mass Balance Closure."
Biotechnology Techniques, 8(2):99-104.
3.2 Moore, W.E., and D.B. Johnson. 1967. Procedures for the Chemical Analysis of Wood
and Wood Products. Madison, WI: U.S. Forest Products Laboratory, U.S. Department
of Agriculture.
3.3 NREL CAT Task Laboratory Analytical Procedure #001, "Standard Method for the
Determination of Total Solids in Biomass".
3.4 NREL CAT Task Laboratory Analytical Procedure #002, "Two Stage Sulfuric Acid
Hydrolysis for Determination of Carbohydrates".
3.5 NREL CAT Task Laboratory Analytical Procedure #004, "Determination of Acid-
Soluble Lignin in Biomass".
3.6 NREL CAT Task Laboratory Analytical Procedure #010, "Standard Method for the
Determination of Extractives in Biomass".
3.7 TAPPI Test Method T222 om-88, "Acid-Insoluble Lignin in Wood and Pulp." In Tappi
Test Methods. Atlanta, GA: Technical Association of the Pulp and Paper Industry.
3.8 TAPPI Test Method T264 om-88, "Preparation of Wood For Chemical Analysis." In
Tappi Test Methods. Atlanta, GA: Technical Association of the Pulp and Paper
Industry.
3.9 Vinzant, T.B., L. Ponfick, N.J. Nagle, C.I. Ehrman, J.B. Reynolds, and M.E. Himmel.
1994. "SSF Comparison of Selected Woods From Southern Sawmills." Appl.
Biochem. Biotechnol., 45/46:611-626.
4. Terminology
3.10 Acid-insoluble lignin is defined to be the residue, corrected for acid-insoluble ash,
retained on a medium porosity filter crucible after the primary 72% and secondary 4%
H2SO4 hydrolysis steps described in this procedure.
3.11 The acid-insoluble lignin content is used in conjunction with other assays to determine
the total composition of biomass samples.

Issue Date: 1/19/95
Supersedes: 8/19/92
6. Interferences
3.12 The results of acid-insoluble lignin analysis are affected by incomplete hydrolysis of
biomass. Unless the sample is hydrolyzed completely, the results will be biased high.
Take care to mix the acid/biomass slurry thoroughly at the beginning and periodically
throughout the concentrated acid hydrolysis.
3.13 The results of acid-insoluble lignin analysis are affected by the timing of the acid
digestion steps. The insoluble lignin will slowly dissolve into solution in an
irreproducible fashion. The timing within this procedure must be followed closely.
3.14 Some proteinaceous materials can also form acid-insoluble substances that are collected
with acid-insoluble lignin.
7. Apparatus
3.16 Convection oven with temperature control of 105 ± 3oC.
3.17 Muffle furnace: an electric furnace is recommended for igniting the sample. The
furnace should be fitted with an indicating pyrometer or thermocouple, so that the
required temperature of 575 ± 25oC can be maintained.
3.18 Autoclave capable of maintaining 121 ± 3oC (Procedure A) or heating manifold

equipped with reflux condensers with 24/40 ground glass joints (Procedure B).
3.19 Water bath set at 30 ± 1oC (Procedure A).
3.20 Filtration set-up including vacuum source and vacuum adapters for crucibles.
3.21 Desiccator containing anhydrous calcium sulfate.
3.22 Reagents
3.22.1 72% w/w H2SO4 (12.00 ± 0.02 M or specific gravity 1.6389 at

15.6oC/15.6oC).

Issue Date: 1/19/95
Supersedes: 8/19/92
3.23 Materials
3.23.1 Glass test tubes, 16x100 mm (Procedure A) or 20x150 mm (ProcedureB).
3.23.2 125 mL glass serum bottles, crimp top style, with rubber stoppers and
aluminum seals to fit (Procedure A).
3.23.3 Erlenmeyer flask, 1000 mL, with 24/40 ground glass joint (Procedure B).
3.23.4 Filtration flask, 250 mL (Procedure A) or 1000 mL (Procedure B).
3.23.5 30 mL (Procedure A) or 50 mL (Procedure B) glass filtering crucible, medium

porosity, nominal maximum pore size of 10 µm.
3.26 Use caution when handling glass bottles after the autoclave step in Procedure A, as they
may have become pressurized.
10. Sampling, Test Specimens, and Test Units
- biomass feedstocks, dried and reduced in particle size if necessary,

- pretreated biomass, washed free of any residual acid or alkali,
3.28 The sample must not contain particles larger than 1 mm in diameter. If milling is
required to reduce the particle size of the test specimen, a laboratory mill equipped with
a 40 mesh (or smaller) screen should be used.
3.29 The total solids content of the "as received" test specimen (prior to any drying or
extraction steps) must be determined by Laboratory Analytical Procedure #001,
"Determination of Total Solids in Biomass", in parallel with the lignin analysis.
Record this value as Tas received.

Issue Date: 1/19/95
Supersedes: 8/19/92
3.30 Material with a total solids content less than 85%, on a 105oC dry weight basis, will
require drying by lyophilization, oven drying, or air drying prior to milling or analysis.
The amount of moisture lost as a result of the preparation procedure must be
determined. This moisture content is used to calculate the total solids content of the
sample based on its preparation and is recorded as Tprep. This value is used to correct
the weight of the prepped material used in the lignin analysis, as described in the
calculations section. The prepared sample should be stored in a manner to ensure its
moisture content does not change prior to analysis.
Note: Preparing samples for analysis by oven drying can produce hard
chunks of material. This material must then be milled to reduce the size of
the large pieces to less then 1 mm in diameter. The sample is then redried
prior to testing.
3.31 Some samples may require extraction prior to analysis, to remove components that may
interfere with the analysis. Laboratory Analytical Procedure #010, "Standard Method
for the Determination of Extractives in Biomass", is used to prepare extractives-free
sample with a moisture content suitable for lignin analysis. As part of this procedure,
the percent extractives in the prepared sample, on a 105oC dry weight basis, is
determined. This value, recorded as % extractives, is used to convert the % lignin
reported on a extractives-free basis to an as received (whole sample) basis.
3.32 The test specimen shall consist of approximately 0.3 g of sample for Procedure A or
approximately 1.0 g of sample for Procedure B. The test specimen shall be obtained
in such a manner to ensure that it is representative of the entire lot of material being
tested.
11. Procedure A - Summative Analysis
3.33 This procedure is suitable for air-dried, lyophilized, and extracted biomass samples, as
well as for samples that have been oven dried at a temperature of 45oC or less. It is not
suitable for samples that have been dried at a temperature exceeding 45oC.
Note: The total solids content of the original sample, Tas received, as well as the
total solids content determined as the sample is prepared, Tprep, must be
known.
3.34 Individually label the crucibles needed for analysis, and ignite them at 575 " 25oC to
achieve a constant weight of " 0.3 mg. Store the ignited crucibles in a desiccator until
needed.

Issue Date: 1/19/95
Supersedes: 8/19/92
Note: In order to determine the absolute amounts of acid-insoluble residue
and acid-insoluble ash, for quality control purposes, it is useful to
weigh and record the ignited crucible to the nearest 0.1 mg.
3.35 Weigh 0.3 ± 0.01 g prepared sample to the nearest 0.1 mg and place in a 16x100 mm
test tube. Record as W1, the initial sample weight. Each sample must be run in
duplicate, at minimum.
3.36 Samples for total solids determination (LAP-001) must be weighed out at the same time
as the samples for the acid-insoluble lignin determination. If this is done later, it can
introduce an error in the calculation because ground biomass can rapidly gain or lose
moisture when exposed to the atmosphere. Record the average total solids value as
Tfinal.
3.37 Add 3.00 ± 0.01 mL (4.92 ± 0.01 g) of 72% H2SO4 and use a glass stirring rod to mix
for 1 minute, or until the sample is thoroughly wetted.
3.38 Place the test tube in the water bath controlled to 30 ± 1oC and hydrolyze for 2 hours.
3.39 Stir the sample every 15 minutes to assure complete mixing and wetting.
3.40 Transfer the hydrolyzate to a glass bottle and dilute to a 4% acid concentration by
adding 84.00 ± 0.04 mL water, or by bringing the combined weight of sample, acid,
and water up to 89.22 ± 0.04 g. Be careful to transfer all the residual solids along with
the hydrolysis liquor.
3.41 Stopper each of the bottles and crimp aluminum seals into place.
3.42 Set the autoclave to a liquid vent cycle to prevent loss of sample from the bottle in the
event of a loose crimp seal. Autoclave the samples in their sealed bottles for 1 hour at
121 ± 3oC.
3.43 After completion of the autoclave cycle, allow the samples to cool for about 20 minutes
at room temperature before removing the seals and stoppers.
3.44 Vacuum filter the hydrolysis solution through one of the previously ignited filtering
crucibles.
3.45 If a carbohydrate analysis (LAP-002) and/or an acid-soluble lignin analysis (LAP-004)

is desired, decant 15-25 mL of filtrate into a resealable container. If this aliquot is not
used immediately for further analysis, store in refrigerator at 4oC.

Issue Date: 1/19/95
Supersedes: 8/19/92
Note: Acid-soluble lignin should be analyzed within 24 hours, preferably
within 6 hours of hydrolysis.
3.46 Use hot deionized water to wash any particles clinging to the glass bottle into the
crucible and to wash the filtered residue free of acid using vacuum filtration.
3.47 Dry the crucible and contents at 105 ± 3oC for 2 hours or until constant weight is
achieved ("0.3 mg upon reheating).
3.48 Cool in desiccator and record the weight, W2, the weight of the crucible, acid-insoluble
lignin, and acid-insoluble ash to the nearest 0.1 mg.
3.49 Place the crucible and contents in the muffle furnace and ignite at 575 ± 25oC for a
minimum of 3 hours, or until all the carbon is eliminated. Heat at a rate of 10oC/min
to avoid flaming. If the sample tends to flare up, the container should be partially
covered during this step. Avoid heating above the maximum stated temperature.
Protect the test container from strong drafts at all times to avoid mechanical loss of the
test specimen.
3.50 Cool in desiccator and record the weight, W3, the weight of the crucible and acid-
insoluble ash, to the nearest 0.1 mg.
Note: The amount of acid-insoluble ash remaining in the crucible is not

equal to the total ash content of the sample. Refer to Laboratory Analytical
Procedure #005 if total ash is to be determined.
12. Procedure B - Klason Lignin Determination
3.51 This procedure is suitable for oven-dried samples (including those dried at temperatures
between 45oC and 105oC) as well as air-dried, lyophilized, and extracted biomass
samples.
Note: The total solids content of the original sample, Tas received, as well as the
total solids content determined as the sample is prepared, Tprep, must be
known.
3.52 Individually label the crucibles needed for analysis, and ignite them at 575 ± 25oC to
achieve a constant weight of ± 0.3 mg. Store the ignited crucibles in a desiccator until
needed.
Note: In order to determine the absolute amounts of acid-insoluble residue

and acid-insoluble ash, for quality control purposes, it is useful to weigh and
record the ignited crucible to the nearest 0.1 mg.

Issue Date: 1/19/95
Supersedes: 8/19/92
3.53 Weigh 1.0 ± 0.05 g prepared sample to the nearest 0.1 mg and place in a 20x150 mm
test tube. Record as W1, the initial sample weight. Each sample must be run in
3.54 Samples for total solids determination must be weighed out at the same time as the
samples for the acid-insoluble lignin determination. If this is done later, it can
introduce an error in the calculation because ground biomass can rapidly gain or lose
moisture when exposed to the atmosphere. Record the average total solids value as
Tfinal.
3.55 Add 15.00 ± 0.02 mL of 72% H2SO4, chilled to 4oC in the refrigerator. Use a glass
stirring rod to mix for 1 minute, or until the sample is thoroughly wetted.
3.56 Hydrolyze the sample for 2 hours at room temperature (approximately 20oC), stirring
every 15 minutes to assure complete mixing and wetting.
3.57 Transfer the hydrolyzate to a 1000 mL Erlenmeyer flask and dilute to a 3% acid
concentration with 560 mL of deionized water. Be careful to transfer all the residual
solids along with the hydrolysis liquid.
3.58 Place the flask on the heating manifold and attach to the reflux condenser. Heat the
liquid to a gentle boil. Start timing at the onset of boiling, and reflux for 4 hours ± 5
minutes.
3.59 At the end of 4 hours, rinse the condenser with a small amount of deionized water
before disassembling reflux apparatus.
3.60 Vacuum filter the hydrolysis solution through one of the previously ignited filtering
crucibles.
3.61 If an acid-soluble lignin determination (LAP-004) is to be run, record the weight of the
collected filtrate. Decant 15-25 mL of filtrate into a resealable container. If this
aliquot is not used immediately for further analysis, store in refrigerator at 4oC.
Note: Acid-soluble lignin should be analyzed within 24 hours, preferably

within 6 hours of hydrolysis.
3.62 Use hot deionized water to wash any particles clinging to the glass bottle into the
crucible and to wash the filtered residue free of acid using vacuum filtration.
3.63 Dry the crucible and contents at 105 ± 3oC for 2 hours or until constant weight is
achieved (±0.3 mg upon reheating).

Issue Date: 1/19/95
Supersedes: 8/19/92
3.64 Cool in desiccator and record the weight, W2, the weight of the crucible, acid-insoluble
lignin, and acid-insoluble ash to the nearest 0.1 mg.
3.65 Place the crucible and contents in the muffle furnace and ignite at 575 ± 25oC for a
minimum of 3 hours, or until all the carbon is eliminated. Heat at a rate of 10oC/min
to avoid flaming. If the sample tends to flare up, the container should be partially
covered during this step. Avoid heating above the maximum stated temperature.
Protect the test container from strong drafts at all times to avoid mechanical loss of the
test specimen.
3.66 Cool in desiccator and record the weight, W3, the weight of the crucible and acid-
insoluble ash, to the nearest 0.1 mg.
Note: The amount of acid-insoluble ash remaining in the crucible is not

equal to the total ash content of the sample. Refer to Laboratory Analytical
Procedure #005 if total ash is to be determined.
13. Calculations
3.67 For lyophilized, air dried, or oven dried samples, or samples requiring no preparation,
calculate % acid-insoluble lignin on an as received 105oC dry weight basis as follows:
W 2 -W 3 W 2 -W 3
% acid - insoluble lignin = x 100% = x 100%
T asreceived T final
W1 x W1 x %
T prep 100
Where:
W1 = initial sample weight.
W2 = weight of crucible, acid-insoluble lignin, and acid-insoluble ash.
W3 = weight of crucible and acid-insoluble ash.
Tas received = % total solids content of the original sample (prior to any
preparation steps) on a 105oC dry weight basis, as determined by the
LAP-001.
Tprep = % total solids content of the sample as determined during the
preparation of the sample for analysis (by lyophilization, oven-drying,
or air drying).
Tfinal = % total solids content of the prepared sample used in this lignin
analysis, on a 105oC dry weight basis, as determined by the LAP-001.
Note: If the sample used in the acid-insoluble lignin analysis required no

preparation (analyzed as received), then Tprep=100% and Tfinal=Tas received. If
a sample was prepared by drying at 105oC prior to analysis, then Tprep=Tas
received and Tfinal=100%.
3.68 For an extracted sample, it may be necessary to report the results on an extractives-free
Issue Date: 1/19/95
Supersedes: 8/19/92
105oC dry weight basis or on an as received (whole sample) 105oC dry weight basis,
or both.
3.68.1 Calculate % acid-insoluble residue on an extractives-free basis as follows:
W 2 - W 3 x 100%
% acid - insoluble residueextractives - free =
%T final
W1 x
100%
Where:
W1 = initial weight of extracted sample
W2 = weight of crucible, acid-insoluble residue, acid-insoluble ash
W3 = weight of crucible and acid-insoluble ash
%Tfinal = % total solids of the extracted sample determined at 105oC
as described by the Standard Method for the Determination of
Total Solids in Biomass.
3.68.2 Correct the acid-insoluble residue value calculated above on an extractives-

free basis, to an as received (whole sample) 105oC dry weight basis:
% acid - insoluble residuewhole sample = % AIRextractives - free x
100%
Where:
% AIRextractives-free = % acid-insoluble residue on an extractives-free
105oC dry weight basis, as determined in the previous step
% extractives = % extractives in the sample extracted as described in
the Standard Method for the Determination of Extractives in
Biomass.
14. Report
3.69 Report the percent acid-insoluble lignin, to two decimal places, on a 105oC dry weight
basis, and cite the reporting basis.
relative percent difference (RPD).

Issue Date: 1/19/95
Supersedes: 8/19/92
3.71 Data obtained by replicate testing of a hybrid poplar in one laboratory gave a standard
deviation in Klason lignin content of 0.32% and a CV% of 1.26%.
3.72 Data obtained by replicate testing of a hybrid poplar sample in six different laboratories
gave a standard deviation of 2.37% and a CV% of 9.92%.
16. Quality Control
3.73 Reported significant figures: The acid-insoluble lignin results will be reported with
two decimal places, on a 105oC dry weight basis.
3.74 Replicates: All samples and all method verification standards are to be analyzed in
3.75 Blank: A blank crucible is to be run through the analysis. The dish is to be weighed
empty, ashed and reweighed. The difference in weight must be less than the equivalent
of a 0.5% error.
3.76 Relative percent difference criteria: The RPD must be less than 3.4%. If the RPD is
too large, the sample will be rerun.
with every batch.
3.79 Sample size: Approximately 0.6 grams of sample is required for conducting duplicate
analyses by Procedure A. Two grams will be required for Procedure B. If there is
insufficient sample, the result will be flagged and the lack of precision will be noted.
3.80 Sample storage: Wet samples, prior to preparation, must be stored in the refrigerator.
Samples that have been prepped by extraction, lyophilization, or oven drying must be
stored in tightly sealed containers or in a desiccator.
3.83 Definition of a batch: Any number of samples which are analyzed together and

Issue Date: 1/19/95
Supersedes: 8/19/92
recorded together. Samples within a batch must be of the same matrix. The maximum
size of a batch would be limited by the equipment constraints. A batch cannot be larger
than what is practical with the equipment.
standard is recorded along with the average, RPD, and a laboratory book/page
standard is to be control charted.

Issue Date: 1/19/95
Supersedes: 8/19/92
Ethanol Project

Procedure
Determination of Acid-Soluble Lignin in Biomass
Procedure Title:
Author: Tina Ehrman Date: 9/9/96
Determination of Acid-Soluble Lignin in Biomass
1. Introduction
1.1 The residue remaining after extensive acid hydrolysis of a biomass sample, corrected
for its ash content, is referred to as acid-insoluble lignin. This value, however, does not
represent the total lignin content of the sample. A small portion of the lignin is
solubilized during the hydrolysis procedure. This lignin fraction is referred to as acid-
soluble lignin (ASL) and may be quantified by ultraviolet spectroscopy.
2. Scope
2.1 This procedure describes a spectrophotometric method for determining the amount of
lignin solubilized upon hydrolysis of a biomass sample. The protocol utilizes the
hydrolyzate generated by LAP-002, "Determination of Carbohydrates in Biomass by
High Performance Liquid Chromatography", or by LAP-003, "Determination of Acid-
Insoluble Lignin in Biomass".
2.2 Sample material suitable for this procedure include hard and soft woods, herbaceous
materials (such as switchgrass and sericea), agricultural residues (such as corn stover,
wheat straw, and bagasse), waste-paper (such as office waste, boxboard, and
newsprint), washed acid- and alkaline-pretreated biomass, and the solid fraction of
fermentation residues. All results are reported relative to the 105±C oven-dried weight
of the sample. In the case of extracted materials, the results may also be reported on
an extractives-free basis.
2.3 Liquid process samples may also be analyzed by this technique to give an estimate of
their acid-soluble lignin content. The values generated must be viewed as estimates
only since many other components present in liquid process samples will also absorb
at the analysis wavelength and will bias the results high.
2.4 All analyses shall be preformed according to the guidelines established in the Ethanol
3. References
3.1 Ethanol Project Laboratory Analytical Procedure #002, "Determination of

Carbohydrates in Biomass by High Performance Liquid Chromatography".
Procedure #004
Issue Date: 9/25/96
Supersedes: 8/19/92 Page 2 of 7
3.2 Ethanol Project Laboratory Analytical Procedure #003, "Determination of Acid-
3.3 Ethanol Project Laboratory Analytical Procedure #010, "Determination of Extractives

in Biomass".
3.4 Kaar, W.E., and D.L. Brink. 1991. "Simplified Analysis of Acid Soluble Lignin."
Journal of Wood Chemistry and Technology, 11(4):465-477.
3.5 TAPPI Test Method T250, "Acid-Soluble Lignin in Wood and Pulp." In Tappi Test
4.1 The acid-soluble lignin determination is used in conjunction with other assays to
determine the total composition of biomass samples.
5. Interferences
5.1 Any component besides acid-soluble lignin which is present in the hydrolyzate and
which absorbs at the analytical wavelength of 205 nm will cause the results to be
biased high. This problem will be most severe with liquid process samples which have
been shown to contain components which absorb at 205 nm.
6. Apparatus
6.1 Spectrophotometer, suitable for measuring absorbance at 205 nm.
7.1 Sulfuric acid, 4% w/w, prepared by diluting 3.00 ± 0.01 mL of 72% w/w H2SO4 with
84.00 ± 0.04 mL of deionized water.
Note: For hydrolyzates generated from solid biomass samples taken through
LAP-002 or LAP-003, the sulfuric acid used to prepare the 4% acid solution
must be from the same prepared batch of 72% w/w H2SO4 used to prepare the
sample.
7.2 Water, 18 megohm deionized.
7.3 Matched pair of quartz cuvettes with a 1-cm path length.
Procedure #004
Issue Date: 9/25/96
7.4 Glass test tubes, of a size suitable for making dilutions.
7.5 Adjustable pipettors of various sizes with the appropriate disposable tips.
9. Procedure
9.1 Set up and calibrate the spectrophotometer following the protocols recommended in
the instrument manual.
9.2 Measure the absorbance of the hydrolyzate at 205 nm, using the 1-cm light path cuvette.
A 4% solution of H2SO4 should be used as a reference blank.
9.3 If the absorbance reading exceeds 0.7, the sample must be diluted. Dilute the sample
so the resulting absorbance reading falls between 0.2 and 0.7. The 4% H2SO4 must be
diluted in the same ratio as the sample and used as the reference blank for this repeat
analysis.
9.4 Repeat the analysis on a second aliquot of the hydrolyzate. Each sample must be
analyzed in duplicate, including the hydrolyzates generated from the analysis of the
LAP-002 or LAP-003 method verification standard.
10. Calculations
10.1 An absorptivity (extinction coefficient) value of 110 L/g-cm is used to calculate the
amount of acid-soluble lignin present in the hydrolyzate. The 205 nm absorptivities
reported for most woods fall in the range of 88 to 113 L/g-cm. The value of 110 L/g-
cm used in this protocol is consistent with the value used in the TAPPI procedure and
represents an average of values found for different woods and pulps.
Note: An absorptivity for a given type of biomass may be determined using

the "reKlasonation" protocol described in Kaar and Brink (1991).
Procedure #004
Issue Date: 9/25/96
10.2 For a liquid process sample, an estimate can be made of the amount of acid-soluble
lignin present as follows:
A
ASL, estimated (g/L) = x df
bxa
Where:
A= absorbance at 205 nm.
df = dilution factor.
b = cell path length, 1 cm.
a = absorptivity, equal to 110 L/g-cm unless experimentally
determined for a given biomass material.
10.3 For a solid biomass sample, the percent acid soluble lignin on a 105oC dry weight or
on an extractives free basis is calculated as follows:
A L
x df x V x
% ASL = b x a 1000 mL x 100
W x T final
100
Where: A = absorbance at 205 nm.

df = dilution factor.
b = cell path length, 1 cm.
a = absorptivity, equal to 110 L/g-cm unless experimentally
determined for a given biomass material.
V = filtrate volume, this volume will either be 87 mL, if the sample
is the hydrolyzate from the carbohydrate analysis (LAP-002)
or the LAP-003 summative acid-insoluble lignin protocol, or
will be equal to the weight of the filtrate obtained in the LAP-
003 Klason lignin protocol expressed in mL.
W = initial biomass sample weight in grams (from LAP-002 or LAP-
003).
%Tfinal = % total solids content of the biomass sample (as received or
after extraction), on a 105°C dry weight basis, as determined
during the LAP-002 or LAP-003 analysis.
Procedure #004
Issue Date: 9/25/96
10.4 For an extracted biomass sample, the percent acid-soluble lignin value, calculated
above, can be converted to an as received (whole sample) 105°C dry weight basis as
follows:
% ASL whole sample = % ASLextractives - free x
100%
Where: % ASLextractives-free = % acid-soluble lignin on an extractives-free

105°C dry weight basis, as determined in the previous step.
% extractives = % extractives in the extracted sample as described in
LAP-010, “Standard Method for the Determination of
Extractives in Biomass”.
11. Report
11.1 For solid biomass samples, report the percent acid-soluble lignin present in the sample,
to two decimal places, on a whole sample 105°C dry weight basis or on an extractives-
free basis. For liquid process samples, report the acid-soluble lignin content as an
estimate, to two decimal places. Cite the basis used in the report.
12. Precision
deviation of 0.12% and a CV of 5.41%.
12.2 Data obtained by replicate testing of a hybrid poplar sample in six different laboratories
gave a standard deviation of 0.98% and a CV of 41%.
13. Quality Control
13.1 Reported significant figures: For solid biomass samples, report the percent acid-soluble
lignin present in the sample, to two decimal places, on a whole sample 105°C dry
weight basis or on an extractives-free basis. For liquid process samples, report the
acid-soluble lignin content as an estimate, to two decimal places. Cite the basis used
in the report.
Procedure #004
Issue Date: 9/25/96
13.3 Blank: Dilute the 4% sulfuric acid solution in the same manner as the sample and use
as the reference blank in the spectrophotometer.
13.4 Relative percent difference criteria: The RPD must be less than 15.5%. If the RPD
is too large, the sample must rerun.
with every batch. This method utilizes the hydrolyzate generated by LAP-002 or LAP-
003 from a well characterized standard material suitable for analysis. For example,
NIST 8492 (Populus deltoides) is used as the MVS in compositional analysis of
hardwoods.
13.7 Sample size: A minimum of 5 mL of hydrolyzate generated by LAP-002 or LAP-003

are required for duplicate analyses. If there is insufficient sample, the result will be
flagged and the lack of precision will be noted.
13.8 Sample storage: Samples shall be stored in an airtight container and refrigerated. They
must be analyzed within 24 hours, and preferably within 6 hours, of the hydrolysis step
of LAP-002 or LAP-003.
Procedure #004
Issue Date: 9/25/96
Ethanol Project

Procedure
Standard Method for Ash in Biomass
Procedure Title:
4/28/94
ISSUE DATE:4-28-94 SUPERSEDES: 8/18/92

Standard Method for Ash in Biomass
1 Introduction
1.1 This procedure has been adopted by ASTM as an ASTM Standard Test Method for
the determination of ash in biomass.
2. Scope
2.1 This test method covers the determination of ash, expressed as the percentage of
residue remaining after dry oxidation (oxidation at 550 to 600oC), of hard and soft
woods, herbaceous materials (such as switchgrass and sericea), agricultural residues
(such as corn stover, wheat straw, and bagasse), wastepaper (such as office waste,
boxboard, and newsprint), acid and alkaline pretreated biomass, and the solid fraction
of fermentation residues. All results are reported relative to the 105oC oven-dried
weight of the sample.
2.2 All analysis shall be performed according to the Ethanol Project Quality Assurance
Plan (QAP).
3. References
3.1 TAPPI Test Method T211, "Ash in Wood and Pulp." In Tappi Test Methods.
Atlanta, GA: Technical Association of the Pulp and Paper Industry.
3.2 Moore, W., and D. Johnson. 1967. Procedures for the Chemical Analysis of Wood
and Wood Products. Madison, WI: U.S. Forest Products Laboratory, U.S.
Department of Agriculture.
4. Terminology
4.1 Ash - The inorganic residue left after ignition at 575oC.
5.1 The ash content is an approximate measure of the mineral content and other inorganic
matter in biomass.
5.2 The ash content is used in conjunction with other assays to determine the total
composition of biomass samples.

Issue Date: 4/28/94
Supersedes: 8/18/92
6. Apparatus
6.1 Aluminum weighing pans or crucibles, 50 mL - If crucibles are used, platinum

crucibles are preferred, but silica or porcelain crucibles may be used.
6.2 Muffle furnace - An electric furnace is recommended for igniting the wood sample.
A furnace fitted with an indicating pyrometer, so that the desired temperature can be
maintained, is preferable.
6.4 Desiccator.
6.5 Drying oven, with temperature control of 105 ± 2oC.
7 ES&H Considerations
8. Test Specimen
- biomass feedstocks,
- pretreated biomass,
8.2 Samples must be dried at 105oC according to the Laboratory Analytical Procedure
#001, Determination of Total Solids and Moisture in Biomass, prior to ash analysis.
Air-dried material can be used in place of 105oC dried material, but the weight of the
material must be corrected for its moisture content prior to calculating the ash.
8.3 The test specimen shall consist of approximately 0.5 to 1.0 g of sample obtained in
such a manner to ensure that it is representative of the entire lot of material being
tested. For 105oC dried samples containing large particles or chunks, it is
recommended that the sample be ground or milled to reduce the size of the large
pieces to less then 1 mm in diameter. The sample is then redried at 105oC prior to
testing.

Issue Date: 4/28/94
Supersedes: 8/18/92
9. Procedure
9.1 Mark a pan or crucible with a unique identification using a porcelain marker, place
it in the muffle furnace, and bring to constant weight by igniting at 575 ± 25oC.
Remove the pan or crucible from the furnace, cool to room temperature in a
desiccator, and weigh to the nearest 0.1 mg. Record this weight as the tare weight.
Keep the pan or crucible in a desiccator until used.
Note: For an aluminum pan, two hours of heating at 575 ± 25oC will be
sufficient to bring the pan to constant weight. With a crucible, however, the
following procedure is used: Place the crucible in the furnace at 575 ± 25oC
for three hours. Remove the crucible and place in a desiccator. Allow the
crucible to cool to room temperature and then weigh the crucible to the
nearest 0.1 mg. Record this weight. After weighing, return the crucible to
the furnace for one hour at 575 ± 25oC, cool again in the desiccator, and
reweigh. Repeat this step until the weight of the crucible varies by less than
0.3 mg from the previous weighing. Record this final weight as the crucible
tare weight.
9.2 Weigh approximately 0.5 to 1.0 g, to the nearest 0.1 mg, of a test specimen into the
tared pan or crucible. If the sample being analyzed is a 105oC dried test specimen,
the sample should be stored in a desiccator until use. Record the weight (container
plus sample minus tare weight of container) as the initial weight of the test specimen,
W2.
Note: For air dried samples, we recommend that samples for moisture
determination should be weighed out at the same time as the samples for the
ash determination. If this is done at a later time it can introduce an error in the
calculation because ground biomass can rapidly gain or lose moisture when
exposed to the atmosphere.
9.3 Place the container and contents in the muffle furnace and ignite at 575 ± 25oC for
a minimum of three hours, or until all the carbon is eliminated. Heat slowly at the
start to avoid flaming. If the sample tends to flare up, the container should be
partially covered during this step. Avoid heating above the maximum stated
temperature. Protect the test container from strong drafts at all times to avoid
mechanical loss of test specimen.
Note: For test specimens containing high amounts of ash (greater than 5%
by weight), it will be necessary to increase the time in the furnace to
overnight to ensure complete elimination of the carbon. This ignition time
period should not exceed 24 hours.

Issue Date: 4/28/94
Supersedes: 8/18/92
9.4 Remove the pan or crucible with its contents to a desiccator, cool to room
temperature, weigh to the nearest 0.1 mg, and record this weight. Repeat the heating
for one hour periods until the weight after cooling is constant to within0.3 mg.
Record the final weight of the ash, W1, as the container plus ash weight minus
container tare weight.
10. Calculations
10.1 For 105oC dried materials, calculate the percentage of ash, based on the initial weight
of the test specimen, as follows.
Ash, % = (W1/W2) x 100
where: W1 = weight of ash, and

W2 = initial weight of 105oC dried sample.
10.2 For air dried samples, the following calculation may be used to report the results on
a 105oC dried weight basis:
Ash, % = (W1 / (W2 x T/100)) x 100
where:
W1 = weight of ash,
W2 = initial weight of sample, and
T = percent total solids of sample, on a 105oC oven-dried weight
basis, as determined by LAP #001.
11. Report
11.1 Report the result to two decimal places, as a percentage of the sample's 105oC dried
weight, and cite the basis used in the calculation.
deviation in ash content of 0.05% and a CV% of 3.88%. Replicate testing of a
National Institute of Standards and Technology (NIST) #8494 wheat straw gave a
standard deviation of 0.20% and a CV% of 1.95%.
12.2 Data obtained by replicate testing of a hybrid poplar sample in six different
laboratories gave a standard deviation in ash content of 0.11% and a CV% of 9.24%

Issue Date: 4/28/94
Supersedes: 8/18/92
13. Quality Control
13.1 Reported significant figures: The ash results will be reported as a percentage with
two decimal places.
13.2 Replicates: All samples and all method verification standards are to be analyzed in
duplicate.
13.3 Blank: An empty aluminum dish or crucible is to be run through the analysis. The
dish is to be weighed empty, ashed, and reweighed. The difference in weight must
be less than the equivalent of a 0.5% error.
13.4 Relative percent difference criteria: The %RPD must be less than 15.5% if the
average ash content is less than 2%. The %RPD must be less than 5.0% if the
average ash content is greater than 2%. If the %RPD is too large, the sample will be
rerun.
13.5 Method verification standard: A method verification standard must be run in

duplicate with every batch. The results of method verification analyses must be
control charted.
13.7 Sample size: Approximately two grams are required for duplicate analyses. If there
is insufficient sample, the result will be flagged and the lack of precision will be
noted.
13.8 Sample storage: The oven dried sample will be stored in a desiccator.
recorded together. Samples within a batch must be of the same matrix. The
maximum size of a batch would be limited by the equipment constraints. A batch
cannot be larger than what is practical with the equipment.
13.12 Control charts: The results of analysis of method verification standards are recorded.
Each result from the duplicate analysis is recorded along with the average, %RPD,
and a laboratory book/page reference.
13.13 Other: All aluminum pans or crucibles shall be preashed. Because the samples and
containers quickly pick up moisture from the air, all the weights shall be taken
immediately after the items are removed from the desiccator.

Issue Date: 4/28/94
Supersedes: 8/18/92
Ethanol Project

Procedure
Procedure Title: Measurement of Cellulase Activities
Author: Bill Adney and John Baker Date:

7-18-96

Measurement of Cellulase Activities
1. Introduction
1.1 The following method describes a procedure for measurement of cellulase activity
using International Union of Pure and Applied Chemistry (IUPAC) guidelines (1). The
procedure has been designed to measure cellulase activity in terms of "filter-paper
units" (FPU) per milliliter of original (undiluted) enzyme solution. For quantitative
results the enzyme preparations must be compared on the basis of significant and equal
conversion. The value of 2.0 mg of reducing sugar as glucose from 50 mg of filter
paper (4% conversion) in 60 minutes has been designated as the intercept for
calculating filter paper cellulase units (FPU) by IUPAC.
1.2 It is extremely important to keep in mind that the FPU is defined only at this extent of
conversion. Reducing sugar yield is not a linear function of the quantity of enzyme in
the assay mixture; as discussed by Ghose (1987), twice the amount of enzyme would
not be expected to yield twice the reducing sugar in equal time. The assay procedure
therefore involves finding a dilution of the original enzyme stock such that a 0.5 mL
aliquot of the dilution will catalyze 4% conversion in 60 minutes (or, in practical terms,
finding two dilutions that bracket the 4%-conversion point so closely that the required
dilution can be obtained, with reasonable accuracy, by interpolation) and then
calculating the activity (in FPU/mL) of the original stock from the dilution required.
Further comments on the required calculations, and their significance, are to be found
in the Appendix.
1.3 Assay mixtures may in some cases contain reducing sugars unrelated to hydrolysis of
substrate glycosidic bonds by the enzyme. Culture filtrates to be assayed for cellulase
may contain nutrient sugars, and the reducing ends of the cellulose polymers of the
substrate may sometimes be measurable as glucose equivalents before any enzyme
attack. For this reason, controls consisting of (a) enzyme without substrate and b)
substrate without enzyme are included with all enzyme assays and sample values are
corrected for any blank values.
2. Scope
2.1 This procedure is only appropriate for the determination of FPU activity in a cellulase
preparation as defined by the IUPAC procedure as outlined above.
Procedure #006
Revision: 7/18/96
3. References
3.1 Ghose, T.K. 1987. "Measurement of Cellulase Activities." Pure & Appl. Chem. 59:
257-268.
3.2 Miller, G.L. 1959. "Use of Dinitrosalicylic Acid Reagent for Determination of
Reducing Sugar." Anal. Chem. 31:426-428.
4.1 This procedure follows IUPAC guidelines and determines enzyme activity as filter
paper units in a cellulase preparation.
5. Apparatus
5.1 Water bath capable of maintaining 50oC ± 0.1oC.
5.2 Spectrophotometer suitable for measuring absorbance at 540 nm.
6.1 DNS Reagent
Mix: Distilled water 1416 mL

3,5 Dinitrosalicylic acid 10.6 g
Sodium hydroxide 19.8 g
Dissolve above, then add:

Rochelle salts (sodium potassium tartrate) 306 g
Phenol (melt at 50oC) 7.6 mL
Sodium metabisulfite 8.3 g
Titrate 3 mL sample with 0.1 N HCl to the phenolphthalein endpoint. It should take
5-6 mL of HCl. Add NaOH if required (2 g = 1 mL 0.1 N HCL).
6.2 Citrate Buffer: For Trichoderma reesei, cellulase assays are carried out in 0.05 M
citrate buffer pH 4.8. For other cellulase enzymes, the pH and the assay temperature
may be different. The assay conditions must be defined when reporting results.
Citric acid monohydrate 210 g

DI water 750 mL
NaOH - add until pH equals 4.3 50 to 60 g
Procedure #006
Revision: 7/18/96
Dilute to 1 L and check pH. If necessary add NaOH until the pH is 4.5.
When the 1 M stock citrate buffer stock is diluted with water to 50 mM
the pH should be 4.8. After diluting the citrate buffer check and adjust
the pH if necessary to pH 4.8.
7.2 Care must be taken when working with phenol, which is toxic and corrosive.
8. Procedure for the Filter Paper Assay for Saccharifying Cellulase
8.1 The detection of glycosidic bond cleavage by this method involves the parallel and
identical treatment of three categories of experimental tubes (assay mixtures, blanks
and controls, and glucose standards), prepared as detailed below. The substrate is a 50
mg Whatman No. 1 filter paper strip (1.0 x 6.0 cm).
8.2 Enzyme assay tubes:
8.2.1 Place a rolled filter paper strip into each 13 x 100 test tube.
8.2.2 Add 1.0 mL 0.05 M Na-citrate, pH 4.8 to the tube; the buffer should saturate
the filter paper strip.
8.2.3 Equilibrate tubes with buffer and substrate to 50oC.
8.2.4 Add 0.5 mL enzyme diluted appropriately in citrate buffer. At least two
dilutions must be made of each enzyme sample, with one dilution releasing
slightly more than 2.0 mg of glucose (absolute amount) and one slightly less
than 2.0 mg of glucose. Target 2.1 and 1.9 mg glucose, respectively, for
these two dilutions. Depending on the enzyme these targets may be hard to
achieve and additional dilutions must be run.
8.2.5 Incubate at 50oC for exactly 60 min.
8.2.6 At the end of the incubation period, remove each assay tube from the 50oC
bath and stop the enzyme reaction by immediately adding 3.0 mL DNS
reagent and mixing.
Procedure #006
Revision: 7/18/96
8.3 Blank and controls:
8.3.1 Reagent blank: 1.5 mL citrate buffer.
8.3.2 Enzyme control: 1.0 mL citrate buffer + 0.5 mL enzyme dilution (prepare a
separate control for each dilution tested).
8.3.3 Substrate control: 1.5 mL citrate buffer + filter-paper strip.
8.4 Glucose standards:
8.4.1 A working stock solution of anhydrous glucose (10 mg/mL) should be made
up. Aliquots of this working stock should be tightly sealed and stored frozen.
The standard should be vortexed after thawing to ensure adequate mixing.
8.4.2 Dilutions are made from the working stock in the following manner:
1.0 mL + 0.5 mL buffer = 1:1.5 = 6.7 mg/mL (3.35 mg/0.5 mL).

1.0 mL + 1.0 mL buffer = 1:2 = 5 mg/mL (2.5 mg/0.5 mL).
1.0 mL + 2.0 mL buffer = 1:3 = 3.3 mg/mL (1.65 mg/0.5 mL).
1.0 mL + 4.0 mL buffer = 1:5 = 2 mg/mL (1.0 mg/0.5 mL).
8.4.3 Glucose standard tubes should be prepared by adding 0.5 mL of each of the
above glucose dilutions to 1.0 mL of citrate buffer in a 13 x 100 mm test
tube.
8.4.4 Blanks, controls and glucose standards should be incubated at 50oC along
with the enzyme assay tubes, and then "stopped" at the end of 60 minutes by
addition of 3.0 mL of DNS reagent.
8.5 Color development (Miller, 1959):
8.5.1 Boil all tubes for exactly 5.0 minutes in a vigorously boiling water bath
containing sufficient water to cover the portions of the tubes occupied by the
reaction mixture plus reagent. All samples, controls, blanks, and glucose
standards should be boiled together. After boiling, transfer to a cold ice-
water bath.
Procedure #006
Revision: 7/18/96
8.5.2 Let the tubes sit until all the pulp has settled, or centrifuge briefly. Dilute all
tubes (assays, blanks, standards and controls) in water (0.200 mL of color-
developed reaction mixture plus 2.5 mL of water in a spectrophotometer
cuvette works well, use the pipettor to mix by drawing the mixture into the
pipettor tip repeatedly). Determine color formation by measuring absorbance
against the reagent blank at 540 nm. With this dilution the glucose standards
described above should give absorbance in the range of 0.1 to 1.0 A.
9. Calculations
9.1 Construct a linear glucose standard curve using the absolute amounts of glucose
(mg/0.5 mL) plotted against A540. The data for the standard curve should closely fit a
calculated straight line, with the correlation coefficient for this straight line fit being
very near to one. Verify the standard curve by running a calibration verification
standard, an independently prepared solution of containing a known amount of glucose
which falls about midpoint on the standard curve.
9.2 Using this standard curve determine the amount of glucose released for each sample
tube after subtraction of enzyme blank.
9.3 Estimate the concentration of enzyme which would have released exactly 2.0 mg of
glucose by means of a plot of glucose liberated against the logarithm of enzyme
concentration (refer to the example in Appendix B, which uses semilogarithmic graph
paper). To find the required enzyme concentration take two data points that are very
close to 2.0 mg and draw a straight line between them, use this line to interpolate
between the two points to find the enzyme dilution that would produce exactly 2.0 mg
glucose equivalents of reducing sugar. Appendix B presents an example.
Note: In this plot, and in the equation below for calculating FPU, the term "enzyme
concentration" refers to the proportion of the original enzyme solution present in each
enzyme dilution (i.e., the number of mL of the original solution present in each mL of
the dilution).
9.4 Calculate FPU:
0.37
Filter Paper Activity = units/ml
[enzyme] releasing 2.0 mg glucose
Where [enzyme] represents the proportion of original enzyme solution present in the
directly tested enzyme dilution (that dilution of which 0.5 mL is added to the assay
mixture). For the derivation of the FPU see Ghose (1987) and Appendix A.
Procedure #006
Revision: 7/18/96
9.5 Refer to Appendix B for an example for calculating IUPAC-FPU.
Procedure #006
Revision: 7/18/96
10.1 Precision can be measured only by the closeness of repeated measurements of the same
quantity of enzyme. This procedure, if carefully followed, should give the same
approximate numerical readings as obtained by other laboratories using the same
procedure. Precision in filter paper assays may be affected by the inherent physical
properties of cellulase preparations.
11. Quality Control
11.1 Reported significant figures: Typically results are reported as whole integers along
with the standard deviation. The assay conditions must be defined when reporting the
results.
11.2 Replicates: Run each dilution in triplicate.
11.3 Blank: As described in the section “Blank and controls”.
11.4 Relative percent difference criteria: Not defined; dependent on the enzyme being
tested.
11.5 Method verification standard: Not available since enzymes change over time.
11.6 Calibration verification standard: A calibration verification standard shall be

independently prepared and analyzed as described in the section “Calculations”.
11.7 Sample size: Dependant upon enzyme concentration.
11.8 Sample storage: Dependant upon source of enzyme. Manufacturer’s instructions

should be followed.
11.9 Standard storage: Store frozen at -20oC or prepare fresh batch; shake vigorously prior
to use.
11.10 Standard preparation: As described in the section “Glucose standards”.
11.11 Definition of a batch: Run all standards, blanks, and samples together in one batch.
The size of the batch may be limited by instrument constraints and should not be larger
than what is practical to handle together.
11.12 Control charts: Not applicable.
11.13 Others: Not applicable.
Procedure #006
Revision: 7/18/96
12. Appendix A: Numerical Values in Equation Used to Calculate Filter Paper Activity
The practical bottom line is that if the assays are set up according to the instructions, and the
calculations are carried out using the equation presented in the calculations section, the results
obtained will correspond to the generally accepted activities in "filter paper units" that would be
obtained by other laboratories around the world, were these other laboratories to test the same
enzyme solution. For those workers interested in the derivation of this equation, and of the "filter
paper unit", the following comments may be helpful in conjunction with Ghose (1987).
The numerator (0.37) in the equation is derived from the factor for converting the 2.0 mg of
"glucose-equivalents" generated in the assay to mmoles of glucose (2.0 ÷ 0.18016), from the volume
of the enzyme being tested that is used in the assay (0.5 mL), and from the incubation time (60
minutes) required for generation of the reducing equivalents.
(2.0 mg glucose / 0.18016 mg glucose/µmol)

= 0.37 µmol/ minute - mL
(0.5 mL enzyme dilution x 60 minutes)
Thus,
Because the "enzyme concentration" in the denominator of the equation is a dimensionless number
(equal to the ratio of the enzyme concentration in the 0.5 mL of enzyme dilution added to the assay
to the enzyme concentration in the original solution, for which FPU values are desired), the right side
of equation therefore winds up with units (mmol min-1mL-1) that look like "International Units per
mL" (I.U./mL). Ghose himself points out, however, that "because the FPU assay is non-linear, the
use of the International Unit per se is incorrect as this unit is based on initial velocities, i.e., linear
reactions in which the product is produced at the same rate during each and every minute of the
reaction." The author goes on to recommend that FPU values for a given cellulase solution be given
simply as "units/mL".
"Definition" of the "Filter Paper Unit":
As a result of the above choice of numerical values, the "Filter Paper Unit" is not actually explicitly
defined. What is defined is the quantity 0.1875 FPU, which is that quantity of enzyme activity that,
when assayed according to the instructions contained herein, will produce reducing sugar equivalent
to 2.0 mg of glucose. One can verify this from the equation presented in the calculations section by
assuming that the enzyme solution being tested needs no dilution to yield reducing sugar equivalent
to 2.0 mg of glucose (i.e., the "enzyme concentration" ratio in the denominator is equal to 1.0), in
which case the activity of the solution being tested is measured as 0.37 filter paper units per mL.
Inasmuch as 0.5 mL of this solution was used in the assay, the absolute quantity of enzyme activity
that is present in the assay (and to which the observed effect can be ascribed) is 0.1875 FPU.
Procedure #006
Revision: 7/18/96
To put it another way, we have a defined method for measuring the activity of a cellulase solution
containing 0.1875 filter paper units per 0.5 mL assay aliquot (0.37 filter paper units per mL of
enzyme solution) but we do not have method for measuring the filter paper activity of solutions with
any other value. Solutions containing more than 0.37 "units" per mL must therefore be diluted to
this standard value to be measured, and solutions containing less than 0.37 "units" per mL (reducing
sugar produced in 60 minutes is less than that equivalent to 2.0 mg of glucose) cannot be assigned
"filter paper unit” activities at all. These latter "sub-2.0-mg" solutions either must be concentrated
before assay, or the activities should not be reported as "filter paper units" at all, but should be
reported instead as "mmoles glucose equivalents released per minute averaged over 60 minutes."
Ghose (1987) explains the special circumstances involved in measurement of "filter paper activity",
and workers are urged to pay close attention to the text of the paper (especially the text surrounding
the equations on page 263 of the reference) rather than just "lifting" the equations themselves.
13. Appendix B: Example for calculating IUPAC-FPU
13.1 Determination of cellulase activity in a T. reesei enzyme preparation using the methods
outlined by IUPAC. All enzyme dilutions were made in citrate buffer, pH 4.8, as
indicated in the following table from a working enzyme stock solution that had been
diluted 1:20 in citrate buffer.
Dilution # Citrate buffer 1:20 Enzyme (ml) Concentration*

(ml)
1 1650 350 0.00875
2 1700 300 0.00750
3 1800 200 0.00500

4 1850 150 0.00375
5 1900 100 0.00250
*The term "concentration" is used to represent the proportion of the original enzyme
solution present in the dilution added to the assay mixture. For example a 1:10 dilution
of the 1:20 working stock of enzyme will have a "concentration" of 0.005.
Procedure #006
Revision: 7/18/96
13.2 Dilution of glucose standards and construction of standard curve.
Glucose stock Citrate buffer Dilution Concentration Abs. 540 nm

(mL) (mL)
1.0 0.5 1:1.5 3.35 mg/0.5 mL 0.765

1.0 1.0 1:2 2.50 mg/0.5 mL 0.579
1.0 2.0 1:3 1.65 mg/0.5 mL 0.384

1.0 4.0 1:5 1.00 mg/0.5 mL 0.220
13.3 Glucose concentration of samples as determined from standard curve.
Dilution # Abs 540 nm Glucose (ml/0.5 mL)
1 0.603 2.63
2 0.567 2.44
3 0.442 1.93
4 0.346 1.51
5 0.248 1.08
Procedure #006
Revision: 7/18/96
13.4 Determination of the concentration of enzyme which would have released exactly 2.0
mg of glucose by plotting glucose liberated against enzyme concentration.
13.5 Calculation of FPU from graph of dilution vs. glucose concentration.
0.37
= 70 FPU/mL
0.0053
Procedure #006
Revision: 7/18/96
Ethanol Project

Procedure
Preparation of Dilute-Acid Pretreated Biomass
Procedure Title:
Author: Daniel Hsu Date:
5/17/95

Preparation of Dilute-Acid Pretreated Biomass
1. Introduction
1.1 Lignocellulosic biomass feedstocks, typically, contain large quantities (55-75% by

weight) of carbohydrates that are polymers of 5- and 6-carbon sugars. Most or all of
these carbohydrates can be converted to ethanol via biotechnology. For bioconversion
to occur, however, the polymers must first be broken down into low-molecular-
weight, or, essentially, monomeric, sugars, and it is generally accepted that enzymatic
hydrolysis is the preferred pathway. Nevertheless, the native (indigenous) cellulose
fraction of the carbohydrates is recalcitrant to enzymatic breakdown; therefore, a
pretreatment step is required to render it amenable to enzyme attack. Although
various pretreatment techniques are under investigation, a batch, dilute sulfuric acid
prehydrolysis method is currently employed at NREL to prepare pretreated biomass
for fermentation research.
2. Scope
2.1 This procedure describes the batch dilute-acid pretreatment method employed at
NREL to prepare pretreated biomass for fermentation research in the Ethanol Project.
Biomass is pretreated in a pressure reactor at 160°C for approximately 10 minutes
using dilute sulfuric acid. The procedure has been developed as a result of several
years of pretreatment research at NREL and has been found to result in
prehydrolyzate (the liquid phase resulted from a dilute-acid pretreatment run) of
satisfactorily high xylose yield and high pretreated solids enzyme digestibility.
Depending on research goals, the pretreated biomass may or may not need to be
washed. The operating conditions used in the procedure, however, have not been
optimized to maximize the xylose yield, enzyme digestibility, or the ethanol yield
that can be obtained through fermentations. Furthermore, the optimal pretreatment
conditions are expected to be substrate specific.
3. References
3.1 NREL Ethanol Project CAT Task Laboratory Analytical Procedure #001, "Standard
Method for Determination of Total Solids in Biomass".

Issue Date: 5/31/95
Supersedes: 3/17/93
3.2 Grohmann, K., Torget, R., and Himmel, M. 1985. "Optimization of Dilute Acid
Pretreatment of Biomass." Biotech. Bioeng. Symp. No. 15. 59-80.
3.3 Grohmann, K., Himmel, M., Rivard, C., Tucker, M., Baker, J., Torget, R., and
Graboski, M. "Chemical-Mechanical Methods for the Enhanced Utilization of
Straw." 1984. Biotech. Bioeng. Symp. No. 14. 137-157.
4.1 Pretreatment is necessary before the carbohydrates in lignocellulosic biomass can be

converted to ethanol via biotechnology. Dilute-acid pretreatment is one technique
that has been extensively investigated and found to be effective.
5. Apparatus
5.1 Parr Instrument Company pressure reactor system, including either a 2-gal or a 1-L
reactor vessel made of Carpenter 20 Cb-3 material, with a matching mixer, jacket
electrical heater, and temperature/mixing control unit that comprises a thermocouple,
all supported on a movable metal stand. Attached to the head plate of the reactor
vessel are a mixer shaft, a pressure gauge, a vent, a valve for chemical injection, a
pressure rupture disc, and a thermowell.
5.2 Hoist (required only for the 2-gal system).
5.3 High pressure pump (HPLC pump, Beckman model 110, or equivalent).
5.4 Balance(s).
5.5 pH meter. (Note: A pH meter must be used if pretreatment of a new type of

biomass substrate is performed. If pretreatment of the substrate has been
performed previously, pH paper, as specified in Paragraph 6.7, can be used in
place of the pH meter.)
5.6 Stopwatch.
5.7 Tachometer.

Issue Date: 5/31/95
Supersedes: 3/17/93
6.1 Sulfuric Acid, 72% (w/w) (12.00 ± 0.02 M or specific gravity 1.6389 at
15.6°C/15.6°C).
6.2 pH 1.00 and 2.00 calibrating buffers.
6.3 Feedstock substrate: Feedstock substrate is milled to an appropriate size (normally

-3 mm [-1/8 in.]). To prevent possible charring in the reactors, the substrate is often
further sieved to remove -60 mesh particles. Upon preparation, the moisture content
is determined, following Laboratory Analytical Procedure #001, Standard Method for
Determination of Total Solids in Biomass.
6.4 Two 3- to 5-gal pails (required only if the 2-gal system is used) or two 1-L beakers
(required only if the 1-L system is used).
6.5 A sink suitable for an ice bath to be prepared for cooling the 2-gal reactor vessel after
a pretreatment run (required only if the 2-gal system is used) or an 8-L plastic bucket
(required only if the 1-L system is used).
6.6 Two 50-mL graduated cylinders (required only if the 2-gal system is used) or a 10-mL
and a 50-mL graduated cylinder (required only if the 1-L system is used).
6.7 pH paper covering pH range 1 to 2. (Note: If the operator prefers using pH meter
to pH paper, this paragraph can be skipped.)
6.8 Prepare the following four items only if separation of prehydrolyzate and pretreated
solids or washing of pretreated solids is required.
6.8.1 A Buchner funnel of 6-L capacity (required only if the 2-gal system is used)
or 600-mL capacity (required only if the 1-L system is used).
6.8.2 A 2-L vacuum flask and an aspirator (required only if the 1-L system is
used).
6.8.3 A filter cloth (a white cotton bed sheet can be cut into size for use) of
approximately 3 ft x 3 ft (required only if the 2-gal system is used) or a
Whatman No. 5 filter paper (required only if the 1-L system is used).
6.8.4 pH paper covering pH range 2 to 6. (Note: If the operator prefers using

pH meter to pH paper, this paragraph can be skipped.)

Issue Date: 5/31/95
Supersedes: 3/17/93
7.1 Before any work proceeds, review the appropriate SOP on pretreatment system. It is
required that new personnel be trained by an experienced personnel prior to
conducting any pretreatment experiment.
7.4 Apparatus in this procedure are heated to elevated temperatures, use caution to avoid
burns.
7.5 High pressure is generated in the reactor. Take precautions to avoid unexpected
venting of the reactor contents.
8. Procedure for Using 2-gallon Parr Reactor System
8.1 Feedstock Substrate and Chemicals Preparation and Other Preparative Work
8.1.1 Weigh an appropriate amount (approximately 480 g dry weight) of

feedstock substrate in a 3- to 5-gal pail. Refer to Paragraph 10.1.1 for an
example of the feedstock substrate weight.
8.1.2 Targeting a 10% solids level in the reactor, use a 50-mL graduated cylinder
to weigh an appropriate amount of 72% sulfuric acid. The weight of the
acid is determined according to calculations shown in Paragraph 10.1.2.
(Note: The target acid level is to obtain a prehydrolyzate pH of 1.3-1.5.
Since the prehydrolyzate pH is not known prior to the run, the acid
concentration to use has to be preestimated. NREL experience has
shown that, for a hardwood substrate, 0.73 wt% acid in the liquid
phase in the reactor is generally acceptable. For a herbaceous
substrate, 0.88 wt% acid is generally acceptable. Following
pretreatment, the prehydrolyzate pH should be tested to determine if
the acid concentration used is acceptable. If not so, it must be adjusted
and the run repeated until the target pH is met.)
8.1.3 Targeting at the solids level in the reactor specified above, weigh an
appropriate amount of deionized water in the second 3- to 5-gal pail. The
weight is determined according to calculations shown in Paragraph 10.1.3.

Issue Date: 5/31/95
Supersedes: 3/17/93
8.1.4 Thoroughly flush the HPLC pump intake and delivery lines with deionized
water (not the water prepared in Step 8.1.3) and discard the effluent.
Following flushing, turn off the pump.
8.1.5 Prepare 15 mL deionized water in the second 50-mL graduated cylinder.
8.1.6 Place the pump intake tubing in the acid from Step 8.1.2 and run the pump
for approximately 5 minutes at 6 mL/min, directing the effluent from the
delivery line back into the acid graduated cylinder. Turn off the pump at the
end of the 5-minute period.
8.1.7 Quantitatively transfer the weighed feedstock substrate and water,

respectively, from Steps 8.1.1 and 8.1.3 into the reactor (using some of the
water to rinse the feedstock substrate pail).
8.1.8 Manually mix the reactor contents.
8.1.9 Close the vent and the chemical injection valves on the reactor head plate,
followed by closing the head plate.
8.1.10 After the reactor is securely closed, open the vent valve.
8.2 Pretreatment Run
8.2.1 Use the hoist to load the reactor into the heater, making sure the vent line
and the rapture disc relief line on the head plate are facing away from the
operator.
8.2.2 Insert thermocouple in thermowell and attach the drive unit.
8.2.3 Turn on the mixer motor, setting the rpm to be about 220 ± 20 (use
tachometer to verify rpm). Allow reactor contents to mix at ambient
temperature for 5 minutes. Proceed to complete the next step during the 5-
minute mixing time.
8.2.4 While Step 8.2.3 is in progress, attach the HPLC pump delivery line to the
reactor chemical injection valve.
8.2.5 Following the 5-minute mixing time in Step 8.2.3, turn on the heater and
set the temperature controller set-point to 50°C.

Issue Date: 5/31/95
Supersedes: 3/17/93
8.2.6 The heater will be turned off automatically when the reactor temperature
reaches 50°C, but the temperature will continue to rise to about 75°C.
Allow the reactor to be heated at about 75°C and mixed for 10 minutes to
remove trapped air.
8.2.7 At the end of the 10-minute period, close the reactor vent and adjust the
controller set-point to 150°C.
8.2.8 While the reactor is being heated and before the temperature reaches 158°C,
prepare an ice bath in the sink next to the reactor apparatus. (It takes about
20 minutes for the reactor temperature to reach 158°C.)
8.2.9 When the reactor temperature reaches 158°C, adjust the controller set-point
8.2.10 As soon as the reactor temperature reaches 159.5°C, turn on the HPLC
pump at a flow rate of 6 mL/min, start the stopwatch, followed by opening
the chemical injection valve. Monitor time and reactor temperature
throughout this period. Prepare a control chart of the reactor pressure at
160°C. The temperature needs to be within 160 " 1°C or the experiment
must be repeated.
8.2.11 When the acid graduated cylinder is almost empty (after approximately 4
minutes for a hardwood substrate or 5 minutes for a herbaceous substrate),
note the elapsed time and add the deionized water from Step 8.1.5 slowly
(to prevent air bubbles from entering the pump intake line) to the acid
graduated cylinder to flush out acid in the line to the reactor.
8.2.12 As soon as the deionized water in the acid graduated cylinder has all entered
the intake tubing, turn off the pump, close the chemical injection valve, and
disconnect the pump delivery line from the valve.
8.2.13 At about the 11.5-minute mark for a hardwood substrate (or 12-minute
mark for a herbaceous substrate), turn off the mixer motor, disconnect the
drive unit, and move the drive unit and the thermocouple out of way.
8.2.14 Use the hoist to lift the reactor out of the heater and, when the elapsed time
reaches 10 minutes plus half of the acid injection time noted in Step 8.2.11,
lower the reactor into the ice bath to quench the reaction.

Issue Date: 5/31/95
Supersedes: 3/17/93
8.2.15 Rotate the mixer shaft manually to assist in cooling uniformly the reactor
contents.
8.2.16 When the pressure in the reactor drops to below 5 psig, check with the
thermocouple that was removed from the thermowell in Step 8.2.13 to
ensure the temperature in the reactor is 95°C or lower. Then, open the vent
slowly to relieve the remaining pressure in the reactor.
8.2.17 Upon fully relieving the pressure in the reactor, use the hoist to lift the
reactor out of the ice bath and set it upright on the flat surface next to the
sink.
8.2.18 Remove the reactor head plate, making sure little or no solids are attached
to the mixing unit.
8.2.19 (This step can be skipped if the feedstock substrate has previously been
used and the appropriate amount of acid to use is known.) Determine the
pH of the prehydrolyzate with the pH meter (the meter should be calibrated
with the pH 1.00 and 2.00 calibrating buffers). If the pH is not between 1.3-
1.5, repeat the run using an adjusted amount of 72% sulfuric acid.
Otherwise, proceed to the next step.
8.2.20 If it is required to separate prehydrolyzate and pretreated solids or to wash

the pretreated solids, proceed to Step 8.3. Otherwise, transfer the reactor
contents to an appropriate container and label the container properly for
storage in a refrigerated area.
8.3 Post-Pretreatment Sample Handling
8.3.1 Set the 6-L Buchner funnel on a clean, dry 3- to 5-gal pail on the floor.
8.3.2 Place the filter cloth over the funnel.
8.3.3 Filter the contents of the reactor through the funnel. Catch the
prehydrolyzate in the pail and squeeze the residual prehydrolyzate off the
pretreated solids.
8.3.4 Store the prehydrolyzate, sealed and properly labeled, in a refrigerated area.
8.3.5 If washing of the pretreated solids is required, proceed to the next step.
Otherwise, store the pretreated solids, sealed and properly labeled, in an
appropriate container in a refrigerated area.

Issue Date: 5/31/95
Supersedes: 3/17/93
8.3.6 Add deionized water to the Buchner funnel to wash the pretreated solids
and monitor the pH of the filtrate using pH paper until it reaches the desired
pH. Mix the solids with washing water occasionally to ensure effective
washing.
8.3.7 Squeeze the residual water off the pretreated solids.
8.3.8 Store the washed pretreated solids, sealed and properly labeled, in an
appropriate container in a refrigerated or frozen area.
9. Procedure for Using 1-L Parr Reactor System
9.1 Feedstock Substrate and Chemicals Preparation and Other Preparative Work
9.1.1 Weigh an appropriate amount (approximately 60 g dry weight) of feedstock

substrate in a 1-L beaker. Refer to Paragraph 10.2.1 for an example of
calculating the feedstock substrate weight.
9.1.2 Targeting a 10% solids level in the reactor, use the 10-mL graduated
cylinder to weigh an appropriate amount of 72% sulfuric acid. The weight
of the acid is determined according to calculations shown in Paragraph
10.2.2. (Note: The target acid level is to obtain a prehydrolyzate pH of
1.3-1.5. Since the prehydrolyzate pH is not known prior to the run, the
acid concentration to use has to be preestimated. NREL experience has
shown that, for a hardwood substrate, 0.73 wt% acid in the liquid
phase in the reactor is generally acceptable. For a herbaceous
substrate, 0.88 wt% acid is generally acceptable. Following
pretreatment, the prehydrolyzate pH should be tested to determine if
the acid concentration used is acceptable. If not so, it must be adjusted
and the run repeated until the target pH is met.)
9.1.3 Targeting at the solids level in the reactor specified above, weigh an
appropriate amount of deionized water in the second 1-L beaker. The
weight is determined according to calculations shown in Paragraph 10.2.3.
9.1.4 Thoroughly flush the HPLC pump intake and delivery lines with deionized
water (not the water prepared in Step 9.1.3) and discard the effluent.
Following flushing, turn off the pump.
9.1.5 Prepare 15 mL deionized water in the 50-mL graduated cylinder.

Issue Date: 5/31/95
Supersedes: 3/17/93
9.1.6 Place the pump intake tubing in the acid from Step 9.1.2 and run the pump
for approximately 5 minutes at 6 mL/min, directing the effluent from the
delivery line back into the acid graduated cylinder. Turn off the pump at the
end of the 5-minute period.
9.1.7 Quantitatively transfer the weighed feedstock substrate and water,

respectively, from Steps 9.1.1 and 9.1.3 into the reactor (using some of the
water to rinse the feedstock substrate beaker).
9.1.8 Manually mix the reactor contents.
9.1.9 Close the vent and the chemical injection valves on the reactor head plate,
followed by closing the head plate.
9.1.10 After the reactor is securely closed, open the vent valve.
9.2 Pretreatment Run
9.2.1 Place the reactor into the heater, making sure the vent line and the rupture
disc relief line on the head plate are facing away from the operator.
9.2.2 Insert thermocouple in thermowell, attach the drive unit, connect mixer
shaft seal cooling water lines, and allow cooling water to flow through the
lines (with discharge line directed to the drain).
9.2.3 Turn on the mixer motor, setting the rpm to be about 175 ± 20 (use
tachometer to verify rpm). Allow reactor contents to mix at ambient
temperature for 5 minutes. Proceed to complete the next step during the 5-
minute mixing time.
9.2.4 While Step 9.2.3 is in progress, attach the HPLC pump delivery line to the
reactor chemical injection valve.
9.2.5 Following the 5-minute mixing time in Step 9.2.3, turn on the heater and
set the temperature controller set-point to 50°C.
9.2.6 The heater will be turned off automatically when the reactor temperature
reaches 50°C, but the temperature will continue to rise to about 75°C.
Allow the reactor to be heated at about 75°C and mixed for 10 minutes to
remove trapped air.

Issue Date: 5/31/95
Supersedes: 3/17/93
9.2.7 At the end of the 10-minute period, close the reactor vent and adjust the
controller set-point to 150°C.
9.2.8 While the reactor is being heated and before the temperature reaches 158°C,
prepare an ice bath in the 8-L plastic bucket. (It takes about 20 minutes for
the reactor temperature to reach 158°C.)
9.2.9 When the reactor temperature reaches 158°C, change the controller set-
point to 160°C.
9.2.10 As soon as the reactor temperature reaches 159.5°C, turn on the HPLC
pump at a flow rate of 6 mL/min, start the stopwatch, followed by opening
the chemical injection valve. Monitor time and reactor temperature
throughout this period. Prepare a control chart of the reactor pressure at
160°C. The temperature needs to be within 160 " 1°C or the experiment
must be repeated.
9.2.11 When the acid graduated cylinder is almost empty, note the elapsed time
and add the deionized water from Step 9.1.5 slowly (to prevent air bubbles
from entering the pump intake line) to the acid graduated cylinder to flush
out acid in the line to the reactor.
9.2.12 As soon as the deionized water in the acid graduated cylinder has all entered
the intake tubing, turn off the pump, close the chemical injection valve, and
disconnect the pump delivery line from the valve.
9.2.13 At about the 9.5-minute mark, turn off the mixer motor, disconnect the
drive unit, detach the cooling water lines of the mixer shaft seal, and move
the drive unit and the thermocouple out of way.
9.2.14 Lift the reactor out of the heater and, when the elapsed time reaches 10
minutes plus half of the acid injection time noted in Step 9.2.11, lower the
reactor into the ice bath to quench the reaction.
9.2.15 Rotate the mixer shaft manually to assist in cooling uniformly the reactor
contents.
9.2.16 When the pressure in the reactor drops to below 5 psig, check with the
thermocouple that was removed from the thermowell in Step 9.2.13 to
ensure the temperature in the reactor is 95°C or lower. Then, open the vent
slowly to relieve the remaining pressure in the reactor.

Issue Date: 5/31/95
Supersedes: 3/17/93
9.2.17 Upon fully relieving the pressure in the reactor, lift the reactor out of the ice
bath and set it upright on the bench.
9.2.18 Remove the reactor head plate, making sure little or no solids are attached
to the mixing unit.
9.2.19 (This step can be skipped if the feedstock substrate has previously been
used and the appropriate amount of acid to use is known.) Determine the
pH of the prehydrolyzate with the pH meter (the meter should be calibrated
with the pH 1.00 and 2.00 calibrating buffers). If the pH is not between 1.3-
1.5, repeat the run using an adjusted amount of 72% sulfuric acid.
Otherwise, proceed to the next step.
9.2.20 If it is required to separate prehydrolyzate and pretreated solids, or, to wash

the pretreated solids, proceed to Step 9.3. Otherwise, transfer the reactor
contents to an appropriate container and label the container properly for
storage in a refrigerated area.
9.3 Post-Pretreatment Sample Handling
9.3.1 Set the 600-mL Buchner funnel on the 2-L vacuum flask and connect the
aspiration line.
9.3.2 Place a sheet of Whatman No. 5 filter paper in the funnel.
9.3.3 Apply vacuum to filter the contents of the reactor through the funnel. Catch
the prehydrolyzate in the vacuum flask. Wait until dripping of
prehydrolyzate stops.
9.3.4 Store the prehydrolyzate, sealed (in a bottle, for example) and properly
labeled, in a refrigerated area.
9.3.5 If washing of the pretreated solids is required, proceed to the next step.
Otherwise, store the pretreated solids, sealed and properly labeled, in an
appropriate container in a refrigerated area.
9.3.6 Apply deionized water to the Buchner funnel to wash the pretreated solids
and monitor the pH of the filtrate using pH paper until it reaches the desired
pH. Mix the solids with washing water occasionally to ensure effective
washing.

Issue Date: 5/31/95
Supersedes: 3/17/93
9.3.7 Wait until dripping of prehydrolyzate stops.
9.3.8 Store the washed pretreated solids, sealed and properly labeled, in an
appropriate container in a refrigerated or frozen area.
10. Calculations
10.1 Example calculations for the 2-gallon Parr reactor system:
10.1.1 Weight of feedstock substrate: assuming the moisture content of the

feedstock substrate to be 22.0%, then a convenient substrate weight of 615
g can be used. (The dry weight of the substrate is 615 x (1-22.0%) = 480 g.)
10.1.2 Weight of 72% sulfuric acid: since it is known that the target slurry solids
level is 10% and the target acid concentration in the reactor liquid phase is
0.73 wt% (assuming the feedstock to be a hardwood), using the numbers
from Paragraph 10.1.1, the weight of 72% sulfuric acid is obtained as
follows:
615 x (1 − 22%) 1
x (1 − 10%) x 0.73% x = 43.8 g
10% 72%
10.1.3 Weight of dilution water: to obtain a 10% slurry in the reactor using the
numbers from Paragraph 10.1.1, the total weight of the liquid phase in the
reactor needs to be:
615 x (1 − 22%)
x (1 − 10%) = 4320 g
10%
4320 − 615 x 22% − 43.8 − 15 = 4120 g
Since the liquid phase (assuming the dilute-acid extractives present in the
feedstock substrate to be negligible) in the reactor is comprised of (1) the
moisture in the feedstock substrate, (2) the dilution water, (3) the 72%
sulfuric acid added, and (4) the acid line flushing water, the weight of the
dilution water required is:

Issue Date: 5/31/95
Supersedes: 3/17/93
10.2 Example calculations for the 2-L Parr reactor system:
10.2.1 Weight of feedstock substrate: assuming the moisture content of the

feedstock substrate to be 22.0%, then a convenient substrate weight of 80.0
g can be used. (The dry weight of the substrate is: 80.0 x (1 - 22.0%) = 62.4
g.)
10.2.2 Weight of 72% sulfuric acid: since it is known that the target slurry solids
level is 10% and the target acid concentration in the reactor liquid phase is
0.73 wt% (assuming the feedstock to be a hardwood), using the numbers
from Paragraph 10.2.1, the weight of 72% sulfuric acid is obtained as
follows:
80.0 x (1 − 22%) 1
x (1 − 10%) x 0.73% x = 5.69 g
10% 72%
80.0 x (1 − 22%)
x (1 − 10%) = 562 g
10%
10.2.3 Weight of dilution water: to obtain a 10% slurry in the reactor using the
numbers from Paragraph 10.2.1, the total weight of the liquid phase in the
reactor needs to be:
Since the liquid phase (assuming the dilute-acid extractives present in the
feedstock substrate to be negligible) in the reactor is comprised of (1) the
moisture in the feedstock substrate, (2) the dilution water, (3) the 72%
sulfuric acid added, and (4) the acid line flushing water, the weight of the
dilution water required is:
562 − 80.0 x 22% − 5.7 − 15 = 523 g

Issue Date: 5/31/95
Supersedes: 3/17/93
11.1 The precision of Parr reactor operation relies largely on operators strictly adhering
to the procedure described above. It also relies on the stable performance of
instruments, including balance(s), pH meter, pressure gauge, stopwatch, tachometer,
and thermocouple (with display) used in the operation. In addition, the stable
performance of the HPLC pump used for acid injection affects the precision of the
operation. Thus, in addition to the operator's quality skills that must be ensured, the
instruments and equipment involved must be maintained in reliable working
conditions.
Statistical analysis of a limited database of six replicate runs (using the same
substrate and operating conditions by six different operators) shows that the
coefficient of variation for the contents of the major components in the pretreated
solids (glucan and Klason lignin) and in the prehydrolyzate (glucose, xylose, and
acid-soluble lignin) were all below 10%. The coefficient of variation for enzyme
digestibility of the pretreated solids was also below 10%.
11.2 Systematic errors may be incurred in preparing dilute-acid pretreated biomass if any
of the instruments used is not properly calibrated. It is thus of critical importance to
ensure proper calibration of the instruments. Periodic calibration of the various
instruments should be performed in accordance with the time intervals recommended
by the instrument manufacturers.
12. Quality Control
12.1 Reported significant figures: Report prehydrolyzate pH to two decimal places, if

using a pH meter and one decimal place if using pH paper. Monitoring of
temperature to 0.1°C during Parr reactor runs is required. All weight
determinations should include three significant digits.
12.2 Replicates: During normal operations, only properly trained operators will be
involved and only single runs are required. For QA/QC verification or for training
purposes, multiple runs using the same feedstock substrate and the same operating
conditions are required either by the same operator or by different operators. The
compositions of prehydrolyzate and pretreated solids and/or the digestibility or
fermentability of the pretreated solids will then be compared and statistically
analyzed to ensure consistency.
12.3 Blank: Not applicable

Issue Date: 5/31/95
Supersedes: 3/17/93
12.4 Relative percent difference criteria: Not applicable for single runs. When duplicate
runs are conducted, the relative percent difference for glucose, xylose, and Klason
lignin of prehydrolyzate and pretreated solids (where applicable) should be no
greater than 5%. For acid-soluble lignin, ash, digestibility, and fermentability, the
relative percent difference should be no greater than 10%.
12.5 Method verification standard: Not applicable
12.6 Calibration verification standard: Not applicable
12.7 Sample size: Typically, 1-L and 2-gal Parr reactor runs, respectively, use 60 and 480
g dry weight of the prepared feedstock substrate.
12.8 Sample storage:
12.8.1 Feedstock: Depending on total solids level and length of storage, the
feedstock substrate is recommended to be stored in ambient conditions
(when total solids content is above 88% and, thus, no risk of deterioration
is involved) or refrigerated (when total solids content is below 88% for up
to 1 month storage) or frozen (when total solids content is below 88% for
prolonged storage).
12.8.2 Pretreated products: Prehydrolyzate or pretreated solids samples should be

refrigerated. It is further recommended to store pretreated solids in acidified
conditions. If washing and prolonged storage of a washed pretreated solids
sample is required, freezing is recommended.
12.8.3 Standard storage: Not applicable
12.8.4 Standard preparation: Not applicable
12.8.5 Definition of a batch: Not applicable
12.8.6 Control charts: Prepare a control chart of reactor pressure at 160EC for
each pretreatment run.

Issue Date: 5/31/95
Supersedes: 3/17/93
Ethanol Project

Procedure
SSF Experimental Protocols: Lignocellulosic Biomass
Procedure Title: Hydrolysis and Fermentation
Author(s): Tammy Kay Hayward, Nancy S. Combs, Date:

Sherry L. Schmidt, George P. Philippidis 5/31/95
ISSUE DATE: 5-31-95 SUPERSEDES: 6/4/93

SSF Experimental Protocols:
Lignocellulosic Biomass Hydrolysis and Fermentation
1. Introduction
1.1 Ethanol is a promising alternative fuel which can be produced biologically from a
variety of waste materials such as paper products, corn fiber, sawmill waste, straw, and
rice. Ethanol has been made from grapes, barley and potatoes for thousands of years.
The production of ethanol from non-starch, lignocellulosic materials is, however, a
fairly recent development. There are many ways to produce ethanol from
lignocellulosic material. The method discussed here is known as simultaneous
saccharification and fermentation (SSF). It utilizes cellulase enzyme to break down the
cellulose and yeast to ferment the resulting glucose. The ethanol can be blended with
gasoline or used neat in combustion engines. As a fuel, ethanol burns cleaner than
gasoline, is completely renewable, and relatively less toxic to the environment.
2. Scope
2.1 The described protocols have been developed based on the personal experience of
NREL researchers with biomass conversion and may be revised periodically. It is the
sole responsibility of the user of the protocols to obtain updated versions from the
NREL technical monitor. These procedures and their revisions by no means represent
optimal conditions for the described experimentation and are proposed simply as a
means of maintaining consistency. Furthermore, the results may vary depending on the
expertise of the researcher and the quality of the materials employed in the studies.
2.2 This LAP consists of two separate sub-procedures. The first is "Hydrolysis of
Lignocellulosic Biomass". The second is "Simultaneous Saccharification and
Fermentation of Biomass". This procedure is intended to test a variety of
lignocellulosic substrates and provide a consistent method for their evaluation among
NREL subcontractors. The procedures are intended for raw biomass substrates or
washed, pretreated substrates only i.e. pretreated substrates containing acetic acid,
furfural, and/or other inhibitors of yeast metabolism must be extensively washed with
water to remove these inhibitors prior to the experiments.
2.3 All analyses shall be performed according to the Ethanol Project Quality Assurance
Plan (QAP).
3. References
3.1 NREL Ethanol Project CAT Task Laboratory Analytical Procedure #001, "Standard
Method for Determination of Total Solids in Biomass".
3.2 NREL Ethanol Project CAT Task Laboratory Analytical Procedure #002,
"Determination of Carbohydrates in Biomass by High Performance Liquid
Chromatography".
3.3 NREL Ethanol Project CAT Task Laboratory Analytical Procedure #006 "Measurement
of Cellulase Activities".
3.4 NREL Ethanol Project CAT Task Laboratory Analytical Procedure #011,
"Determination of Ethanol Concentration in Biomass to Ethanol Fermentation
Supernatants by Gas Chromatography".
3.5 NREL Ethanol Project CAT Task Laboratory Analytical Procedure #013, "HPLC
Analysis of Liquid Fractions of Process Samples for Soluble Sugars".
3.6 NREL Ethanol Project CAT Task Laboratory Analytical Procedure #015, "HPLC
Analysis of the Liquid Fractions of Process Samples for Organic Acids, Glycerol,
HMF, and Furfural".
3.7 T. Vinzant, L. Ponfick, N. Nagle, C. Ehrman, J. Reynolds, and M. Himmel, "SSF

Comparison of Selected Woods from Southern Sawmills." 1994. Applied Biochemistry
and Biotechnology Vol. 45/46 pp 611-626.
3.8 G. Philippidis, T.K. Smith and C. Wyman, "Study of the Enzymatic Hydrolysis of
Cellulose for Production of Fuel Ethanol by Simultaneous Saccharification and
Fermentation Process." 1993. Biotechnology and Bioengineering Vol. 41 pg 846-853.
4. Terminology
4.1 Saccharification (SAC) or hydrolysis of lignocellulosic biomass: the addition of

enzyme to lignocellulosic biomass which results in the formation of oligomers,
cellobiose and glucose. It is performed under sterile conditions and is also referred to
as digestibility.
4.2 Simultaneous saccharification and fermentation (SSF): a method for producing ethanol
from lignocellulosic biomass in which both enzymatic saccharification of cellulose by
enzymes and fermentation of the resulting sugars by yeast occur at the same time in the
same vessel.

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Supersedes: 6/4/93
5. Apparatus
5.1 In addition to the equipment described in LAPs 01, 02, 06, 011, 013, and 015 the
following are required for this work.
5.2 An autoclave is necessary for the sterilization of media and flasks both prior to and
after experiments.
5.3 A laminar flow hood or biosafety cabinet is necessary for sterile sampling.
5.4 A -70oC freezer is necessary for the storage of frozen yeast cultures.
5.5 A bench top centrifuge is required for SSF sample preparation.
5.6 A shaker incubator is necessary for the SSF's in order to keep the fermentations at
38oC +/- 2oC and 150 rpm.
5.7 Bubble traps, also called gas locks, CO2 traps and water traps, are devices which
prevent air from entering the shake flask and at the same time allow carbon dioxide to
escape. They must be autoclavable. One such device is a rubber stopper through
which a glass tube is inserted. A cotton plug is placed in the tube and the tube is
connected to silicone tubing the end of which is submerged in a test tube with H2O.
The test tube is taped to the side of the flask. Another device that can be inserted into
a rubber stopper is all glass and has a u-tube filled with water. The carbon dioxide can
bubble out, but the water prevents the air from entering. SSF's require a bubble trap.
5.8 An analytical balance is necessary for accurately measuring out biomass samples and
preparing SSF flasks.
5.9 Cell counting chamber slide (for ex. hemocytometer) for yeast cell counts.
5.10 Microscope capable of 1000 times magnification.
5.11 Autoclavable shake flasks, Morton closures (metal caps), and sterile pipets
(disposable with tips that can be broken off conveniently to provide the wide opening
needed for sampling SSF slurries).
5.12 Convection oven, with temperature control of 80 ± 3°C, desiccator, and aluminum
foil weighing dishes for dry cell mass concentration measurements.

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5.13 A glucose analyzer is suggested for rapid analysis of glucose. Manufacturers include
Yellow Springs Instruments.
6. Solutions, Media, and Stock Cultures
6.14 10X YP medium (liquid)

Yeast extract 100 g/L
Peptone 200 g/L
Adjust pH to 5.0 with sulfuric acid. Autoclave for 30 minutes at 121oC.
Yeast Extract, Peptone, Dextrose (YPD) media is a common growth medium for yeast.
It is rich in amino acids, vitamins, and minerals necessary for yeast growth and
fermentation. This complex medium is supplied in excess, so that nutrients are not a
limiting factor. Although this yeast will grow at other pH conditions, pH 5 is chosen
because it is optimal compromise for SSF of most substrates when using common
cellulases.
6.15 YPD Plates (solid medium)

Peptone 20 g/L
Dextrose (glucose) 20 g/L
Agar 15 g/L
Dissolve dextrose in deionized (DI) water. Weigh agar into container, add glucose
solution. Autoclave for 30 minutes at 121oC. Let cool and add sterile 10X YP
medium. Mix solution gently, and aseptically pour the plates. Store plates inverted in
the refrigerator.
6.16 YPD medium (liquid)

Peptone 20 g/L
Dextrose (glucose) 20 g/L
Adjust pH to 5.0 with sulfuric acid and then filter sterilize (do not autoclave glucose
and YP together.)

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6.17 Antibiotics
If desired, use 0.2 mL of penicillin and/or 0.2 mL of streptomycin filter sterilized stock
solutions in the SSF mixture (stock solutions: Penicillin 5 g/L; Streptomycin 5 g/L).
Use of antibiotics is not recommended because of the added ES&H risks. (See ES&H
section)
6.18 Frozen stock culture of Saccharomyces cerevisiae D5A
50 mL sterile 40% glycerol

50 mL inoculum from NREL-supplied plate
Autoclave or filter sterilize a 40% solution of glycerol in DI water. Let cool to room
temperature. Prepare initial inoculum from the plate by transferring culture into 100
mL of YPD media in a sterile 250 mL flask. Incubate in a rotary shaker at 38oC for 24
hours. Test for pH, glucose, and ethanol. The pH should be between 4.5 and 5.0,
glucose should be between 0 and 5 g/L, and ethanol between 8 and 10 g/L. Observe
the culture under the microscope for bacterial contamination and culture purity. Mix
the glycerol and inoculum aseptically. Dispense one milliliter aliquots into sterile
cryovials. Place in a -70oC freezer. Each cryovial will have a standardized number of
yeast cells per vial and subsequent SSF inocula should be prepared using a frozen vial..
Once every three months perform a viability check on the frozen stock. Thaw one vial.
Vortex to resuspend cells. Perform a cell count with a hemacytometer under the
microscope. Then, perform colony forming unit (CFU) tests using YPD plates by
diluting the culture with sterile saline (0.89% NaCl solution) to obtain a spread plate
cell count of 30-300 cells/plate.
The number of cells on the plate multiplied by the dilution factor gives you the
CFU's/mL.
Percent viability is CFUs/mL divided by the hemacytometer count/mL. For example
1.0 x 107 CFU/mL and 1.0 x 108 cells counted/mL gives a viability of 10%. Make a
new frozen stock when the % viability drops below 50%. Maintain a control chart on
the viability.
6.19 Liquid cellulase enzyme preparation.
Filter sterilize all enzyme upon arrival. Use non-cellulosic based filters such as the
0.45 mm VacuCap 90 from Gelman, product number 4624 made of polyethersulfone.
Nylon and glass pre-filters are also suggested. Store enzyme in the refrigerator in
sterile containers. The activity should be monitored using the LAP-006, "Measurement
of Cellulase Activities". Cellulase activity values should be used to track enzyme

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stability over time. A control chart should be created for this purpose. However, the
SSF and SAC loadings need to be based on a consistent and standardized number, that
being the official NREL Filter Paper Units per milliliter number for that preparation.
This standardization allows us to more easily compare SSF and SAC data obtained
from different subcontracts.
6.21 Treat all biological growths with caution. Do not smell flasks as a method of checking
for contamination. Any contaminant microorganism has the potential of being a health
hazard.
6.22 The use of antibiotics in fermentations can lead to antibiotic resistant microbes which
can cause persistent infections in researchers. In addition, prolonged exposure to
antibiotics can cause allergic reactions to a variety of medications.
6.23 Avoid breathing dusts of yeast extract by weighing and transferring the solid in a
chemical fume hood. Yeast extract dust can coat the lungs and cause allergic reactions
and/or breathing problems. Dust masks are also recommended.
6.24 Autoclave all samples from SSF or SAC or inoculum prior to disposal. Treat
unautoclaved glassware, etc., with a 1% Chlorox or 30% hydrogen peroxide solution
to kill organisms prior to washing.
8. Procedure for the Hydrolysis of Lignocellulosic Biomass (SAC)
6.25 The goal of this procedure is to test pretreated or raw biomass substrates by determining
the initial hydrolysis (SAC) rate during saccharification catalyzed by cellulase
enzymes. Microorganisms are not employed in this experiment.
6.26 Based on the biomass moisture content (LAP-001) and cellulose content (LAP-002)
data, determine the quantity of biomass needed. Shake flasks should have stopper or
Morton closure and a 2:5 medium to flask volume ratio. All SAC flasks need to have
a 1% w/w effective cellulose content. Do not dry pretreated substrates; once dry
the pores of the biomass permanently collapse and do not have the same digestibility.
Example: 0.5263 g of alpha-cellulose is the weight needed based on the calculation for
a 50 gram working weight and 95% total solids. 4.167 grams is the weight needed of
a pretreated wood with a 60% cellulose content (LAP-002) and a 20% total solids
content (LAP-001) in a final working weight of 50 grams.

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6.27 Based on the filter paper activity of the cellulase enzyme (FPU/mL) and the working
weight of the flask, determine the volume of enzyme needed. All flasks need to have
an enzyme loading of 25 FPU/g cellulose based on the official NREL value for the
cellulase enzyme preparation. If all flasks in an experiment will use the same lot of
enzyme and the same working weight, then the same volume of enzyme (for example
1.5 mL) will be added to each flask.
Enzyme loading is the most critical factor affecting rates and yields. More accurate
addition of the enzyme can be obtained if it is diluted. Also, the new, diluted enzyme
can be freshly filtered to ensure sterility. Use freshly diluted sterilized enzyme for each
experiment to ensure enzyme activity has not decreased and sterility is maintained. Do
not store the enzyme in diluted form for over one day.
Example calculation: 1.506 mL of 1:10 diluted enzyme is needed for an experiment

where the diluted enzyme solution has an activity of 8.3 FPU/mL and a SAC flask
working volume is 50 mL. Prior to starting an experiment, prepare a fresh, filter sterile
(non-cellulosic filter) 1:10 dilution of enzyme in 1x media. Dilution is performed so
that the volume added can be accurately measured and easily dispersed into the flask.
6.28 Using the substrate weight and diluted enzyme volume determined in the previous
steps, calculate the amount of DI water and 10X YP medium needed. An example of
a SAC flask recipe is as follows:
4.167 g pretreated wood (1% w/w cellulose, substrate has a 60% cellulose
content and a 20% total solids content)
1.5 mL 1:10 diluted NREL-supplied cellulase enzyme
5.0 mL 10X YP solution
39.33 mL DI water (50 - 1.5 - 5 - 4.167)
50.0 grams total (working weight)
6.29 Each experiment should include an alpha-cellulose control substrate. All substrates
should be tested in duplicate at a minimum. A control chart should be set up to
compile the alpha-cellulose final yields over time.
6.30 Record the actual amount of substrate weighed into each flask to at least the nearest
milligram. For example 0.5321 grams of alpha-cellulose in Flask #1 and 0.5200
grams for Flask #2. Percent digestibility will be based on this number. Be as actuate
as possible and re-calculate the actual enzyme loading.
6.31 Add the DI water and 10X YP media. Gently swirl the flask and completely wet the
biomass chips.

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6.32 Check and adjust the pH of the slurry of each flask/vessel to 5.0 ± 0.2 with either lime
or sulfuric acid. Add stoppers to the flasks. Weigh to the nearest milligram the entire
flask set-up and record as pre-autoclave weight.
6.33 Since all the work up to this point has not been done aseptically, autoclave the flasks
and/or vessels within two hours. Autoclave at 121oC for 30 minutes (1 hour for a
fermentor containing 4 L of medium; make sure the vessel can ventilate freely). Let
the flasks/vessels cool to room temperature.
6.34 Reweigh the flasks to the nearest milligram and record the post-autoclave weight. Add
back lost weight as sterile mLs of DI water.
6.35 In a laminar flow hood aseptically add to the first flask or vessel, the required volume
of filter sterilized cellulase enzyme, as determined in step 8.3.
6.36 Mix the flasks by swirling. Aseptically take a time zero slurry sample. (see step 8.15).
6.37 Incubate the flasks in a rotary shaker at 150 rpm and 38oC. For vessels, set the agitation
speed at 150 rpm.
6.38 Repeat steps 8.11-8.13 with the other flasks. Start each flask individually and note the
time of completion for first and last flasks.
6.39 At appropriate sampling times (for example: 0, 3, 6, 24, 48, 72, and 168 hours) take 3
mL slurry samples aseptically with sterile large mouth pipet tips or pipets for flasks,
or through a port of about 0.5" internal diameter for vessels. Store in capped
tubes/vials. Place the samples on ice until all the samples of that specific time point
have been collected. Place the capped tubes/vials in a boiling water bath for exactly
5 minutes to inactivate the enzyme. Chill on ice.
6.40 Centrifuge and filter to remove denatured enzyme and lignocellulosic biomass.
Determine the amount of glucose present in each supernatant sample by YSI or HPLC
(LAP-013). Measure the concentration of cellobiose by HPLC (LAP-013) for at least
3 of the time-points. If the analysis will be done later, freeze the supernatant in sealed
HPLC glass vials.
6.41 For the last time point make samples for YSI and HPLC as in step 8.15-8.16. In
addition, streak a sample from each flask or vessel on a YPD plate to check for
contamination by any microorganism. Observe, under the microscope, a sample of the
slurry for the presence of contaminants. Report the final slurry pH of each flask or
vessel.

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6.42 All test tubes and flasks containing cultures or samples should be autoclaved prior to
disposal. All other items (e.g. pipettes, syringes) that come into contact with the
culture should be placed in containers of biocidal solutions before sterilization and
reuse or disposal. Contaminated pipette tips and microcentrifuge tubes should be
autoclaved before disposal.
6.43 Autoclave the residual hydrolysis material, making sure that the stoppers are loose
enough to allow ventilation. Ensure that fermentors can ventilate freely. Sterilized
liquids may be discharged down the drain after the solids are removed and thrown in
the trash.
6.44 Calculate the glucose yield as % of the theoretical yield (% digestibility) by using the
following formula:
[Glucose] + 1.053 [Cellobiose]
%Yield = X 100%
1.111 f [Biomass]
where:
[Glucose] Residual glucose concentration (g/L)
[Cellobiose] Residual cellobiose concentration (g/L)
[Biomass] Dry biomass concentration at the beginning of
the fermentation (g/L)
f Cellulose fraction in dry biomass (g/g)
The multiplication factor, 1.053, converts cellobiose to equivalent glucose.
6.45 Graph and/or tabulate the collected data (glucose and cellobiose concentration vs. time)
for each experiment.
6.46 Quality Control
6.46.1 Reported significant figures: Report % digestibility to one decimal place.
6.46.2 Replicates: At least duplicate flasks.
6.46.3 Blank: None.
6.46.4 Relative percent difference criteria: 5% digestibility within one set of flasks
run at the same time, by the same person, in the same shaker, with the same
analytical instrument.

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6.46.5 Method verification standard: Alpha-cellulose control. The moisture content
of alpha-cellulose must be measured every 3 months.
6.46.6 Calibration verification standard: None.
6.46.7 Sample size: See flask preparation instructions.
6.46.8 Sample storage: Store wet pretreated biomass in the refrigerator for no more
than 3 weeks, otherwise freeze it. Store dry (88% or more total solids)
biomass at room temperature. Do not use biomass that exhibits signs of
spoilage.
6.46.9 Standard storage: Alpha-cellulose is considered dry biomass and can be
stored at room temperature.
6.46.10 Standard preparation: None.
6.46.11 Definition of a batch: Flasks started at the same time with one set of alpha-
cellulose controls.
6.46.12 Control charts: % digestibility, at time final for alpha-cellulose and enzyme
activity controls. See 8.22.14.
6.46.13 Sterility verification: In all flasks, the final pH should be 5.0 " 0.2, no
microbes should be detected by microscope or plate checks. Flasks that did
not remain sterile must be repeated.
6.46.14 Enzyme activity: The filter paper activity, (LAP-006) FPU/mL, must be
measured every 6 months. Keep an activity control chart, but use the official
NREL cellulase activity number for enzyme loading calculations.
9. Procedure for Simultaneous Saccharification and Fermentation (SSF)
6.47 Inoculum preparation
6.47.1 The goal of this procedure is to prepare a seed culture for SSF. An aerobic
fermentation of glucose is used to produce yeast cell mass.
6.47.2 This inoculum preparation procedure involves two growth stages. The first
stage, pre-inoculum, is a flask in which the frozen stock culture, containing
a standardized number of cells, is inoculated into YPD (liquid medium). This
stage eases the yeast in its transition from stasis to growth phase. The growth

Issue Date: 5/31/95
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phase occurs in a second flask which contains YPD and is inoculated from
the first.
6.47.3 To prepare the first stage flask, transfer 50 mL of sterile YPD into a 125 mL
sterile baffled shake flask with a Morton closure (metal cap).
6.47.4 To prepare the second stage flask, transfer the desired amount of YPD into
the appropriate sterile Morton closure flask. Base flask size on sufficient
inoculum for the SSF experiment (10% v/w transfer) plus at least 20 mL for
sample analysis etc. Maintain a 2:5 liquid to flask volume ratio. Account for
a 10% v/v seed volume from the preinoculum.
Example: for an experiment containing six 100 g SSFs, 60 mL plus 20 mL

is 80 mL needed, then to give a 2:5 ratio, we round off to 100 mL in a 250
mL flask. The inoculum recipe would be as follows: 10 mL of preinoculum
into 90 mL of YPD in a sterile 250 mL baffled flask with a Morton closure.
6.47.5 Inoculate the first-stage YPD flask (pre-inoculum) with one thawed stock vial
of Saccharomyces cerevisiae D5A. Incubate at 38oC and 150 rpm for 6-8
hours. Before transferring to the next stage, check microscopically for
contamination. Only use pure cultures.
6.47.6 Inoculate the second-stage with a 10% v/v transfer from the pre-inoculum.
Incubate at 38oC and 150 rpm for 12-16 hours. Before transferring, check
microscopically for contamination and analyze for residual glucose
concentration. The culture can be transferred once the glucose falls below 2
g/L. Optimally, there should be some residual glucose to ensure cells are still
in the growth phase. Check pH and perform DCM (dry cell mass) analysis.
Prepare samples for HPLC and GC analysis at the end of the inoculation.
Read the ethanol content and use 10% of the value as the time zero ethanol
concentration in the SSF flasks. Use only pure D5A cultures.
6.47.7 Create an inoculum control chart with the final DCM, pH, ethanol, and
glucose concentrations for each inoculum.
6.48 Procedure For Dry Cell Mass Concentration
6.48.1 The goal is to measure the dry cell mass concentration.
6.48.2 Dry aluminum dishes in the oven at 80oC overnight.
6.48.3 Cool the dishes in a desiccator for 30 minutes.

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6.48.4 Record each dish weight to four decimal places using an analytical balance.
6.48.5 Using a sterile pipet, take a 10-mL inoculum sample, centrifuge, and wash the
cell pellet twice with 10 mL of DI water (2 volume wash). After the second
wash and centrifuge cycle, resuspend the pellet in 5 mL of DI water.
6.48.6 Transfer pellet by repeated vortex washes with DI water to a weighed dish.
6.48.7 Dry the dishes and cells in the oven at 80oC overnight.
6.48.8 Cool the dishes in the desiccator for 30 minutes.
6.48.9 Record the weight of the dishes plus dried cells.
6.48.10 Calculate the dry cell mass concentration of the inoculum in g/L by using the
following formula:
weight of dish plus dried cells − weight of dish
DCM =
0.01 L
6.49 Procedure for the Simultaneous Saccharification and Fermentation of Biomass
6.49.1 The goal of this procedure is to assess the conversion of lignocellulosic

biomass into ethanol using the SSF process. This procedure is almost
identical to the saccharification protocol differing only in the following ways:
(1) yeast is used to convert the glucose into ethanol, (2) cellulose content is
higher at 3% w/w, (3) bubble traps are used to maintain anaerobic
conditions and (4) carbon dioxide, ethanol, glycerol, lactic and acetic acid are
formed and residual glucose and cellobiose levels remain low.
6.49.2 Determine the amount of biomass needed for each SSF flask based on the
biomass moisture and cellulose content. All flasks should have a 3% w/w
effective cellulose concentration. Do not dry pretreated substrates. Once
dry, the pores of the biomass permanently collapse. Shake flasks should have
a 2:5 medium to flask volume ratio and should be equipped with water
traps.

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6.49.3 Based on the filter paper activity of the cellulase enzyme (FPU/mL) and the
working weight of the flask, determine the volume of enzyme needed. All
flasks should use an enzyme loading of 25 FPU/g cellulose using the
official NREL FPU/mL for the enzyme preparation. The volume of
enzyme added will be the same for all SSF flasks with the same working
weight. Prior to starting an experiment, prepare a fresh, filter sterile (non-
cellulosic filter) 1:10 dilution of enzyme in media. Dilution is performed so
that the volume added can be accurately measured and easily dispersed into
the flask. Do not store the enzyme in diluted form for over one day.
Enzyme loading is the most critical factor affecting rates and yields. More
accurate addition of the enzyme can be obtained if it is diluted. Also, the
new, diluted enzyme can be freshly filtered to ensure sterility.
6.49.4 Using the above substrate weight and diluted enzyme volume, calculate the
amount of DI water and 10X YP medium needed. Below is an example SSF
recipe for a 250 mL water-trap flask:
For example:
3.14 g alpha-cellulose (LAP-001, 95% moisture)
10.0 g 10X YP
10.0 mL S. cerevisiae D5A inoculum (second stage)
9.04 mL NREL supplied 1:10 diluted cellulase enzyme assuming an
undiluted volumetric activity of 83 FPU/mL
67.82 g DI water
--------
100.0 grams total
note: It is easier to add the enzyme and inoculum via a sterile pipet. The
density of both ingredients is assumed to be 1 g/L.
6.49.5 Each experiment should include an alpha-cellulose control substrate. All SSF
should be performed in duplicate at a minimum. A control chart should be
set up to include the ethanol yields from a standard final time point (for
example 168 hours) of alpha-cellulose SSF.
6.49.6 Tare the first flask, weigh in substrate and 10X YP media. Record the actual
amount of substrate weighed into each flask to at least the nearest milligram.
For example 3.167 grams of alpha-cellulose in Flask #1 and 3.1743 grams
for Flask #2. Percent theoretical ethanol will be based on this number. Be
as accurate as possible and re-calculate the actual enzyme loading.

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6.49.7 Check and/or adjust the pH of the slurry to 5.0 ± 0.2 with either lime or
sulfuric acid. Account for any change in weight by adding less DI water.
6.49.8 Add DI water. Gently swirl flask to completely wet the biomass chips in the
liquid.
6.49.9 Add water trap, autoclave tape, label etc. to flask (do not place any water in
the traps at this time.) Weigh the whole flask to the nearest milligram
assembly and record this weight as pre-autoclave. Repeat for each SSF.
6.49.10 Since all the work up to this point has not been done aseptically, autoclave
the flasks and/or vessels as soon as possible. Autoclave at 121oC for 30
minutes (1 hour for a fermentor containing 4 L of medium; make sure the
vessel can ventilate freely). Let the flasks/vessels cool to room temperature.
Re-weigh each flask assembly to the nearest milligram and add back lost
weight as mL of sterile DI water.
6.49.11 In a laminar flow hood aseptically add to the first flask or vessel:
(a) 10% v/w yeast inoculum (in this case 10 mL)

(b) Required mL of filtered sterilized 1:10 diluted cellulase enzyme (in
this case 9.04 mL)
(c) Swirl to mix flask ingredients well.
6.49.12 Add water to the water/CO2 trap of the flask and incubate the flasks in a
shaker at 150 rpm and 38oC. For vessels, set the agitation speed at 150 rpm.
6.49.13 Repeat with the other flasks. Start each flask separately and record the time
of completion for first and last flasks.
6.49.14 At appropriate sampling times (for example 24, 48, 72, 96, 120, 144, and 168
hours) take 4 mL slurry samples aseptically with sterile large mouth pipet tips
or pipets for flasks, or through a port of about 0.5" internal diameter for
vessels. Store in capped tubes/vials. Chill on ice, centrifuge, collect and filter
the supernatant. Analyze for glucose and cellobiose (LAP-013), glycerol,
lactic acid, and acetic acid (LAP-015) by HPLC, and ethanol (LAP-011) by
GC. If the analysis will be done later, freeze the filtered supernatant in
HPLC/GC glass vials.

Issue Date: 5/31/95
Supersedes: 6/4/93
6.49.15 For the last time point make samples for YSI, GC and HPLC. In addition,
streak a sample from each SSF flask or vessel on a YPD plate. Plates should
show viable yeast with no contaminant organisms. Observe slurry under the
microscope for presence of biomass fibers and yeast cells. The presence of
foreign organisms is cause for repeating the SSF. Read and record the slurry
pH of each flask or vessel. Final pH should be 5.0 +/- 0.7. Drop in pH to
less than 4.0 is cause for repeating the SSF. If possible, perform
compositional analysis (LAP-002) of the SSF residue and close mass balance.
6.49.16 All test tubes and flasks containing cultures or samples should be autoclaved
prior to disposal. All other items (e.g. pipettes, syringes) that come into
contact with the culture should be placed in biocidal solution before washing,
reuse and/or disposal.
6.49.17 Autoclave the residual SSF material, making sure that the CO2 traps are dry,
so that the flasks can ventilate. For fermentors, ensure that they can ventilate
freely. Sterilized liquids may be discharged down the drain after the solids
(biomass, dead yeast cells, and denatured enzyme) are removed and thrown
in the trash.
6.49.18 Calculate the ethanol yield as % of the theoretical yield by using the following
formula:
[EtOH ] f − [EtOH ] o
%Yield = X 100%
0.568 f [Biomass]
where:
[EtOH] f Ethanol concentration at the end of the
fermentation (g/L)
[EtOH] o Ethanol concentration at the beginning of the
fermentation (g/L)
[Biomass] Dry biomass concentration at the beginning of
the fermentation (g/L)
f Cellulose fraction of dry biomass (g/g)
0.568 Conversion Factor for cellulose to ethanol
based on stoichiometric biochemistry of yeast.

Issue Date: 5/31/95
Supersedes: 6/4/93
6.49.19 Report, graph, and/or tabulate information about each experiment including:
• Any observations about the experiment (e.g. foaming, color, etc.).

• Any deviations from the standard protocol.
• Residual and produced ethanol concentration, as well as theoretical
yield vs. SSF time (expressed in hours.)
• If possible, residual concentration of glycerol and other metabolites.
• Final pH of SSF.
• Contamination assessment and description of the morphology (shape,
color, size, and texture) of the colonies appearing on the plates,
including S. cerevisiae D5A.
6.50 Quality Control
6.50.1 Reported significant figures: Report % theoretical yield to one decimal place.
6.50.2 Replicates: At least duplicate flasks.
6.50.3 Blank: None.
6.50.4 Relative percent difference criteria: 5% yield within one set of flasks run at
the same time, by the same person, in the same shaker, with the same
analytical instrument.
6.50.5 Method verification standard: Sigma alpha-cellulose control. The moisture

content of alpha-cellulose needs to be measured every 3 months.
6.50.6 Calibration verification standard: None.
6.50.7 Sample size: Not applicable.
6.50.8 Sample storage: Store wet pretreated biomass in the refrigerator for no more
than 3 weeks, otherwise freeze it. Store dry (88% or more total solids)
biomass at room temperature. Do not use biomass that exhibits signs of
spoilage.
6.50.9 Standard storage: Sigma alpha-cellulose can be stored at room temperature.
6.50.10 Standard preparation: None.
6.50.11 Definition of a batch: Flasks started at the same time by the same researcher.

Issue Date: 5/31/95
Supersedes: 6/4/93
6.50.12 Control charts: Make a control chart of alpha-cellulose SSF results (final-day
ethanol (g/L), final pH, and % theoretical yield) for each experiment. Make
inoculum control chart, enzyme activity control chart, frozen stock viability
control chart.
6.50.13 Other: Verification of a pure D5A culture--all flasks--final pH should be 5.0

+/- 0.7, no microbes other than D5A should be detected by microscope or
plate checks. Contaminated flasks need to be repeated.
6.50.14 Enzyme activity: Measure enzyme activity using LAP-006, (Measurement of

Cellulase Activities) every 6 months. Tabulate and make a control chart.
Again, use the official NREL number for the SSF. Enzyme loading is the
most critical factor in rates and yields of ethanol production via SSF.

Issue Date: 5/31/95
Supersedes: 6/4/93
Ethanol Project

Procedure
Enzymatic Saccharification of Lignocellulosic
Procedure Title: Biomass
Author: Larry Brown and Robert Torget Date:

8/19/96

Issue Date: 8/26/96
Supersedes: 8/11/93
Enzymatic Saccharification of Lignocellulosic Biomass
1. Introduction
1.1 This procedure describes the enzymatic saccharification of cellulose from native or
pretreated lignocellulosic biomass to glucose in order to determine the maximum
extent of digestibility possible (a saturating level of a commercially available or in
house produced cellulase preparation and hydrolysis times up to one week are used).
2. Scope
2.1 This procedure is appropriate for lignocellulosic biomass. If the biomass is suspected
to have some starch content, dry weight percent cellulose calculated from total glucan
(LAP-002) must be corrected to subtract the starch contribution to total dry weight
percent glucose.
3. References
3.1 Grohmann, K., Torget, R., and Himmel, M. (1986), Biotech. Bioeng. Symp. No. 17,
135-151.
3.2 Ghose, T.K. (1987), Pure & Appl. Chem., 59, 257-268.
3.3 Stockton, B.C., Mitchell, D.J., Grohmann, K., and Himmel, M.E. (1991), Biotech. Let.,
13, 57-62.
3.4 Adney, B. and Baker, J. (1993), Ethanol Project Laboratory Analytical Procedures,
LAP-006, National Renewable Energy Laboratory, Golden, CO, 80401.
3.5 Ehrman, C. I. (1996), Ethnaol Project Laboratory Analytical Procedures, LAP-016,

National Renewable Energy Laboratory, Golden, CO, 80401.

Issue Date: 8/26/96
Supersedes: 8/11/93
4. Terminology
4.1 Pretreated biomass - Biomass that has been subjected to milling, chemical treatment
with water or steam, strong or dilute acid or alkali, or other physical or chemical
methods to render the cellulose content of the material more accessible to enzymatic
action.
4.2 Cellulase enzyme - an enzyme preparation exhibiting all three synergistic cellulolytic
activities: endo-1,4-β-D-glucanase, exo-1,4-β-glucosidase, or β-D-glucosidase
activities, which are present to different extents in different cellulase preparations.
5.1 The maximum extent of digestibility is used in conjunction with other assays to
determine the appropriate enzyme loading for saccharification of biomass.
6. Interferences
6.1 Test specimens not suitable for analysis by this procedure include acid- and alkaline-
pretreated biomass samples that have not been washed. Unwashed pretreated biomass
samples containing free acid or alkali may change solution pH to values outside the
range of enzymatic activity.
7. Apparatus
7.1 VWR model 1540 incubator set at 50o ± 1oC.
7.2 Cole-Parmer model 7637-20 "Roto-Torque" Fixed Speed Rotator.
7.3 A 24-slot large-holed test tube rack that can be attached to the "Roto-Torque" Rotator.
7.4 Eppendorf model 5414 microcentrifuge.
7.5 pH meter.
7.6 Analytical balance, sensitive to 0.0001 g.
7.7 Yellow Springs Instrument, Inc., Model 27 Glucose Analyzer or Model 2700 Select
Biochemistry Analyzer.
7.8 Drying oven adjusted to 105oC ± 2oC.
7.9 A 200 µL and a 1000 µL Eppendorf Pipetman pipet with tips.

Issue Date: 8/26/96
Supersedes: 8/11/93
8.1 Tetracycline (10 mg/mL in 70% ethanol).
8.2 Cycloheximide (10 mg/mL in distilled water).
8.3 Sodium citrate buffer (0.1M, pH 4.80).
8.4 Cellulase enzyme of known activity, FPU/mL.
8.5 -glucosidase enzyme of known activity, pNPGU/mL.
8.6 Solka Floc 200 NF, FCC (microcrystalline cellulose) from Brown Company
with ash, moisture, and xylan contents determined (see Ethanol Project
Laboratory Analytical Procedures, LAP-001, -002, and -005).
8.7 Eppendorf Safe-Lock 1.5-mL microcentrifuge tubes.
8.8 20-mL glass scintillation vials equipped with plastic-lined caps.
9.1 Cycloheximide and tetracycline are hazardous and must be handled with appropriate
care.
10. Procedure
10.1 Total solids must be determined for all cellulose containing samples to be digested
(LAP-001).
Note: all lignocellulosic materials which have undergone some aqueous

pretreatment must never undergo any drying whatsoever prior to enzyme
digestibility, since irreversible pore collapse can occur in the micro-structure
of the biomass leading to decreased enzymatic release of glucose from the
cellulose. Additionally, all frozen lignocellulosic materials which are to be
subjected to digestibility tests can not have been frozen for more than one
month prior to analysis, since, depending on the environment, sublimation
could have occurred, leading to possible irreversible collapse of micropores
in the biomass.

Issue Date: 8/26/96
Supersedes: 8/11/93
10.2 Weigh out a biomass sample equal to the equivalent of 0.1 g of cellulose on a 105oC
dry weight basis (the cellulose content of the sample is initially determined as glucose
by LAP- 002, minus the contribution of any starch present, LAP-016) and add to a 20
mL glass scintillation vial. Also, weigh out 0.1 g of the Solka Floc MVS and add to
another vial.

Issue Date: 8/26/96
Supersedes: 8/11/93
10.3 To each vial, add 5.0 mL 0.1 M, pH 4.8 sodium citrate buffer.
10.4 To each vial, add 40 µL (400 Fg) tetracycline and 30 µL (300 µg) cycloheximide to
prevent the growth of organisms during the digestion.
10.5 Calculate the amount of distilled water needed to bring the total volume in each vial
to 10.00 mL after addition of the enzymes specified in the following step. Add the
appropriate calculated volume of water to each vial. All solutions and the biomass are
assumed to have a specific gravity of 1.000 g/mL. Thus, if 0.200 g of biomass is added
to the vial, it is assumed to occupy 0.200 mL and 9.733 mL of liquid is to be added.
10.6 Bring the contents of each vial to 50oC by warming in the incubator set at 50o ± 1oC.
To each vial is added an appropriate volume of the cellulase enzyme preparation to
equal approximately 60 FPU/g cellulose and the appropriate volume of β-glucosidase
enzyme to equal 64 pNPGU/g cellulose.
Note: If the rate of enzymatic release of glucose is to be measured, all

contents of the vial prior to the addition of the enzyme must be at 50oC. The
enzymes are always added last since the reaction is initiated by the addition
of enzyme.
10.7 Prepare a reaction blank for the substrate. The substrate blank contains buffer, water,
and the identical amount of substrate in 10.00 mL volume.
10.8 Prepare enzyme blanks for cellulase and β-glucosidase with buffer, water, and the
identical amount of the enzyme.
10.9 Close the vials tightly and place them in the "Roto-Torque" fixed speed rotator set at
an approximate angle of 45oC that has been placed in the VWR incubator set at 50oC.
Incubate with gentle rotation (68 RPM) for a period of 72 to 168 hours or until the
release of soluble sugars from the sample(s) becomes negligible when measured by
YSI, as described in the next step.
10.10 If the progress of the reaction is to be measured, a 0.3-0.5 mL aliquot is removed at

each predetermined time interval after the vial contents have been well mixed by
shaking. This is accomplished by using a 1.0-mL Eppendorf Pipetman pipet with the
tip of the plastic 1.0-mL tip slightly cut off (to allow solids, as well as liquid, to be
withdrawn into the orifice). The sample is expelled into a 1.5-mL microcentrifuge tube
and centrifuged for 1.5 minutes. The supernatant is subjected to glucose analysis using
the YSI glucose analyzer.

Issue Date: 8/26/96
Supersedes: 8/11/93
11. Calculations
11.1 To calculate the percent digestibility of the cellulose added to the scintillation vial,
determine glucose concentration in the centrifuged supernatant by YSI. Subtract the
glucose concentrations, if any, from the substrates and enzyme blanks.
11.2 Correct for hydration (multiply the glucose reading by 0.9 to correct for the water
molecule added upon hydrolysis of the cellulose polymer) and multiply by 10 mL total
volume of assay.
Example: If the glucose analyzer reading (corrected with blanks) is 9.9 mg/mL, then
the amount of cellulose digested is:
0.0099 g/mL x 10 mL x 0.9 = 0.0891 g
11.3 Calculate percent digestion:
grams cellulose digested

% digestion = x 100
grams cellulose added
12. Report
12.1 Report the percent cellulose digested in the sample, to two decimal places, on a 105°C
dry weight basis. Cite the basis used in the report.
13.1 The precision of this protocol has not been defined because it is dependent upon
cellulase source and substrate composition. Not only will different preparations of
cellulase hydrolyze identical substrates to different extents, but different preparations
of pretreated biomass exhibit different amounts of homogeneity.
14. Quality Control
14.1 Reported significant figures: Typically results are reported as percentages, calculated
to two decimal places, along with the standard deviation and RPD. The assay
conditions, specifically digestion time, must be defined when reporting the results.

Issue Date: 8/26/96
Supersedes: 8/11/93
14.2 Replicates: It is recommended the samples be run in duplicate to verify reproducibility.
14.3 Blank: Enzyme and substrate blanks are run to correct for glucose contributions other
than that produced by cellulose hydrolysis.
14.4 Relative percent difference criteria: Not defined; dependent on the substrate being
tested. Different preparations of pretreated biomass will exhibit different amounts of
homogeneity, which will influence the extent to which they are hydrolyzed.
14.5 Method verification standard: Solka Floc 200 NF is digested alongside the samples.
Hydrolysis is expected to be in the range of 94.00 - 96.00%.
14.7 Sample size: Dependent upon percent dry weight cellulose composition. Typically
between 0.10 and 1.00 grams of sample will be required.
14.8 Sample storage: Pretreated samples should be stored moist, or frozen not longer than
one month.
14.11 Definition of a batch: A batch is defined as the sample replicates and method
verification standard hydrolyzed with an identical cellulase preparation and incubated
during the same time.
14.12 Control charts: Percent hydrolysis of Solka Floc 200 NF will be charted, use of
different preparations of cellulase enzyme and total hydrolysis time will be noted.

Issue Date: 8/26/96
Supersedes: 8/11/93
Ethanol Project

Procedure
Standard Method for the Determination of Extractives
Procedure Title: in Biomass
Author: Tina Ehrman Date: 4/22/94
ISSUE DATE: 4/22/94 SUPERSEDES: n/a

Revision: 4/22/94
Supersedes: n/a
Standard Method for the Determination of Extractives In Biomass
1. Introduction
1.1 With many types of biomass feedstocks it is necessary that the extractives be removed
from the sample prior to analysis to prevent interference with the analytical procedure.
Historically, ethanol-benzene has been used to extract waxes, fats, some resins, and
portions of wood gums. Subsequent hot water extractions were then used to remove
tannins, gums, sugars, starches, and coloring matter. Soxhlet extraction with 95%
ethanol has been found to be an effective, non-toxic alternative to extractions
employing benzene.
1.2 This procedure has been accepted by ASTM as an ASTM Standard Test Method for
the determination of extractives in biomass feedstocks.
2. Scope
2.1 This test method covers the determination of ethanol soluble extractives, expressed as
the percentage of the oven-dried biomass, of hard and soft woods, herbaceous
materials, agricultural residues, and wastepaper.
2.2 All analyses shall be performed in accordance with the Ethanol Project Quality
Assurance Plan (QAP).
3. References
3.1 ASTM D1105-84, "Method for Preparation of Extractive-Free Wood." In 1993 Annual
Book of ASTM Standards, Volume 04.09. Philadelphia, PA: American Society for
Testing and Materials.
3.2 Moore, W., and D. Johnson. 1967. Procedures for the Chemical Analysis of Wood and
Agriculture.
3.3 NREL CAT Task Laboratory Analytical Procedure #001, "Determination of Total
Solids and Moisture in Biomass."
3.4 NREL Chemical Technologies Research Branch Procedure #001c, "Determination of

Extractives Content."
3.5 TAPPI Test Method T204, "Solvent Extractives of Wood and Pulp." In Tappi Test

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Supersedes: n/a
4.1 Extractives, as defined by this procedure, are the fraction of a biomass sample soluble
in ethanol and that are left as a residue following exhaustive Soxhlet extraction.
Extractives include non-structural components of biomass samples which potentially
could interfere with the analysis of the biomass sample, and as such must be removed
prior to compositional analysis.
4.2 This method gives results comparable to ASTM Method D1105-84.
5. Apparatus
5.1 Soxhlet extraction apparatus - A glass Soxhlet extraction apparatus of suitable size
(100 mL) for containing the sample and a 250 mL collection flask is required for the
conventional Soxhlet procedure. An automated extraction apparatus (Brinkmann
Buchi B-810 or equivalent) with circulating oil bath and associated glassware is
required for the automated Soxhlet procedure.
5.2 Alundum extraction thimbles - Medium porosity (10 - 15 µm pore), sized to fit the
Soxhlet extractor.
5.3 Analytical balance - Sensitive to 0.1 mg.
5.4 Rotary evaporator with vacuum and water bath - Rotary evaporator equipped with a
"bump" trap, condenser, receiving vessel, and vacuum source sufficient to pull a
vacuum of less than 150 torr.
5.5 Vacuum oven or drying oven - Vacuum oven should be controllable to a temperature
of 40 ± 1°C and vacuum of between 75 to 100 torr. If drying oven is used in place of
the vacuum oven, the drying oven must be able to maintain 45 ± 2EC.
6.1 Ethyl alcohol, 95% in water (190 proof), USP grade.
6.2 Boiling chips.
6.3 Glass wool.
6.4 Buchner funnel.
6.5 Desiccator.

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Supersedes: n/a
7.1 Ethanol is a flammable reagent.
8. Sampling and Test Specimens
8.1 The test specimen shall consist of approximately 10 grams of milled sample obtained
in such a manner as to ensure that it is representative of the entire lot of material being
tested.
8.2 If the sample requires milling prior to extraction, pass the sample through a 40 mesh
screen (a laboratory scale Wiley mill is recommended for this milling step). Wet
samples will require air drying prior to milling.
9. Procedure
9.1 Dry the Soxhlet extraction thimble at 105°C to constant weight. Remove, cool to room
temperature in a desiccator, and weigh to the nearest 0.1 mg.
9.2 Carefully add the sample to the extraction thimble. Do not overfill the thimble, leave
at least a 1 cm gap between the sample and the top of the thimble. Weigh the filled
thimble to the nearest 0.1 mg. Place a plug of glass wool on top of the sample to
prevent sample loss during the extraction.
Note: Samples for total solids determination (following Laboratory

Analytical Procedure #001, Determination of Total Solids and Moisture in
Biomass) must be weighed out at the same time as the samples for the
extractives determination. If this determination is done at a later time, an
error in the calculation of the amount of extractives will be introduced, since
the moisture content of a biomass sample can change rapidly when exposed
to air.
9.3 Place several boiling chips into a clean, dry receiving flask or beaker. Weigh the
container, with chips, to the nearest 0.1 mg and record as the tare weight of the
container.
9.4 For a conventional Soxhlet extraction (this procedure was reproduced from the
Chemical Technologies Research Branch Procedure #001c, Determination of
Extractives Content):
9.4.1 Assemble the Soxhlet apparatus using at least 160 mL of 95% ethanol. Insert
the thimble and heat at reflux for 24 hours. Periodically check the reflux rate
and adjust the heating rate to give four to five solvent exchanges per hour in

Revision: 4/22/94
Supersedes: n/a
the Soxhlet thimble. Approximately 100-120 solvent exchanges are required
during the 24 hour period.
9.4.2 When the extraction time is complete, remove the thimble and carefully
transfer the sample to a Buchner funnel. Remove any residual solvent by
vacuum filtration and wash the sample thoroughly with 95% ethanol,
collecting all of the filtrate. Allow the biomass to air dry in the Buchner
funnel while it is still attached to the vacuum system.
9.4.3 Combine the filtrate from the previous step and any solvent from the upper
section of the Soxhlet apparatus with the solvent in the 250 mL flask. Place
the flask on the rotary evaporator and remove the solvent under vacuum. Use
a water bath temperature of 45 ± 5°C to heat the flask during evaporation.
9.4.4 After all of the visible solvent is removed by the rotary evaporator, place the
flask in a vacuum oven (75-100 torr) at 40 ± 1°C for 24 ± 1 hour. Remove
the flask at this time and allow to cool to room temperature in a desiccator.
Weigh the flask and record this total weight to the nearest 0.1 mg.
9.5 For an automated Soxhlet extraction:
9.5.1 Turn on the circulating oil bath, and set to 170oC.
9.5.2 Add approximately 100 mL of 95% ethanol to the receiving beaker. Place the
thimble containing the sample inside the extractor tube. Finish assembling
the automated Soxhlet extractor as directed in the instrument manual.
9.5.3 Begin the extraction, verifying that the reflux rate is ten to twelve solvent
exchanges per hour. Reflux for eight hours, giving a total of 80 to 100
solvent exchanges.
Note: An overnight extraction may be used. Select a circulating oil

bath temperature (and, indirectly, a solvent exchange rate) that will
produce approximately 100 solvent exchanges during the extraction
period. Verify that the apparatus is free of leaks so that there is no
loss of solvent during the period of unattended operation.
9.5.4 At the end of the extraction, remove the thimble and transfer to a Buchner
funnel. Remove any residual solvent by vacuum filtration and wash the
sample thoroughly with 95% ethanol, collecting all of the filtrate. Allow the
biomass to air dry in the Buchner funnel while it is still attached to the
vacuum system.

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9.5.5 Combine the filtrate, the solvent from the extractor tube, and the solvent in
the beaker. Evaporate to dryness using a rotary evaporator, as described in
the conventional Soxhlet extraction section, or alternatively by using the
automated extraction apparatus, as described in the next two steps.
9.5.6 Leaving the beaker in place on the heating block, decrease the temperature
of the circulating oil bath to 100oC. Evaporate away the solvent until only
about 10 mL remains.
9.5.7 Place the beaker in a drying oven set (45oC) or vacuum oven (75-100 torr and
40°C) for 24 ± 1 hour. Remove the beaker, cool to room temperature in a
desiccator, and weigh to the nearest 0.1 mg.
10. Calculations
10.1 Calculate the oven dry weight of the sample, using the average total solids content as
determined by the Laboratory Analytical Procedure #001, Determination of Total
Solids and Moisture in Biomass.
(Weight, thimble plus sample - Weight, thimble) x % Total solids

ODW =
100
Weight container plus residue - Tare wt. container

% Extractives = x 100
ODW
10.2 Calculate the amount of extractives in the sample, on a percent dry weight basis.
11. Report
11.1 Report the average percent extractives in the sample on an 105oC dried weight basis,
along with the standard deviation and the relative percent difference.
12.1 Data obtained by replicate testing of a hybrid poplar sample in one laboratory gave a
standard deviation in extractive content of 0.15% and a CV% of 7.6%. Replicate
testing of a National Institute of Standards and Technology (NIST) #8494 wheat straw
gave a standard deviation of 0.20% and a CV% of 1.6% and NIST #8493 Pinus radiata
gave a standard deviation of 0.20% and a CV% of 8.0%.
12.2 Prolonged heating of the extractive residue may bias the reported results low because
of evaporation of semivolatile constituents. Insufficient heating or using inadequate
vacuum can bias the results high because of incomplete removal of the ethanol solvent.

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13 Quality Control
13.1 Reported significant figures: All results shall be reported as a percentage with two
decimal places.
13.2 Replicates: All samples and method verification standards shall be analyzed in
duplicate.
13.3 Blank: It is recommended that a solvent blank be run with every batch of samples.
13.4 Relative percent difference criteria: The %RPD must be less than 10%. If the %RPD
is too large, the sample will be rerun.
13.5 Method verification standard: It is recommended that a method verification standard

be run with every batch. This standard is a material of known extractives content that
is run in parallel with the samples to track the reproducibility of the analysis.
13.7 Sample size: The sample, added to an extraction thimble, shall be a minimum of three
grams or the results will be flagged as having compromised precision.
13.8 Sample storage: Store the extracted sample in the refrigerator until needed for further
analysis.
recorded together. The maximum size of a batch is limited by the equipment
constraints. A batch cannot be larger than what is practical with the available
equipment.
13.12 Control charts: All method verification standards shall be control charted.

Revision: 4/22/94
Supersedes: n/a
Ethanol Project

Procedure
Determination of Ethanol Concentration in Biomass to
Procedure Title: Ethanol Fermentation Supernatants by Gas
Chromatography
Author: David W. Templeton Date: 5/5/94

Determination of Ethanol Concentration in Biomass to Ethanol
Fermentation Supernatants by Gas Chromatography
1 Introduction
1.1 Biomass is thought to be a good substrate for bioconversion to ethanol. This ethanol
could be used as a more environmentally benign transportation fuel. Various
processes have been developed to convert biomass to ethanol. The efficiency of a
process can be determined by measuring the yield of ethanol. Gas Chromatography
(GC) is a fast and accurate means of determining volatile components such as
ethanol.
2 Scope
2.1 This procedure details a method to prepare biomass to ethanol fermentation

supernatants for analysis and quantify their ethanol concentration. This packed
column GC method utilizes isopropanol as an internal standard to determine
concentration, in g/L, of ethanol in the fermentation supernatant. This method is
capable of analyzing fermentation supernatant ethanol concentrations ranging from
1 to 50 g/L.
2.2 This procedure describes an associated QA/QC program to demonstrate that the
results comply with the Ethanol Project Quality Assurance Plan. Adherence to the
Quality Assurance Plan is assumed for all work associated with this procedure.
3 Terminology
3.1 Biomass to ethanol fermentation-A process accomplished by enzymatically breaking

down biomass structural polymers to monomeric sugars, and utilizing yeast to
ferment these sugars to ethanol. Simultaneous saccharification and fermentation
(SSF) is one method used to convert biomass to ethanol.
3.2 Fermentation supernatant-The liquified portion of a biomass to ethanol fermentation,

including cells and biomass debris. For the purposes of the procedure, this refers to
slurry samples prior to preparation for analysis.
Procedure #011
Revision: 5/5/94
Supersedes: n/a 2 of 10
3.3 Analytical sample-A fermentation supernatant that has been filtered and diluted with
internal standard for GC analysis.
4.1 Ethanol is the desired product of the biomass to ethanol fermentation process.
Accurate quantitation of ethanol concentration is crucial to the design, assessment,
and improvement of the process.
5. Interferences
5.1 Biomass to ethanol fermentation supernatants contain high concentrations of organic

and inorganic residue. Much of this residue is not volatile and remains in the
injection port leading to fouling and activation. An activated injection port can cause
the catalytic breakdown of the analytes of interest, before they are detected, adversely
affecting the quantitation.
5.2 The biomass to ethanol fermentation process can produce a wide variety of volatile
compounds some of which may co-elute with ethanol or isopropanol. This can
adversely affect the quantitation.
6. Apparatus
6.1 Gas Chromatograph (HP 5890 or equivalent)-Utilizing a flame ionization detector

(FID), removable, deactivated glass, injection port liner, associated regulators, gas
lines, and septa.
6.2 Packed GC Column-6'x1/8" stainless steel Porapak Q (HP 19001A-Q00 or

equivalent).
6.3 Autoinjector (HP 7673 or equivalent)-Recommended.
6.4 Data handling system (HP Chemstation or equivalent)-Typically computer

controlled, but may be an integrator or chart recorder.
6.5 Centrifuge.
6.6 Pan balance-Readable to 0.01g.
Procedure #011
Revision: 5/5/94
7.1 Isopropanol-Reagent grade.
7.2 Ethanol-200 proof.
7.3 Ethanol-Separate source, either a 200 proof ethanol standard from a different
manufacturer than (7.2), or a commercially prepared solution of known
concentration.
Note: Store isopropanol and ethanol reagents appropriately, protect from

evaporation and moisture.
7.4 UHP nitrogen-GC carrier gas.
7.5 UHP hydrogen-FID fuel.
7.6 Zero air-FID oxidant.
7.7 Water-Reagent grade.
7.8 Repeat pipette(s)-Capable of delivering appropriate volumes.
7.9 Autosampler vials (2 ml) with associated caps.
7.10 Crimper-If necessary.
7.11 Capped centrifuge tubes.
7.12 0.45 µm filter-Either in line syringe filter unit or insert for centrifuge tube.
7.13 Assorted class A volumetric pipette and flasks.
7.14 Paper towel(s).
8.1 Utilize caution when handling compressed gasses (especially compressed

hydrogen).
8.2 Ethanol and isopropanol are flammable reagents.
Procedure #011
Revision: 5/5/94
9.1 Sample the fermentation so as to collect a representative slurry sample.
9.2 If collecting several samples, chill collected supernatants on ice while continuing to
collect more.
9.3 Fermentation slurries are to be centrifuged within one hour of sampling. This can be
accomplished by spinning at 6000 RPM for five minutes.
9.4 Filter the liquid portion of the sample through a 0.45 µm filter.
9.5 Dilute each filtered fermentation supernatant into a labeled autosampler vial. For
typical fermentation supernatants, a tenfold dilution brings the ethanol concentration
in the analytical sample within the linear range of the calibration curve. In this case,
100 µL of filtered fermentation supernatant is added, using a suitable repeat pipette,
to 900 µL internal standard spiking solution. If the concentration of the analytical
sample falls outside of the linear range, re-dilute the sample accordingly.
9.6 For every 25 analytical samples, prepare a sample in duplicate. Duplicates are used
to analyze the method precision.
9.7 For every 25 analytical samples, prepare a method verification standard by pipetting
100 µL intermediate calibration verification solution into 900 µL internal standard
spiking solution. This method verification standard is compared to the concentration
verification standard to verify confidence in the pipetting accuracy and technique.
Note: Regularly check the accuracy of repeat pipettes; they can be the source
of large errors.
9.8 Unless the same internal standard spiking solution is used for both analytical
standards and samples, prepare an internal standard check sample one for every 25
samples. Prepare by pipetting 100 µL intermediate calibration verification solution
into 900 µL of the internal standard spiking solution used for analytical samples. The
internal standard check sample is used to compare the different internal standard
spiking solutions.
9.9 Cap the autosampler vial snugly.
Procedure #011
Revision: 5/5/94
9.10 Refrigerate all samples. Analyze samples as soon as practicable. Do not store
analytical samples, prior to analysis, for more then one month.
10. Preparation of Apparatus
10.1 Clean the injection syringe before starting the analysis. Biomass to ethanol
fermentation supernatants tend to leave a sticky film on the plunger which affects the
precise operation of the syringe.
10.1.1 Remove the plunger from the syringe and wipe down using a paper towel
soaked in reagent grade water.
10.1.2 Place a few drops of reagent grade water on top of syringe and force several
volumes through with the plunger.
10.2 If possible, utilize any autosampler's self cleaning ability to extensively clean the
syringe between injections.
10.3 Change the deactivated glass injection port liner frequently. Typically the isopropanol
internal standard area counts begin to fall soon after running fermentation samples.
After the first twenty or so analytical sample injections, these values tend to stabilize
at a lower sensitivity. It is in this stabilized region that analysis takes place. After
approximately 100 additional injections the isopropanol internal standard area counts
become unstable and analytical precision is compromised. The injection port liner
must be changed at this time.
10.4 Change the GC septum after every 50 injections or so.
11. Calibration and Standardization
11.1 Prepare appropriate concentration internal standard spiking solution, using reagent
grade isopropanol. The internal standard spiking solution must be added in the same
proportion to every standard or sample analyzed by this method. This procedure
specifies nine parts of a 1 g/L internal standard spiking solution be added to one part
sample or standard. Therefore, the internal standard concentration is 0.9 g/L
universally throughout this procedure.
11.2 Prepare 3 to 6 ethanol analytical standards, using 200 proof ethanol, ranging from 0.1
to 5.0 g/L and all containing 0.9 g/L isopropanol.
Procedure #011
Revision: 5/5/94
11.3 Prepare 1-3 intermediate calibration verification solutions from the separate ethanol
source. Dilute 1:10 with internal standard spiking solution, using a volumetric pipette
and volumetric flask. When diluted in this manner, the intermediate calibration
verification solution becomes a calibration verification standard. The concentration
of the calibration verification standard(s) are to be within the range of the analytical
standards, but not equal to any of them.
11.3.1 Refrigerate analytical and calibration verification standards in sealed

autosampler vials for storage. Properly sealed, the analytical and calibration
standards are good for three months.
11.4 Prepare several solvent washes by dispensing reagent grade water into an autosampler
vial and seal.
12. Conditions
12.1 Oven temperature: 155oC (isothermal).
12.2 Inlet temperature: 175oC.
12.3 Detector temperature: 250oC.
12.4 Run time: 5.5 minutes.
12.5 Ethanol retention time: About 2.3 minutes.
12.6 Isopropanol retention time: About 4.1 minutes.
12.7 Carrier gas flow rate: 30 ml/min.
12.8 FID flow rates: Per manufacturer's recommendations.
12.9 Injection volume: 1 µl.
13. Procedure
13.1 Warm all standards and samples to room temperature and lightly mix the contents of
all the vials.
Procedure #011
Revision: 5/5/94
13.2 Set up analytical run as follows:
13.2.1 Solvent wash.
13.2.2 Analytical standards from low concentration to high.
13.2.3 Calibration verification standard.
13.2.5 A batch of samples in randomized order. A batch, typically, is a set of

samples from one time point, including associated method verification
standards, replicates, and internal standard check standards. A batch usually
does not exceed 20 samples.
13.2.6 Calibration verification standard.
13.2.8 Repeat 13.2.5-13.2.7, if necessary.
13.3 Recalibrate the standard curve after every 6 hours of analysis.
13.4 Do not reuse analytical or calibration verification standards.
14. Calculations
14.1 This method utilizes an internal standard which corrects for variations in the injection
volume.
14.2 Typically analytical software is utilized to perform concentration calculations. The

chromatograms are integrated based upon peak area. The calibration curve is
generated by a linear regression of the analytical standards, and ignores the origin as
a calibration point. Confirm the linearity of the calibration curve; the coefficient of
variation of the linear regression must be greater than 0.99.
14.3 If analytical software is not utilized, the calculation may be done manually. From the
standards data, create a calibration curve of amount ratio, plotted on the abscissa,
versus response ratio, plotted on the ordinate. The amount ratio is the ethanol
concentration divided by the isopropanol internal standard concentration. The
response ratio is the ethanol area divided by the isopropanol area.
Procedure #011
Revision: 5/5/94
Calculate a linear regression through the standard points, excluding 0,0 as a data
point. The equation of the resulting line should take the form of :
response ratio = slope (amount ratio) + (yintercept) For the analytical samples, the
response ratio is determined and the internal standard concentration is known. Use
the equation, above, to solve for the ethanol concentration in the samples.
14.4 The calculated fermentation supernatant concentration needs to account for any
dilution that occurred during the preparation of the analytical sample. In this case,
multiply the ethanol concentration of the analytical sample by ten.
15. Report
15.1 Report all results to the nearest 0.1 g/L.
16. Precision and Bias and Quality Control
16.1 Analysis in one laboratory of a calibration verification standard at the lower end of
the analytical range showed a recovery of 100.32% with a coefficient of variation of
3.60%.
16.2 Analysis in one laboratory of a calibration verification standard at the higher end of
the analytical range showed a recovery of 97.93% with a coefficient of variation of
4.67%.
17 Quality Control
17.1 Reported significant figures: Report all results to the nearest 0.1 g/L according to
section 15.1.
17.2 Replicates: Prepare one replicate for every 25 samples prepared according to section
9.6.
17.3 Blank: Prepared according to section 11.4. The solvent washes are used check for
any carryover problems. There should be no ethanol or isopropanol peaks in the
solvent wash runs.
17.4 Relative percent difference (rpd) criteria: Calculate the rpd for each duplicate set.
Limits for rpd have not been determined precisely, but a rpd greater than 15% is
cause for reanalysis.
Procedure #011
Revision: 5/5/94
17.5 Method verification standard: Prepared one for every 25 samples prepared according
to section 9.7.
17.6 Calibration verification standard: Prepared according to section 11.3.
17.7 Sample size: 100 µL of filtered fermentation supernatant prepared according to

section 9.5.
17.8 Sample storage: Refrigerated in autosampler vials for no more than a month
according to section 9.10.
17.9 Standard preparation: Per section 11.
17.10 Batch size: Defined in section 13.2.5.
17.11 Control Charts: Calculate the percent recovery for each calibration verification
standard run and control chart these values. Control chart the slope of the analytical
curve.
17.12 Matrix effects: It is assumed that the matrix (fermentation media) for these sample
will not vary greatly. If the matrix is considerably changed, it must be shown that the
new matrix does not affect the accuracy of this method.
Procedure #011
Revision: 5/5/94
Ethanol Project

Procedure
Standard Test Method for Moisture, Total Solids, and
Procedure Title: Total Dissolved Solids in Biomass Slurry and Liquid
Process Samples

6/13/94

Standard Test Method for Moisture, Total Solids, and Total Dissolved Solids
in Biomass Slurry and Liquid Process Samples
1. Introduction
1.1 A critical component of evaluating processes involved in the conversion of biomass is

the accurate compositional analysis of samples from those process streams. Although
these process samples can be analyzed as a whole sample (slurry), often they are
separated into solid and liquid fractions and analyzed separately. To be meaningful,
the results of chemical analyses of biomass are typically reported on a dry weight basis.
The following procedure describes the method used to determine the total solids (or
moisture) content of a slurry or the liquid fraction of a biomass process sample. This
procedure can also be used to determine the total dissolved solids content of such
samples.
2. Scope
2.1 This test method covers the determination of total solids (or moisture) in slurries or the
liquid fraction of samples generated during the pretreatment, fractionation, and
fermentation of biomass.
3. References
3.1 Moore, W.E., and D.B. Johnson. 1967. Procedures for the Chemical Analysis of Wood
and Wood Products. Madison, WI: USDA Forest Products Laboratory, U.S.
Department of Agriculture.
3.2 Technical Association of the Pulp and Paper Industry (TAPPI) Standard Method T OS-
63.
3.3 NREL Chemical Analysis and Testing Task Laboratory Analytical Procedure #001,
"Standard Test Method for the Determination of Total Solids in Biomass".
4. Terminology
4.1 The total solids content of a sample is the amount of material left as a residue upon
drying at 105oC to constant weight.
4.2 The total dissolved solids content applies to liquid or slurry samples and is defined in
this procedure as the amount of residue from the filtrate of a 0.2 µm filtered sample
that has been dried at 105oC to constant weight.
5.1 The total solids content is a measure of the amount of solids suspended or dissolved
in a process liquid or slurry. Conversely the moisture content is a measure of the
amount of water (and other components volatilized at 105oC) present in such samples.
5.2 The results of chemical analyses of processed biomass samples are typically reported
on a dry weight basis. The total solids content of a sample is used to convert the
analytical results obtained on another basis to that of a dry weight basis.
6. Apparatus
6.1 Automatic infrared moisture analyzer (such as Denver Instrument Company IR-100 or
equivalent) or a convection oven, with temperature control of 105 ± 2oC.
6.3 Desiccator.
7.1 Syringe filter, 0.8 µm prefilter over 0.2 µm final filter (such as Gelman Acrodisc µF
or equivalent).
7.2 Aluminum foil weighing dishes.
7.3 Quartz pads for weighing dishes.
7.4 Transfer pipettes.
9.1 Test specimens suitable for analysis by this procedure are slurries and the liquid
fraction of samples generated during the pretreatment, fractionation, or fermentation
of biomass. If the total solids (or moisture) contents of the solid fraction of these
process samples are to be determined, the Laboratory Analytical Procedure #001,
"Standard Method for the Determination of Total Solids in Biomass", must be used
instead.
9.2 The test specimen shall consist of approximately 3 to 10 g of sample obtained in such
a manner as to ensure that it is representative of the entire lot of material being tested.
Thorough mixing of slurries and liquid fractions with precipitates is of particular
importance.
10. Convection Oven Procedure
10.1 Accurately weigh a predried aluminum foil weighing dish to the nearest 0.1 mg and
then tare the balance.
10.2 For a total solids or moisture determination, thoroughly mix the sample and then weigh
out 3 to 10 g, to the nearest 0.1 mg, into the tared weighing dish. For total dissolved
solids, the sample added to the tared weighing dish should first be passed through a
0.8/0.2 µm syringe filter.
10.3 Place the sample into a convection oven at 105 ± 2oC and dry to constant weight
(±0.1% change in the amount of moisture present upon one hour of reheating).
Typically overnight drying is required for very wet samples.
10.4 Remove the sample from the oven and place in a desiccator; cool to room temperature.
10.5 Weigh the dish containing the oven-dried sample to the nearest 0.1 mg.
11. Infrared Moisture Analyzer Procedure
11.1 Program the automatic moisture analyzer for an analysis temperature of 105oC and for
a pre-determined end of analysis criteria of a rate of weight change that does not exceed
0.05% in one minute.
11.2 Verify that the instrument has reached the analysis temperature of 105oC and then place
an aluminum foil weighing dish with quartz pad on the balance pan. Wait five minutes
to ensure that the dish and pad are completely dry and then tare the balance.

Issue date: 7/11/94
Supersedes: none
11.3 For a total solids or moisture determination, quickly transfer 3 to 10 g of the thoroughly
mixed sample to the quartz pad in the weighing dish. For total dissolved solids, the
sample added to the quartz pad should first be passed through a 0.8/0.2 µm syringe
filter.
11.4 As soon as the instrument balance produces a stable weight, proceed with the analysis.
11.5 Once the sample has been dried to constant weight, as determined by the programmed
analysis parameters, the analysis will be automatically terminated by the instrument.
12. Calculations
12.1 Calculate the percent total solids or the percent moisture on a 105oC dry weight basis
as follows (the automated moisture analyzer will provide the selected calculated value
as part of the instrument printout):

% Total solids = x 100
(weight dried sample plus dish - weight dish)

% Moisture = [1 - ] x 100
12.2 Calculate the percent total dissolved solids on a 105oC dry weight basis as follows (the
automated moisture analyzer will provide the selected calculated value as part of the
instrument printout):

% Total dissolved solids = x 100
weight sample after 0.8/0.2 um filtration
13. Report
13.1 Report the result as a percentage with two decimal places.
relative percentage difference.

Issue date: 7/11/94
Supersedes: none
14.1 The precision of this analysis is determined by evaluation of %RPD data from samples
of many different types and assumes a 95% confidence interval. Based on this
evaluation, the precision of the infrared moisture analyzer procedure is 3% using a
Denver Instrument IR-100. The precision for the convection oven procedure is 1%.
14.2 An inherent error in any oven drying procedure is that volatile substances other than
water are removed from the sample during drying.
15. Quality Control
15.1 Reported significant figures: All data is reported with two decimal places.
15.2 Replicates: All samples are run in duplicate.
15.3 Relative percent difference criteria: For the infrared drying method the maximum
%RPD for duplicate analysis of a liquid samples is 9%. For the oven method the
maximum %RPD is 3%. If the %RPD is exceeded, the sample should be rerun.
15.4 Blank: This gravimetric analysis utilizes a balance blank with every batch of samples,
consisting of a weighing dish passed through all steps of the procedure.
15.5 Method verification standard: A method verification standard should be run with every
batch of samples. A solution containing 2% (w/v) sodium chloride can be prepared and
used for this purpose, provided it is stored in a tightly sealed container. Process 5.0 mL
of this solution in the same manner as a sample.
15.8 Sample size: 10 mL sample minimum, 15 mL recommended.
15.9 Sample storage: Samples should be refrigerated.

Issue date: 7/11/94
Supersedes: none
15.12 Control charts: The results of the method verification standard are to be control
charted.
16. Keywords
16.1 Moisture, total solids, biomass.

Issue date: 7/11/94
Supersedes: none
Ethanol Project

Procedure
HPLC Analysis of Liquid Fractions of Process
Procedure Title: Samples for Monomeric Sugars and Cellobiose
Author: Raymond Ruiz and Tina Ehrman Date:

6/28/96

Samples for Monomeric Sugars and Cellobiose
1. Introduction
1.1 Carbohydrates make up a major portion of biomass samples. These carbohydrates are
polysaccharides constructed primarily of glucose, xylose, arabinose, galactose, and
mannose monomeric subunits. During the processing of the biomass, such as dilute
acid pretreatment, a portion of these polysaccharides are hydrolyzed and soluble sugars
released into the liquid stream. Fermentation samples, whether they are time point
samples or end point residues, will also contain soluble sugars. The soluble sugars in
the liquid fraction of process samples can be quantified by HPLC with refractive index
detection.
2. Scope
2.1 This procedure is used to determine the soluble monosaccharide content of the liquid
fractions of biomass to ethanol process streams, including pretreatment liquors, liquid
fermentation time point samples, and the liquid fraction of fermentation residues. The
soluble sugar content indicates the amount of fermentable sugars available for
conversion to ethanol at specific process steps.
3. References
Agriculture.

Issue Date: 8/26/96
Supersedes: 11/01/94
4.1 The concentrations of monomeric sugars and cellobiose are used in conjunction with
other assays to determine the total composition of process stream samples.
5. Apparatus
5.1 Analytical balance, accurate to 0.1 mg.
5.2 pH meter, readable to 0.01 pH unit.
5.3 HPLC system equipped with refractive index detector.
5.4 Biorad Aminex HPX-87C and/or HPX-87P columns (or analytical HPLC columns
shown to give equivalent separations as the Biorad columns). equipped with the
appropriate guard columns.
Note: Deashing guard column cartridges from BioRad, of the ionic form
H+/CO3−, are recommended when using an HPX-87P column. These
cartridges have been found to be effective in eliminating baseline ramping.
6.1 High purity sugars for standards - cellobiose, glucose, xylose, arabinose, galactose, and
mannose.
6.2 Second set of the high purity sugars listed above, from a different source (manufacturer
or lot) for preparation of calibration verification standards (CVS).
6.3 Calcium carbonate, ACS reagent grade.
6.4 Water, HPLC grade or better, 0.2 µm filtered.
6.5 pH paper (range 2-9).
6.6 Disposable syringes, 3 mL, fitted with 0.2 µm syringe filters.
6.7 Autosampler vials with crimp top seals to fit.
6.8 Volumetric pipets, class A, of appropriate sizes.
6.9 Volumetric flasks, class A, of appropriate sizes.

Issue Date: 8/26/96
6.10 Adjustable pipettors, covering ranges of 10 to 1000 µL.
8.1 This analysis uses a multipoint calibration as described in the procedure.
9. Procedure
9.1 Thoroughly mix the sample and then measure and record the pH of a small aliquot to
the nearest 0.01 pH unit. It will be necessary to adjust the pH of the sample if this
reading falls outside of the operating pH range (5-9) of the HPLC column to be used.
9.2 Dilute the samples as needed, so the concentration of each sugar falls within the
validated range of the analytical method. Prepare each dilution in duplicate.
Note: It may be useful to determine initial glucose concentrations of the

samples using an alternative technique, such as YSI glucose analyzer, in
order to predict whether or not the sugars in the sample will fall within the
linear range of the analysis. In samples such as pretreatment liquors, xylose
typically is present at high concentrations, often three to five times the level
of glucose. In other samples, such as the liquid fractions of fermentation
samples, the levels of all the soluble sugars can be relatively low, and the
samples therefore will not require dilution.
9.3 Prepare method verification standards (MVS) by selecting representative samples to

be used to determine spike recoveries. Spike an accurately measured volume of each
selected sample with a known amount of the components of interest, such that the final
concentrations still fall within the linear range of the analysis. Process these spiked
samples along with the rest of the samples.
9.4 If the pH of the sample is less than 5, neutralize duplicate 10 mL aliquots with calcium
carbonate to a pH between 5 and 6. Avoid neutralizing to a pH greater than 6 by
monitoring with a pH strip. Add the calcium carbonate slowly after about pH 4 and
swirl frequently.
Note: Samples of pH greater than 9, such as alkaline pretreatment liquors,

should not be analyzed using the HPX-87C or HPX-87P columns unless the
sample pH can be brought into the operating range of the column, pH 5 to 9.
9.5 Pass the appropriately diluted and/or neutralized samples through 0.2 µm syringe filters
into autosampler vials in preparation for HPLC analysis. Seal and label the vials.
Reserve a portion of the undiluted sample in case repeat analyses are required.

Issue Date: 8/26/96
Store the reserved samples in the refrigerator.
9.6 Prepare a series of sugar calibration standards in HPLC grade water at concentrations
appropriate for creating a calibration curve for each sugar of interest. A suggested
scheme for the HPX-87C column is to prepare a set of multi-component standards
containing glucose, xylose, and arabinose in the range of 0.2 - 12.0 mg/mL. For the
HPX-87P column, cellobiose, galactose, and mannose should be included as additional
components in the standards. Extending the range of the calibration curves beyond
12.0 mg/mL will require validation.
9.7 Prepare an independent calibration verification standard (CVS) for each set of
calibration standards, using sugars obtained from a source other than that used in
preparing the calibration standards. The CVS must contain precisely known amounts
of each sugar contained in the calibration standards, at a concentration that falls in the
middle of the validated range of the calibration curve. The CVS is to be analyzed after
each calibration curve and at regular intervals in the HPLC sequence, bracketing groups
of samples. The CVS is used to verify the quality of the calibration curve(s)
throughout the HPLC run.
9.8 Analyze the calibration standards, the calibration verification standards, the samples,
and the method verification (spiked) samples by HPLC using a Biorad Aminex HPX-
87C or HPX-87P column for glucose, xylose, and arabinose. If cellobiose, mannose,
and galactose are also to be determined, only the Biorad Aminex HPX-87P column
should be used. For many analyses, it is useful to run the same samples on both
columns and compare the results. The following instrumental conditions are used for
both the HPX-87C and the HPX-87P columns:

Eluant: 0.2 µm filtered and degassed HPLC grade water.
Column temperature: 85oC.
10. Calculations
10.1 Create a calibration curve for each sugar to be quantified using linear regression. From
these curves, determine the concentration in mg/mL of the sugars present in each
sample analyzed by HPLC, corrected for dilution.

Issue Date: 8/26/96
10.2 Calculate and record the percent recoveries, %CVS, for each sugar contained in the
calibration verification standards analyzed by HPLC.
conc. detected by HPLC, mg/ml

% CVS recovered = x 100
known conc. of CV before HPLC analysis, mg/ml
10.3 Calculate and record the percent spike recoveries (% recovery MVS) for each sugar
used to prepare the method verification standards analyzed by HPLC.
10.3.1 Correct the initial sample concentration for the dilution resulting from the
addition of a known volume of spike solution.
V sample
C corrected = x C sample
V final
Where:Vsample = volume of sample prior to spiking, in mL.

Vfinal = final volume of solution (spike plus sample), in mL.
Csample = initial concentration of sample prior to spiking in
mg/mL, as determined by HPLC.
Ccorrected = concentration of sample after being corrected for
dilution, in mg/mL.
10.3.2 Calculate the percent recovery of the spike.
C actual - C corrected
% Recovery MVS = x 100
C spike
Where: Cactual = actual concentration of spiked sample, as determined

by HPLC, in mg/mL.
Ccorrected = concentration of sample after correcting for
dilution, in mg/mL, as calculated above.
Cspike = known concentration of spike solution added to
sample prior to analysis, in mg/mL.

Issue Date: 8/26/96
11.1 In the determine the cellobiose and monomeric sugar contents of process samples, the
neutralized samples are routinely analyzed using the HPX-87P column. When these
samples are known not to contain galactose and mannose, the HPX-87C column may
be used instead. Based on a root mean square evaluation of duplicate data, there is a
95% certainty that the "true value" will be within the range of the average plus or
minus:
- glucose 3.37% (HPX-87C) and 3.12% (HPX-87P),
- xylose 1.92% (HPX-87C) and 5.02% (HPX-87P).
Analytes at or near the detection limit could have significantly higher precision errors.
11.2 Samples containing significant amounts of protein, oligosaccharides, or other

compounds which elute early on the HPX-87C and HPX-87P columns will exhibit
baseline disturbances which may interfere with the quantification of the analytes.
12. Quality Control
12.1 Reported significant figures: Report all results in mg/mL with two decimal places. The
standard deviation and relative percent difference are also to be reported.
12.2 Replicates: All samples are to be run in duplicate. For replicate analyses of the same
sample, report the average, standard deviation, and relative percent difference (RPD).
12.3 Relative percent difference criteria: The maximum RPD for duplicate samples is as
follows: glucose, 5.8%, and xylose, 8.1%. If the stated RPD is exceeded, the sample
should be rerun. However, analytes at or near the detection limit could have
significantly higher RPDs.
12.4 Blank: The only requirement is an instrumental blank, consisting of the HPLC grade
water analyzed by HPLC in the same manner as the samples.
12.5 Method verification standard: This method will utilize a matrix spike as the method
verification standard, as indicated in the procedure.
12.6 Calibration verification standard: Calibration verification standards shall be

independently prepared and analyzed as described in the procedure.
size of a batch will be limited by the equipment constraints. A batch cannot be larger
12.8 Sample size: 30 mL minimum.

Issue Date: 8/26/96
12.10 Standard storage: Standards should stored frozen. Upon thawing, the standards should
be vortexed p and then shaken after thawing and again prior to use.
12.11 Standard preparation: Standards are prepared according to section 8.6 of this
procedure.
12.12 Control charts: All spike recoveries and calibration verification standards are control
charted.

Issue Date: 8/26/96
Ethanol Project

Procedure
Dilute Acid Hydrolysis Procedure for Determination of
Procedure Title: Total Sugars in the Liquid Fraction of Process Samples
Author: Raymond Ruiz and Tina Ehrman Date: 3/6/96

Dilute Acid Hydrolysis Procedure for Determination of Total Sugars
in the Liquid Fraction of Process Samples
1. Introduction
1.1 Carbohydrates make up a major portion of biomass samples. These carbohydrates are
polysaccharides constructed primarily of glucose, xylose, arabinose, galactose, and
mannose monomeric subunits. During dilute acid pretreatment of biomass, a portion
of these polysaccharides are hydrolyzed and soluble sugars released into the liquid
stream. These sugars, if present in oligomeric form, cannot easily be quantified
without further processing into their monomeric units.
2. Scope
2.1 This procedure is used to determine the total sugar content, including both
monosaccharides plus oligosaccharides, of the liquid fractions of biomass to ethanol
process streams, including pretreatment liquors, liquid fermentation time point
samples, and the liquid fraction of fermentation residues.
3. References
Agriculture.
4.1 The total sugar content is used in conjunction with other assays to determine the total
composition of process stream samples.

Issue Date: 8/12/96
4.2 The difference between the total sugar content determined by this procedure and the
monosaccharide content, determined by LAP #013, can be used to assess the
competence of specific process steps and is an indicator of the amount of non-
fermentable sugars present at that specific phase of the process.
5. Apparatus
5.2 pH meter, readable to 0.01 pH unit.
5.3 Pan balance, accurate to 0.01 g.
5.4 Autoclave, set to 121o ± 3oC.
5.5 HPLC system equipped with refractive index detector.
5.6 Biorad Aminex HPX-87C with corresponding guard column and/or HPX-87P column
with deashing guard column (or analytical HPLC column shown to give equivalent
separations as the Biorad columns).
6.1 High purity sugars for standards - glucose, xylose, arabinose, galactose, and mannose.
6.2 Second set of the high purity sugars listed above, obtained from a different source
(manufacturer or lot) for preparation of calibration verification standards.
6.3 Sulfuric acid, 72% w/w (specific gravity 1.6389 at 15.6oC/15.6oC).
6.4 Calcium carbonate, ACS reagent grade.
6.5 Water, HPLC grade or better, 0.2 µm filtered.
6.6 Erlenmeyer flasks, 25 mL.
6.7 Glass bottles, crimp top style, with rubber stoppers and aluminum seals to fit.
6.8 Pasteur pipettes.

Issue Date: 8/12/96
6.10 0.2 µm syringe filters.
6.11 Disposable syringes, 3 mL.
6.13 Volumetric pipets, class A, 20 mL.
6.15 Adjustable pipettors, coverings ranges of 10 to 1000 FL.
8.1 This analysis uses a multipoint calibration as described in the procedure.
9. Procedure
9.1 Thoroughly mix and then accurately measure out duplicate 20.0 mL portions of each
sample into labeled crimp-top bottles. If the available amount of sample is limited, this
procedure can by scaled back by using 10.0 mL portions. Other procedural steps must
then be scaled by accordingly.
Note: If the specific gravity of the sample is close to 1.0, the sample can be
measured out by accurately weighing 20.00 g portions on a pan balance
instead of by pipetting.
9.2 Dispense a separate aliquot of each sample into an Erlenmeyer flask, measure and
record the pH of each sample to the nearest 0.01 pH unit.

selected sample with a known amount of the sugars of interest, such that the final
concentrations of each component still falls within the linear range of the analysis.
Process these spiked samples along with the rest of the samples.

Issue Date: 8/12/96
9.4 Calculate the amount of 72% w/w sulfuric acid required to bring the initial acid
concentration of each sample to a 4% final acid concentration (refer to the example
presented in the calculation section). Swirl the sample while carefully adding the
required amount of acid.
9.5 Stopper the sample bottles and crimp aluminum seals into place.
9.6 Prepare a set of sugar recovery standards (SRS) that will be taken through the complete
analytical procedure to correct for losses due to the destruction of sugars during the
dilute acid hydrolysis. Weigh out the required amounts of each sugar to the nearest 0.1
mg, transfer the sugars to a crimp-top bottle, and add 20.00 g HPLC grade water. A
typical protocol for preparing the necessary sugar recovery standards is presented in the
following table:
Sugar recovery Target weights (for 20.00 mL total volume)

standard (SRS) Column
glucose xylose galactos arabinos mannos
e e e
G/X/A low either column 25 mg 25 mg --- 25 mg ---
G/X/A mid 100 mg 100 mg --- 100 mg ---
G/X/A high 200 mg 200 mg --- 200 mg ---
G/X/Gal/A/M HPX-87P 25 mg 25 mg 25 mg 25 mg 25 mg
low only
G/X/Gal/A/M 100 mg 100 mg 100 mg 100 mg 100 mg
mid
G/X/Gal/A/M 200 mg 200 mg 200 mg 200 mg 200 mg
high
9.7 To each sugar recovery standard, add 697 µL of 72% sulfuric acid (refer to example in
the calculation section). Stopper the bottles, label, and crimp aluminum seals into
place.
9.8 Autoclave the sealed samples, method verification standards, and sugar recovery
standards for one hour at 121oC. After completion of the autoclave cycle, allow the
hydrolyzates to cool somewhat before removing the seals and stoppers.

Issue Date: 8/12/96
9.9 Neutralize the hydrolyzates with calcium carbonate to a pH of 5 to 6. Add the calcium
carbonate slowly with frequent mixing to avoid problems with foaming. Monitor the
pH of the solutions with pH paper and, as the pH begins to approach 4.0, slow down
the rate of calcium carbonate addition and swirl frequently to avoid over-neutralizing.
Keep these neutralized samples cold.
9.10 Dilute the neutralized samples as needed, so the concentration of each sugar falls within
the validated range of the analytical method.
Note: It may be useful to determine initial glucose concentrations of the

samples using an alternative technique, such as YSI glucose analyzer, in
order to predict whether or not the sugars in the sample will fall within the
linear range of the analysis. The results from the LAP-013 analysis will also
be helpful in determining if dilution will be needed. In samples such as
pretreatment liquors, xylose typically is present at high concentrations, often
three to five times the level of glucose. In other samples, such as the liquid
fractions of fermentation samples, the levels of all the soluble sugars can be
relatively low, and the samples may not require dilution.
9.11 A portion of each appropriately diluted hydrolyzate is prepared for HPLC analysis by
passing the sample through a 0.2 µm syringe filter into an autosampler vial. The vial
is then sealed and labeled.
9.12 Prepare a series of sugar standards in HPLC grade water at concentrations appropriate
for creating a calibration curve for each of the sugars of interest. A suggested scheme
for the HPX-87C column is to prepare a set of multi-component standards containing
glucose, xylose, and arabinose in the range of 0.2 -12.0 mg/mL. For the HPX-87P
column, galactose and mannose should be included as additional components in the
standards.
Note: Extending the range of the calibration curves beyond 12.0 mg/mL will
require validation.
calibration standards. This CVS should contain precisely known amounts of each
sugar contained in the calibration standards, at a concentration that falls in the middle
of the validated range of the calibration curve. The CVS is to be analyzed at regular
intervals during the HPLC sequence and is used to verify the validity of the calibration
curves throughout the HPLC run.

Issue Date: 8/12/96
9.14 Analyze the calibration standards, the calibration verification standards, the hydrolyzed
sugar recovery standards, the hydrolyzed samples, and the hydrolyzed method
verification (spiked) samples by HPLC using an Biorad Aminex HPX-87C or HPX-
87P column for glucose, xylose, and arabinose. If mannose and galactose are also to
be determined, only the Biorad Aminex HPX-87P column should be used. For many
analyses it is useful to run the same samples on both columns and compare the results.
The following instrumental conditions are used for both the HPX-87C and the HPX-
87P columns:

Eluant: 0.2 µm filtered and degassed HPLC grade water.
9.15 Samples containing sugar levels falling outside the validated range of the calibration
curves must be rerun after appropriately diluting.
10. Calculations
10.1 For each sample and standard, calculate the volume of 72% sulfuric acid required to
bring the acid concentration to a 4% final acid concentration. The molar concentration
of hydrogen ions, [H+], in a sample can be calculated from its pH:
pH = -log[H+], therefore, [H+] = antilog(-pH).
The volume of 72% sulfuric acid to be added is then calculated from the following
[( C 4% x V s ) - ( V s x [ H + ] x 98.08g H 2 SO4 /2 moles H + )]

V 72% =
C72%
equation:
where: V72% is the volume of 72% acid to be added, in mL
Vs is the initial volume of sample or standard, in mL, which
includes the volume of spike added (if applicable)
C4% is the concentration of 4% w/w H2SO4, 41.0 g/L
C72% is the concentration of 72% w/w H2SO4, 1176.3 g/L
[H+] is the concentration of hydrogen ions, in moles/L

Issue Date: 8/12/96
Example #1: Calculate the amount of 72% H2SO4 needed to prepare a sample with a
pH of 2.41 for 4% acid hydrolysis. If the pH is 2.41, then [H+]=0.00389 M. Therefore:
[(41.0 g/L )(20 mL) - (20 mL)(0.00389 moles/L)(98.08 g/2 moles)]

= 0.694 mL
1176.3 g/L
Example #2: Calculate the amount of 72% H2SO4 needed to prepare a sugar recovery
standard for 4% acid hydrolysis. The standard itself is prepared in water with no added
acid, so the pH can be assumed to be about 7. Therefore [H+]=0.0000001 M, a value
small enough to be ignored in the following calculation.
(41.0 g/L x 20 mL) / 1176.3 g/L = 0.697 mL
10.2 Create a calibration curve for each sugar to be quantified using linear regression. From
these curves, determine the concentration in mg/mL of the sugars present in each
solution analyzed by HPLC.
10.3 Calculate and record the amount of each calibration verification standard (CVS)
conc. detected by HPLC, mg/mL

% CVS recovery = x 100
known conc. of standard, mg/mL
recovered following HPLC analysis.
10.4 For all three sugar recovery standards (SRSs), calculate the amount of each component
sugar recovered after being taken through the dilute acid hydrolysis procedure.
Average the % Rsugar values obtained for each individual sugar and report as % Rave,
sugar.

% R sugar = x 100
known conc. of sugar before hydrolysis, mg/mL
10.5 Use the percent hydrolyzed sugar recovery values calculated in the previous step to
correct the corresponding sugar concentration values obtained by HPLC for each of the
hydrolyzed samples (Ccor. sample) and each of the hydrolyzed spiked samples (Ccor. spiked
sample), accounting for any dilution made to the sample prior to HPLC analysis.

Issue Date: 8/12/96
C HPLC x dilution factor
Cx=
% R ave. sugar / 100
Where: CHPLC = conc. of a sugar as determined by HPLC, mg/mL.

% Rave. sugar = average recovery of a specific SRS component.
Cx = Ccor. sample or Ccor. spiked sample, concentration in mg/mL of
a sugar in the hydrolyzed sample or spiked sample
after correction for loss on 4% hydrolysis.
10.6 Calculate and record the percent spike recoveries (% MVS recovery) for each sugar
used to prepare the method verification standards analyzed by HPLC.
V sample
C cor. spiked sample - C cor. sample x
V final
% MVS recovery = x 100
C spike
Where: Ccor. = concentration of the hydrolyzed spiked

spiked sample
sample after being corrected for loss on 4%
hydrolysis, in mg/mL.
Ccor. sample = concentration in mg/mL of the hydrolyzed sample
after correction for loss on 4% hydrolysis.
Vsample = volume of sample prior to spiking, in mL.
Cspike = known concentration in mg/mL of the spike solution
added to a sample prior to analysis.
11.1 In the determination of the total sugar contents of process samples, the neutralized
hydrolyzates are routinely analyzed using the HPX-87P column. When these samples
are known not to contain galactose and mannose, the HPX-87C column may be used
instead. Based on a root mean square evaluation of duplicate data, there is a 95%
certainty that the "true value" will be within the range of the average plus or minus:
- glucose 4.86% (HPX-87C) and 2.90% (HPX-87P),
- xylose 2.73% (HPX-87C) and 3.09% (HPX-87P).

Issue Date: 8/12/96
12. Quality Control
12.1 Reported significant figures: All results are reported in mg/mL with two decimal
places. The standard deviation or relative percent difference are also to be reported.
12.2 Replicates: All samples are to be run in duplicate. For replicate analyses of the same
sample, report the average, standard deviation, and relative percent difference (RPD).
follows: glucose, 7.5%, and xylose, 5.0%. If the stated RPD is exceeded, the sample
should be rerun. However, analytes at or near the detection limit could have
significantly higher RPDs.
12.4 Blank: The only requirement is an instrumental blank, consisting of the HPLC grade
water analyzed by HPLC in the same manner as the samples.

12.8 Sample size: 50 mL per sample.
12.10 Standard storage: Standards should be frozen and then shaken vigorously upon
thawing.
12.11 Standard preparation: Standards are prepared according to instructions given in the
Procedure section of this protocol.
charted.

Issue Date: 8/12/96
Ethanol Project

Procedure
Procedure Title: Samples for Byproducts and Degradation Products
Author: Raymond Ruiz and Tina Ehrman Date:

8/22/96

Samples for Byproducts and Degradation Products
1. Introduction
1.1 During processing of biomass samples, such as in acid pretreatment of biomass, a liquid
portion is produced which may contain carbohydrate degradation products, such as
HMF and furfural, as well as other components of interest, such as organic acids and
sugar alcohols. These components are analyzed by HPLC with refractive index
detection to determine optimal production process parameters or to monitor ongoing
processes.
2. Scope
2.1 This procedure is used to determine the concentration of carbohydrate degradation

products as well as selected organic acids and sugar alcohols present in the liquid
fractions of biomass to ethanol process streams. These process streams include
pretreatment liquors, liquid fermentation time point samples, and the liquid fraction of
fermentation residues.
3. References
Agriculture.
4.1 This procedure is used to determine the amount of ethanol, selected organic acids and
sugar alcohols, and carbohydrate degradation products (such as HMF and furfural) in
the liquid fraction of biomass to ethanol process streams. Several of the compounds
being measured are potential inhibitors of the process, and are therefore important to
monitor.

Issue Date: 9/25/96
Supersedes: 11/1/94
4.2 The concentrations of these byproducts and degradation products are used in
conjunction with other assays to determine the total composition of process stream
samples.
5. Interferences
5.1 Arabitol coelutes with xylitol. If the sample is thought to contain arabitol, the
experimentally determined xylitol concentration should be flagged as potentially being
biased high due to the suspected arabitiol component.
5.2 The HPLC column used in this protocol is only partially capable of resolving the
monomeric sugars of importance in biomass analysis. Glucose, xylose, and arabinose
will be resolved, but galactose and mannose will coelute with xylose. If monomeric
sugars are present in concentrations far exceeding the concentrations of the analytes to
be quantified by this protocol, some of these analytes will appear as small humps on
the shoulders of larger peaks, leading to difficulties when integrating.
5.3 In addition to the glycerol, arabitol, and xylitol, some samples may contain sorbitol.
This sugar alcohol elutes about a minute earlier than xylitol on the Aminex HPX-87H
column, and will appear as a peak in between the xylose and arabinose peaks.
6. Apparatus
6.2 HPLC system equipped with a refractive index detector and a Biorad Aminex HPX-
87H analytical column (or equivalent) with corresponding guard column.
7.1 High purity standards - including xylitol, succinic acid, lactic acid, glycerol, formic
acid, acetic acid, ethanol, 5-hydroxy-2-furaldehyde (HMF), and furfural.
7.2 Second set of the high purity standards listed above, from a different source
(manufacturer or lot), to be used to prepare calibration verification standards (CVS).
7.3 Sulfuric acid, concentrated, ACS reagent grade.
7.4 Water, HPLC grade or better.
7.5 0.2 µm syringe filters.

Issue Date: 9/25/96
Supersedes: 11/1/94
7.6 Disposable syringes, 3 mL.
7.7 Solvent filtration system equipped with 0.2 µm filter.
7.9 Volumetric pipettes, class A, of appropriate sizes.
7.11 Adjustable pipettors, covering ranges of 10 to 1000 µL.
8.1 Sulfuric acid is corrosive and should be handled with care.
9.1 Care must be taken to ensure a representative sample is taken for analysis.
9.2 Store sample in a sealed container to ensure the concentration of its volatile
components do not change.
10. Procedure
10.1 Prepare 0.01N sulfuric acid for use as mobile phase in this analysis. In a one liter
volumetric flask, add 278 µL concentrated sulfuric acid and bring to volume with
HPLC grade water. Filter through a 0.2 µm filter and degas thoroughly before use.
10.2 Prepare the sample for HPLC analysis by passing it through a 0.2 µm syringe filter into
an autosampler vial. Seal and label the vial. Prepare each sample in duplicate.

selected sample with a known amount of the components of interest, such that the final
concentrations of each component still falls within the linear range of the analysis.
Process these spiked samples along with the rest of the samples.
10.4 Prepare a series of calibration standards containing the compounds that are to be
quantified, referring to Table 1 for suggested concentration ranges and special
considerations. If standards are prepared outside of the suggested ranges, the new
range for these calibrations curves must be validated. Since the retention times of two

Issue Date: 9/25/96
Supersedes: 11/1/94
pairs of components, xylitol/succinic acid and glycerol/formic acid, are so close, before
standards are prepared the column should be tested to verify adequate separation and
quantification can be achieved. If the separation is not adequate, either replace or
regenerate the column and then confirm improved separation has been achieved. If
separation is still not adequate, prepare standards without one of the components from
each pair (succinic acid for the first case, formic acid for the second). An second set
of standards must then be prepared containing the deleted component(s).
Table 1.
Expected Suggested *Special

Component retention time concentration range considerations
xylitol 11.6 min 0.2 - 6.0 mg/mL a
succinic acid 12.0 min 0.2 - 10.0 mg/mL a
lactic acid 13.2 min 0.2 - 12.0 mg/mL
glycerol 14.2 min 0.2 - 8.0 mg/mL a
formic acid 14.4 min 0.2 - 6.0 mg/mL a
acetic acid 15.5 min 0.2 - 12.0 mg/mL b
ethanol 22.7 min 1.0 - 15.0 mg/mL b
HMF 29.4 min 0.02 - 5.0 mg/mL c
furfural 42.8 min 0.02 - 5.0 mg/mL b,c
calib. verification -- middle of linear range --
meth. verification -- within linear range d
*Special considerations:
a = confirm xylitol/succinic acid and glycerol/formic acid can be adequately

separated before preparing standards.
b = samples containing volatile components must be submitted in sealed
containers.
c = the linear range for HMF and furfural is limited by their solubility; when
preparing a standard, add these components after the ethanol has been added
to make it easier to solubilize the HMF and furfural.
d = representative sample(s) should be chosen for spiking.

Issue Date: 9/25/96
Supersedes: 11/1/94
calibration standards, using components obtained from a source other than that used
in preparing the calibration standards. This CVS must contain precisely known
amounts of each compound contained in the calibration standards, at a concentration
that falls in the middle of the validated range of the calibration curve (refer to Table 1).
The CVS is to be analyzed after each calibration curve and at regular intervals in the
HPLC sequence, bracketing groups of samples. The CVS is used to verify the quality
of the calibration curve(s) throughout the HPLC run.
10.6 Analyze the calibration standards, calibration verification standards, method

verification standard, and the samples by HPLC using a Biorad Aminex HPX-87H
column. The following instrumental conditions are to be used for the HPX-87H
column:
Eluant: 0.2 µm filtered and degassed 0.01 N sulfuric acid.
Run time: 50 minutes.
10.7 If an analyzed sample or method verification standard (spiked sample) falls outside the
validated calibration range, dilute as needed and rerun the sample. The value can then
be reported after correcting for dilution.
11. Calculations
11.1 Create a calibration curve for each analyte to be quantitated using linear regression.
From these curves, determine the concentration in mg/mL of each component present
in the samples analyzed by HPLC, correcting for dilution if required.
11.2 Calculate and record the amount of each calibration verification standard (CVS)
recovered following HPLC analysis.

% CVS recovered = x100%
expected conc. of standard, mg/mL
11.3 Calculate and record the percent spike recoveries (% recovery MVS) for each
component used to prepare the method verification standards analyzed by HPLC.
11.3.1 Correct the initial sample concentration for the dilution resulting from the
addition of a known volume of spike solution.

Issue Date: 9/25/96
Supersedes: 11/1/94
V sample
C corrected = x C sample
V final
Where:Vsample = volume of sample prior to spiking, in mL.
Csample = initial concentration of sample prior to spiking in
mg/mL, as determined by HPLC.
Ccorrected = concentration of sample after being corrected for
dilution, in mg/mL.
11.3.2 Calculate the percent recovery of the spike.
C actual - C corrected
% Recovery MVS = x 100
C spike
Where: Cactual = actual concentration of spiked sample, as determined

by HPLC, in mg/mL.
Ccorrected = concentration of sample after correcting for
dilution, in mg/mL, as calculated above.
Cspike = known concentration of spike solution added to
sample prior to analysis, in mg/mL.
12.1 Based on a root mean square evaluation of duplicate data, there is a 95% certainty that
the "true value" will be within the range of the average plus or minus:
-glycerol, 5.52%; acetic acid, 4.58%;
-HMF, 6.15%; furfural, 5.61%;
-lactic acid, 5.85%; and succinic acid, 3.18%.
13. Quality Control
13.1 Reported significant figures: All results are reported in mg/mL, with two decimal
places. Also report the standard deviation and relative percent difference.
13.2 Replicates: All samples are run in duplicate.

Issue Date: 9/25/96
Supersedes: 11/1/94
follows: glycerol, 8.8%; acetic acid, 7.1%; HMF, 9.3%; furfural, 8.9%; lactic acid,
9.4%; and succinic acid, 4.5%. If the RPD exceeds the stated value, the sample should
be rerun. However, analytes at or near the detection limit could have significantly
higher RPDs.
13.4 Blank: The only requirement is an instrument blank, consisting of the HPLC grade
water analyzed by HPLC in the same manner as a sample.

size of a batch would be limited by the equipment constraints.
13.8 Sample size: 5 mL minimum.
13.9 Sample storage: Samples should be refrigerated. Those containing volatile

components must be stored in sealed containers.
13.10 Standard storage: Standards should be frozen in sealed containers or vials. Shake
vigorously upon thawing.
13.11 Standard preparation: Standards are prepared as described in the 'Procedure' section
of this protocol.
charted.

Issue Date: 9/25/96
Supersedes: 11/1/94
Ethanol Project

Procedure
Determination of Starch in Biomass Samples by
Procedure Title: Chemical Solubilization and Enzymatic Digestion

5/29/96

Determination of Starch in Biomass Samples by Chemical Solubilization
and Enzymatic Digestion
1. Introduction
1.1 Starch is a high molecular weight polymer consisting of glucose units linked by α-
glucosidic bonds. Starch consists of two glucose polymers, amylose and amylopectin.
Amylose is a linear polymer of glucose linked through α-D-1,4-glucosidic bonds while
amylopectin is a branched polymer consisting of α-D-1,4-glucosidic bonds with a small
number of α-D-1,6-glucosidic linkages present as interchain branch points. The
relative proportions of these polymers varies with the source, but typically contains 15
to 25% amylose and 75 to 85% amylopectin. Upon hydrolysis, starch is broken down
to a spectrum of higher and lower molecular weight oligosaccharides. Complete
enzymatic hydrolysis yields D-glucose, which can be analyzed using an immobilized
enzyme (glucose oxidase) technique.
starch + H2O amyloglucosidase

β-D-glucose (dextrose)
glucose oxidase
glucose + O2 ' H2O2 + D-glucono-δ-lactone
1.2 Amyloglucosidase (glucoamylase) is an exoglucosidase which catalyzes the hydrolysis

of both the α-D-1,6-glucosidic branch points and the predominating α-D-1,4-glucosidic
linkages of starch. Amyloglucosidase removes glucose units successively from the
nonreducing ends of starch chains and dextrins. The rate of hydrolysis of the α-D-1,6-
glucosidic branch points is much slower than the rate for the α-D-1,4-glucosidic
linkages.
2. Scope
2.1 This method covers the determination of starch in biomass samples. Sample material
suitable for this procedure include hard and soft woods, herbaceous materials,
agricultural residues, waste-paper, washed acid- and alkaline-pretreated biomass, and
the solid fraction of fermentation residues. All results are reported relative to the
105EC oven-dried weight of the sample. In the case of extracted materials, the results
may also be reported on an extractives-free basis.
Procedure #016
Issue Date: 9/25/96
Supersedes: n/a Page 2 of 9
2.2 This procedure is suitable for air-dried, lyophilized, and extracted biomass samples, as
well as for samples that have been oven dried at a temperature of 45oC or less. The
assay results will be biased slightly low for samples dried at 105oC. If sample
availability is limited, it may be necessary to run this analysis on a 105oC dried sample
but the results must be flagged as being biased low.
2.3 The assay is also suitable for wet samples if the particle size is known to be small and
if the moisture content of the sample can be estimated accurately enough to predict the
amount of sample needed to give 0.5 g of solids.
2.4 All analyses shall be preformed according to the guidelines established in the Ethanol
3. References
3.1 NREL Ethanol Project Laboratory Analytical Procedure #001, "Standard Method for
the Determination of Total Solids in Biomass".
3.2 Solvay Enzymes. 1996. Fungal glucoamylase for Starch Hydrolysis. Diazyme L-200
Technical Notes.
3.3 YSI Incorporated. 1994. Determination of Cook in Extruded Cereal Products.

Application Note #319.
4.1 The percent starch content is used in conjunction with other assays to determine the
total composition of biomass samples.
5. Interferences
5.1 Interference by free glucose and cellobiose present in samples is not a problem because
both glucose and cellobiose are destroyed during the NaOH solubilization step.
6. Apparatus
6.2 YSI 2700 Select Biochemistry Analyzer - equipped with a YSI 2365 dextrose
membrane and YSI 2357 buffer and calibrated with YSI 2776 2.5 g/L calibrator
solution.
Procedure #016
Issue Date: 9/25/96
7.1 Glucose calibration verification standards, such as YSI 2.0 and 9.0 mg/mL glucose
standards.
7.2 Amyloglucosidase (suggested source, Sigma A-3042).
7.3 Amylopectin (suggested source, Sigma A-7780).
7.4 Methanol, ACS reagent grade.
7.5 Hot plate or a water bath set at 90o ± 2oC.
7.6 Graduate cylinders of appropriate sizes.
7.7 100 mL, 500 mL, and 1000 mL volumetric flasks, class A.
7.8 5 and 10 mL pipets or adjustable pipettor.
7.9 Timer.
7.10 Water bath set at 40o " 1oC.
7.11 Serum bottles or Erlenmeyer flasks, 125 mL.
7.12. Prepared reagents:
7.12.1 2N NaOH - weigh 40 grams of sodium hydroxide pellets into a 500 mL

volumetric flask. Add 300 mL of reagent grade water and mix. Cool, dilute
to volume and mix.
7.12.2 2N HCl - measure 82.4 mL of concentrated hydrochloric acid and transfer to

a 500 mL volumetric flask. Let cool, dilute to volume with reagent grade
water and mix.
7.12.3 Acetate buffer (pH 4.2) - weigh 9.1 grams of sodium acetate into 500 mL
volumetric flask. Add about 300 mL of reagent grade water and mix until all
the solid is dissolved. Add 22.3 mL (23.4 grams) of glacial acetic acid.
Dilute to volume with water and mix.
Procedure #016
Issue Date: 9/25/96
7.12.4 Amyloglucosidase working solution - prepare a fresh working solution of the
enzyme such that it contains 60 units of activity per milliliter. If using the
Sigma A-3042 amyloglucosidase, dilute the solution one hundred-fold into
cold reagent grade water. Prepare daily and store in the refrigerator.
7.12.5 25% TCA - dissolve 50.0 grams of trichloracetic acid in 200 mL reagent
grade water.
7.12.6 Phosphate buffer - dissolve 40 g NaH2PO4 and 10 g Na2HPO4 in reagent

grade water, bring to volume in a 1000 mL volumetric flask.
8.2 Trichloroacetic acid is a hazardous chemical; appropriate precautions must be taken.
9. Procedure
9.1 The sample must not contain particles larger than 0.25 mm in diameter. If milling is
required to reduce the particle size of the test specimen, a laboratory mill equipped with
a 40 mesh, or smaller, screen should be used. If the sample size is too small to allow
the use of a laboratory mill, a coffee grinder may be used instead.
Note: If pretreated samples are to be analyzed, the sample must be

thoroughly washed to remove any residual acid or alkali prior to drying.
9.2 Duplicate portions of each sample must be weighed out for a total solids determination
(following LAP-001) at the same time as the portions for the starch determination. If
this is done later, it can introduce an error in the calculation because ground biomass
can rapidly gain or lose moisture when exposed to the atmosphere. Record the average
total solids value as Tfinal.
9.3 Weigh out approximately 0.50 g of sample to the nearest 0.0001 g and transfer to a 125
mL serum bottle or Erlenmeyer flask. Record as Wsample, the initial sample weight.
Each sample must be run in duplicate, at minimum.
Procedure #016
Issue Date: 9/25/96
9.4 A standard reference material, amylopectin, is run in parallel with each batch of
samples and its calculated recovery used to correct the sample results for losses due to
the procedure. Weigh 0.5 g portions of amylopectin to the nearest 0.0001 g and transfer
to serum bottles or Erlenmeyer flasks. Record the weight as Wstandard, the initial
standard reference material weight. The standard must be run in duplicate, at
minimum. As with the samples, the total solids content, Tfinal, of the standard reference
material must also be determined.
9.5 Add 25 mL of reagent grade water to each bottle. Swirl to ensure the sample is wetted
and evenly dispersed.
Note: A few drops of methanol may be used to prewet the sample which will
aid in its dispersion once the water is added.
9.6 Add 10 mL 2N NaOH to the solution in each bottle. Place bottles on a heating unit or
in a water bath preheated to 90oC. Heat for 20 minutes, swirling periodically to wet
any sample that may be clinging to the side of the bottle. A glass stirring rod may be
needed to break up clumps of material.
9.7 Add 10 mL 2N HCl to each bottle and swirl to mix. Cool the bottles to below 50oC.
9.8 Add 10 mL of acetate buffer to each bottle and swirl to mix.
9.9 Add 5.0 mL amyloglucosidase working solution to each bottle. Mix well and place the
bottles in a 40oC water bath for 60 minutes.
9.10 After 60 minutes incubation, remove the bottles from the water bath. Immediately add
5 mL of 25% TCA to each bottle to stop hydrolysis.
9.11 Cool to room temperature and transfer each hydrolyzate to a 100 mL volumetric flask.
Rinse out the bottle with small volumes of phosphate buffer and transfer all the rinses
to the volumetric flask. Bring to volume with phosphate buffer and mix well.
9.12 Since the enzyme solution may contain free glucose, an enzyme blank must be run in
parallel with the samples. Dilute duplicate 5.0 mL portions of the amyloglucosidase
working solution to 100 mL with reagent grade water in a volumetric flask. These
enzyme blanks will be analyzed in the same manner as the sample, with the averaged
results used to correct the glucose contents of the samples.
Procedure #016
Issue Date: 9/25/96
9.13 The sample itself may contain free glucose, which normally would be analyzed as
starch. However in this procedure the glucose, and also cellobiose, is destroyed in the
NaOH solubilization step. Therefore no correction for free glucose is needed when
calculating the total starch content of a sample.
9.14 Set up and calibrate the YSI as described in the manufacturer's manual using the
dextrose membrane, YSI 2357 system buffer, and YSI 2776 2.5 g/L calibrator solution.
Program the instrument to autocalibrate every fourth sample or every fifteen minutes,
set the sample size to 25 µL, and use the following probe parameters:
Chemistry - dextrose
Units - g/L
Calibrator - 2.50 g/L
End point - 30 seconds
Cal station # - 1
9.15 Verify the calibration of the YSI using the glucose calibration verification standards
before starting the run. Reverify the calibration periodically during the analysis and
at the end of the run.
9.16 Measure the glucose levels in the enzyme blanks and in all the samples. The
validated linear range of the instrument is 0 - 9.0 g/L dextrose. If the value reported
exceeds the validated range, the hydrolyzate must be diluted appropriately and rerun.
10. Calculations
10.1 Calculate the amount of starch recovered from each analysis of the amylopectin
standard reference material as follows, on a 105oC dry weight basis, and then average
the recoveries:
( YSI standard , g/L - YSI enzyme blank , g/L) x total volume, L

% Standard recovered = x 0.9 x 100%
% total solids, T final
standard weight, g,W standard x
100
Note: Amylopectin recoveries of 93 to 95% have routinely been achieved

with this protocol. Recoveries less than 90% indicate the data generated for
the batch of samples should be rejected and the analysis repeated.
Procedure #016
Issue Date: 9/25/96
10.2 Calculate the amount of starch present in each sample, on a 105oC dry weight basis:
( YSI sample , g/L - YSIsubenzyme blank , g/L) x total volume, L

% Starch = x 0.9 x 100%
% total solids, T final
sample weight, g,W sample x
100
Note: The factor 0.9 converts grams of glucose to grams of the anhydrosugar
(starch, in this case). The factor can be calculated by dividing the molecular
weight of glucose less one molecule of water (180 - 18) by the molecular
weight of glucose.
10.3 The calculated percent starch in each sample can be corrected for assay losses using
the percent recovery of the standard reference material, amylopectin, as follows:
% starch
% Starch, corrected = x 100%
average % standard recovered
11. Report
11.1 Report the percent starch present in the sample, to two decimal places, on a 105oC
dry weight basis.
12.1 Data obtained by replicate testing of a corn stover in one laboratory gave a standard
deviation of 0.28% and a CV of 1.31%. Data obtained by replicate testing of a
amylopectin reference material in three laboratories gave a standard deviation of
0.58% and a CV of 4.61%.
12.2 This procedure has been validated for materials which have been air-dried,
lyophilized, or oven dried at a temperature of 45oC or less. The assay results will be
biased slightly low for samples dried at 105oC. If sample availability is limited, it
may be necessary to run this analysis on a 105oC dried sample but the results must
be flagged.
Procedure #016
Issue Date: 9/25/96
13. Quality Control
13.1 Reported significant figures: Report the percentage of starch present in the sample to two
decimal places, on a 105°C dry weight basis, or extractives-free basis. Cite the basis used
in the calculation.
13.3 Blank: As described in the “Procedure” section, enzyme blanks are prepared in duplicate
and analyzed in the same manner as the samples.
13.4 Relative percent difference criteria: The RPD must be less than 8.0%. If the RPD is too
large, the sample must rerun.
13.5 Method verification standard: In this procedure, amylopectin is used as both a method
verification standard and as a means for estimating and correcting for assay losses. At
minimum, the amylopectin must be run in duplicate with every batch of samples. If the
recovery of amylopectin is less than 90%, the data generated for the whole batch of
samples must be rejected and the analysis repeated.
13.6 Calibration verification standard: Calibration verification standards may be purchased

from YSI or independently prepared. The CVSs are analyzed periodically through the
YSI run as described in the “Procedures” section.
13.7 Sample size: A minimum of 3.6 grams of sample (on a dry weight basis) are required for
duplicate analyses, which includes both the starch and the total solids assays. If there is
insufficient sample, the result will be flagged and the lack of precision noted.
13.8 Sample storage: Dried samples shall be stored in an airtight container at room
temperature. Samples with moisture contents greater than 10% shall be stored in an
airtight container and refrigerated for not longer than one week. If longer periods of
storage are required, these samples must be stored frozen.
13.9 Standard storage: YSI standards should be stored refrigerated and should not exceed the
manufacturer’s stated expiration data.
13.10 Standard preparation: Purchase the YSI 2776 2.5 g/L calibrator solution from YSI.
13.11 Definition of a batch: Any number of samples which are analyzed and recorded together.
The maximum size of a batch would be limited by the equipment constraints.
13.12 Control charts: The result of each replicate analysis of the method verification standard
(amylopectin) is recorded along with the average, RPD, and a laboratory book/page
Procedure #016
Issue Date: 9/25/96
E t h a n o l Project

Procedure
Determination of O-Acyl Groups in Biomass by High
Procedure Title: Performance Liquid Chromatography
Author: Tina Ehrman and Raymond Ruiz Date:

07/31/98
ISSUE DATE: 07-31-98 SUPERSEDES: n/a

Determination of O-Acyl Groups in Biomass by High Performance Liquid Chromatography
1. Introduction
1.1 Aliphatic groups in wood and herbaceous feedstocks are acetyl and formyl groups
which can be combined as O-acyl groups with the polysaccharide portion. A number
of different approaches can be used to analyze for acetyl, including acid hydrolysis,
saponification, transesterification, spectrophotometirc, and aminolysis. Acid hydrolysis
was selected as the approach of choice since the hydrolyzate produced during the
routine compositional analysis of cellulosic samples could also be used in the analysis
of O-acyl groups. In this approach, dilute acid is used to split O-acyl groups from the
polysaccharides. The resulting acetic and formic acids are then quantified by HPLC.
2. Scope
2.1 This procedure describes a HPLC method for determining the amount of acetyl and
formyl groups cleaved upon hydrolysis of a biomass sample. The protocol utilizes the
hydrolyzate generated by LAP-002, "Determination of Carbohydrates in Biomass by
High Performance Liquid Chromatography". Special handling during the post-
autoclave steps of LAP-002 is required to ensure the volatile components are not lost.
2.2 Sample material suitable for this procedure include hard and soft woods, herbaceous
materials (such as switchgrass and sericea), agricultural residues (such as corn stover,
wheat straw, and bagasse), and waste-paper (such as office waste, boxboard, and
newsprint). Pretreated biomass may also be analyzed by this method, although most
pretreatment conditions will have already removed the acetyl and formyl groups. All
results are reported relative to the 105EC oven-dried weight of the sample. In the case
of extracted materials, the results may also be reported on an extractives-free basis.
3. References
3.1 Solar, R., F. Kacik, and I. Melcer. 1987. Simple Semimicro Method for the
Determination of O-Acetyl Groups in Wood and Related Materials. Nordic Pulp and
Paper Research Journal, 4:139-141.
Procedure #017
Issue Date: 07/31/98
3.2 Ethanol Project Laboratory Analytical Procedure #001, "Standard Method for the
Determination of Total Solids in Biomass".
3.3 Ethanol Project Laboratory Analytical Procedure #002, "Determination of

Carbohydrates in Biomass by High Performance Liquid Chromatography".
3.4 Ethanol Project Laboratory Analytical Procedure #003, "Determination of Acid-

3.5 Ethanol Project Laboratory Analytical Procedure #004, "Determination of Acid-Soluble

Lignin in Biomass".
3.6 NREL Ethanol Project Laboratory Analytical Procedure #010, "Standard Method for
the Determination of Extractives in Biomass".
4. Terminology
4.1 Prepared Biomass - Biomass that has been prepared by lyophilization, oven drying, air
drying, and in some instances by extraction, to reduce the moisture content of the
sample so it is suitable for O-acetyl group analysis.
4.2 Oven-Dried Weight - The moisture-free weight of a biomass sample as determined by

LAP-001, "Standard Method for Determination of Total Solids in Biomass".
5.1 The percent acetyl and formyl group contents are used in conjunction with other assays
to determine the total composition of biomass samples.
6. Interferences
6.1 Formic acid is produced not only from the cleaving of O-formyl groups but also from
the hydrolysis of HMF. The amount of levulinic acid present may be used as a rough
indicator of the source of the formic acid, since levulinic acid is produced in equal
molar quantities with formic acid when HMF is hydrolyzed.
7. Apparatus
7.1 Hewlett Packard Model 1090 HPLC, or equivalent, with refractive index detector.
7.2 HPLC column, BioRad Aminex@ HPX-87H (or equivalent).
Procedure #017
7.3 Guard column, cartridges appropriate for the HPLC column used.
8.1 Reagents
8.1.1 High purity chemicals for standards (98%+) - two sets of acetic acid, formic
acid, and levulinic acid from different lots or manufacturers.
8.1.2 0.01 N H2SO4.
8.2 Materials
8.2.1 Disposable nylon syringe filters, 0.2 µm.
8.2.2 Disposable syringes, 3 mL.
8.2.3 Autosampler vials, with crimp top seals to fit.
9.2 Use caution when handling hot glass bottles after the autoclave step, as they may have
become pressurized.
10.1 The hydrolyzates generated as part of LAP-002 or LAP-003 are to be utilized in this
method. Sample weight, total solid content, and extractive content (if needed) are
determined as specified in the LAP-002 or LAP-003 protocols. The unused portion of
the hydrolyzate can be taken through the LAP-002 and LAP-003 as desired.
Procedure #017
11. Procedure
11.1 Upon removal of the hydrolyzed biomass samples from the autoclave (as part of the
LAP-002 or LAP-003 procedure), allow the contents of the bottles to cool for 20
minutes at room temperature. Swirl the contents of each bottle vigorously and allow
to cool further, until the contents are at room temperature and the solids have settled.
11.2 Working with one bottle at a time, remove the seal and stopper, taking care not to stir
up any solids which have settled to the bottom.
11.3 Using a disposable syringe, immediately remove about 1.5 mL of sample, again taking
care not to disturb the settled solids. Quickly pass this aliquot through a 0.2 µm filter
into an autosampler vial. Immediately cap the vial.
11.4 Prepare a series of calibration standards in deionized water at concentrations

appropriate for creating a calibration curve for each component of interest. A
suggested scheme for the HPX-87H column is to prepare a set of multi-component
standards containing acetic acid, formic acid, and levulinic acid in the range of 0.02 to
0.5 mg/mL.
11.5 Prepare an independent calibration verification standard (CVS) using chemicals

obtained from a source other than that used in preparing the calibration standards. The
CVS must contain precisely known amounts of each component contained in the
calibration standards, at a concentration that falls in the middle of the validated range
of the calibration curve. The CVS is to be analyzed after each calibration curve and at
regular intervals in the HPLC sequence, bracketing groups of samples. The CVS is
used to verify the quality of the calibration curve(s) throughout the HPLC run.
11.6 Analyze the calibration standards, the CVS, the MVS, and the samples by HPLC using
a Biorad Aminex@ HPX-87H column. The following instrumental conditions are
used:
Sample volume: 50 FL.

Eluant: 0.2 µm filtered and degassed 0.01 N H2SO4.
Column temperature: 55°C.
Run time: 20 minutes (refer to precautionary note which follows).
Procedure #017
Note: The hydrolyzates being tested will contain low levels of HMF and/or furfural.
These components will appear as peaks in the chromatogram of the following sample.
It is important to verify the HMF and furfural peaks are not interfering with the peaks
of interest. With the instrument set up used to develop this method, use of a 20 minute
run time resulted in the HMF peak appearing at about 10 minutes into the following
chromatogram, and the furfural peak appearing at about 19 minutes. Neither peak
interfered with the analytes of interest.
12. Calculations
12.1 Create a calibration curve by linear regression analysis for each component to be
quantified. From these curves, determine the concentration in mg/mL of the organic
acids present in each solution analyzed by HPLC.
12.2 For lyophilized, air dried, or oven dried samples, or for samples requiring no
preparation, calculate the percentage of each organic acid present in the sample on an
as received 105°C dry weight basis follows:
1g 1g
Cx xV F Cx xV F
1000 mg 1000 mg
% Analyte = x 100% = x 100%
% T as received % T final
W1 x W1 x
% T prep 100%
Where: W1 = initial weight of sample, in grams

C = concentration of component in hydrolyzed sample in mg/mL
%Tas received = % total solids content of the original sample (prior to any
preparation steps) on a 105°C dry weight basis, as determined by the
LAP-001
%Tprep = % total solids content of the sample as determined during the
preparation of the sample for analysis (by lyophilization, oven-drying,
or air drying)
%Tfinal = % total solids content of the prepared sample used in this analysis,
on a 105°C dry weight basis, as determined by the LAP-001
Note: If the sample used in the analysis required no preparation (analyzed as received),
then %Tprep = 100% and %Tfinal = %Tas received.
Procedure #017
12.3 If the biomass was prepared according to the "Standard Method for the Determination
of Ethanol Extractives in Biomass" (LAP-010), first calculate the percentage of each
organic acid on an extractives-free 105°C dry weight basis and then correct this value
to an as received (whole sample) 105°C dry weight basis.
12.3.1 Calculate the percentage of each component on an extractives-free basis as

follows:
1g
Cx xV F
1000 mg
% Analyteextractives - free = x 100%
% T final
W1 x
100%
Where: C = concentration of component in hydrolyzed sample in mg/mL

W1 = initial weight of extracted sample, in grams
%Tfinal = % total solids content of the prepared sample used in this
analysis (in this case, extracted sample), on a 105°C dry weight
basis, as determined by LAP-001
12.3.2 Correct the % organic acid value on an extractives-free basis, calculated

above, to an as received (whole sample) 105EC dry weight basis as follows:
% Analytewhole sampleW
= % Analyteextractives - free x
100%
h
W
Where: % Analyteextractives-free = % analyte on an extractives-free

105°C dry weight basis, as determined in the previous step
% extractives = % extractives in the extracted sample as
described in the Standard Method for the
Determination of Extractives in Biomass (LAP-010)
Procedure #017
13. Report
13.1 Report the percent of each analyte to two decimal places, on a whole sample 105°C dry
weight basis or on an extractives-free basis. Cite the basis used in the report.
14.1 Data obtained by replicate testing of a hybrid poplar in two laboratories, using an HPX-
87H column, gave a standard deviation in acetic-acid content of 0.08% and a CV% of
1.77%.
15. Quality Control
15.1 Reported significant figures: Report the percentage of each analyte present in the
hydrolyzed sample to two decimal places, on a whole sample 105°C dry weight basis
or on an extractives-free basis. Cite the basis used in the calculation.
15.3 Blank: The only requirement is a reagent blank specified in LAP-002 and LAP-003,
which starts out as an empty 16x100 mm test tube (ie, no sample) which is taken
through all the procedural steps.
15.4 Relative percent difference criteria: The RPD for acetic-acid must be less than
2.75%. If the RPD is too large, the sample must be rerun.
with every batch. This method utilizes a well characterized standard material suitable
for analysis. For example, NIST 8492 (Populus deltoides) is used as the MVS in O-
acyl analysis of hardwoods.

independently prepared and analyzed as described in section 11.5 of this procedure.
15.7 Sample size: As specified in LAP-002 and LAP-003, a minimum of 0.6 grams of
sample (on a dry weight basis) are required for duplicate analyses. If there is
insufficient sample, the result will be flagged and the lack of precision will be noted.
Procedure #017
15.8 Sample storage: Samples shall be stored in an airtight container and refrigerated.
15.9 Standard storage: Standards should be kept frozen in airtight vials or test tubes.
Vortex the standards vigorously upon thawing to ensure thorough mixing.
15.10 Standard preparation: Standards are prepared as described in section 11.4 of this
method.
Procedure #017
Ethanol Project

Procedure
Determination of Insoluble Solids of Pretreated Biomass
Procedure Title: Material
Author: Fannie Posey Eddy, Jody Okafor, Candic Date: 9/23/98

Roberson
ISSUE DATE:9/18 SUPERSEDES:

Determination of Insoluble Solids in Pretreated Biomass
1. Introduction
1.1 Pretreated biomass samples are composed of water-soluble and water insoluble
components. These two fractions are analyzed separately. Analytical results are then
mathematically recombined in the computation of the mass balance on the pretreatment
process. To separate the water soluble portion from the sample a thorough water
extraction is performed leaving a portion of solid material called the insoluble solids
fraction of the sample. This method describes two reliable procedures for determining
both the percent insoluble solids and percent fraction insoluble solids in a sample of
hydrolyzate slurry from pretreated biomass.
2. Scope
2.1 This procedure is intended to determine the percentage of water insoluble solids in a
pretreated biomass sample after all soluble components have been extracted with
aggressive water washing.
2.2 All analyses shall be performed according to guidelines established by the Ethanol
Quality Insurance Plan.
3. References
3.1 NREL Ethanol Project Laboratory Analytical Procedure #012, "Standard Test Method
for Moisture, Total Solids, and Total Dissolved Solids in Biomass Slurry and Liquid
Process Samples.
4. Terminology
4.1 Pretreated Biomass-Biomass which has been chemically and/or thermally altered to
change the structural composition.
4.2 Hydrolyzate slurry-The liquid and solid material in a sample resulting from biomass
pretreatment.

Issue Date:
Supersedes:
4.3 Hydrolyzate Liquor- Liquid portion of hydrolyzate slurry.
4.4 Washed Solids-Water insoluble portion of hydrolyzate slurry.
4.5 Pressate-Liquid product from pretreated biomass pressed via centripetal force, manual
or hydraulic pressure.
4.6 Filtrate-Hydrolyzate liquid product from hydrolyzate slurry which has been placed in
a Buchner funnel and vacuum filtered.
4.7 Oven Dried Weight-The moisture-free weight of a biomass sample as determined by

Lap-001, Standard Method for Determination of Total Solids in Biomass.
4.8 Insoluble Solids (IS)-The oven dried weight of water insoluble solids divided by the
weight of whole hydrolyzate slurry sample (as received).
4.9 Fraction Insoluble Solids (FIS)-The oven dried weight of water insoluble solids
divided by the oven dried weight of the whole hydrolyzate slurry.
5.1 The percent insoluble solids and the percent fraction insoluble solids are used to
combine the liquid and solid compositions of the pretreated biomass in the mass
balance determination.
6. Interference
6.1 Technique is critical to minimizing the amount of material lost during the wash steps.
Care should be taken when separating the liquid from the solids or results will not meet
standard quality assurance requirements.
6.2 Hydrolyzate slurries separate quickly. Special attention is recommended when taking
samples .To obtain a representative sample thoroughly mix prior to taking a sample.
7. Apparatus Procedure A (Centrifugation)

Issue Date:
Supersedes:
7.2 Convection ovens with temperature control to 45 °C ± 3 and 105 °C ± 3.
7.3 Analytical balance readable to 0.01g.
7.4 Desiccator.
7.5 Centrifuge refrigerated to 4°C and rotor specified to hold 300 ml. capacity bottles and
rated at least 9,000 rpm.
7.6 Aluminum foil weighing dish.
7.7 Centrifugation bottles with wide opening, caps with seals and 300 ml
capacity/reservoir.
7.8 Biochemical analyzer (YSI) for measurement of glucose (optional).
7.9 Eppendorf microcentrifuge tubes and transfer pipettes (optional).
8. Apparatus Procedure B (Filtration)
8.1 Whatman GF/D 2.7um glass microfiber FilterCup (1600R823) with FilterCup stem
(1600R900). An alternate is Buchner funnels (two-part, polypropylene) with GF/D
glass microfiber filter paper.
8.2 Glass microfiber filter paper sized for the Buchner funnel chosen.
8.3 1000 ml vacuum flask.
8.4 Filtration set-up including vacuum source and vacuum adapters for Buchner funnels.
8.5 Items 1-4 as described in section 7.
8.6 Teflon coated spatulas.

Issue Date:
Supersedes:
9.2 Electronic pipette and disposable pipettes in the range of 25 ml.
9.3 2,000 ml flask or beaker.
9.4 YSI dextrose standards 2.0 g/L, 9.0 g/L (optional).
9.5 Water 18 megaohm deionized.
11. Procedure A (Centrifugation)
11.1 Record weight of centrifuge bottle and cap to nearest the 0.01g on an analytical balance
and tare.
11.2 Add 25-50 grams of sample. Record weight of sample as received to the nearest 0.01g.
11.3 Add 175-200 grams of deionized water. Shake vigorously for 60 seconds. (Note: If
collecting liquor for further analysis, do so prior to this step).
11.4 Cap and spin in centrifuge at 4°C for 20 minutes at 9,000 rpm.
11.5 Carefully remove bottles from centrifuge to minimize disturbance of sample pellet.
11.6 Remove liquid portion by decanting without disturbing sample pellet. Use electronic
or manual pipetter to remove small amounts of liquid until small traces are left.
11.7 Repeat steps 10.3-10.6 three more times or until sample pH is at least 4.5-7.0. (For
some applications it may be necessary to test the amount of glucose present by
biochemistry analyser in determining if another wash is necessary. Glucose remaining
in the last wash should not exceed more that 0.05 g/L.)
11.8 Record weight of bottle, cap and sample to the nearest 0.01g. Then subtract weight of
bottle and cap from this recorded weight to caluculate and record the weight of washed

Issue Date:
Supersedes:
sample.
11.9 Perform total solids on the as received and washed material as described in Lap-001.
11.10 Store remaining sample in refrigerator or freezer as appropriate to maintain sample

integrity.
12. Procedure B (Filtration)
12.1 Record weight of buchner funnel filter cup with glass filter paper and record to the
nearest 0.01g on analytical balance and tare.
12.2 Add 25-50 grams of sample. (Record weight of sample as received).
12.3 Gradually pour 200-250 ml of deionized water. Stir gently to mix sample. Allow
vacuum filtration to proceed slowly.
12.4 Use pH sticks to test the pH of the filtrate and sample.Repeat steps 12.2-12.3 three
more times or until sample pH is at least 4.5-7.0. (For some applications it may be
necessary to test the amount of glucose present by biochemistry analyser in determining
if another wash is necessary. Glucose remaining in the last wash should not exceed
more than 0.5 g/L.)
12.5 Let process sit under vacuum for 5 minutes to remove excess water. Remove filter cup
and place on paper towel to allow water accumulation on filter cup bottom to escape.
12.6 Weigh filter cup with glass fiber filter paper and sample. Record weight. Subtract the
weight of the filter cup and filter to get the wet weight of the washed sample. Then
place in 105°C oven for overnight.. (If alternate Buchner funnel is used check with
manufactuer for temperature limitations. Use 45°C as oven temperature if necessary
and dry to a constant weight.)
12.7 Remove filter cup with sample from oven and place in desiccator for five minutes.
Weigh filter cup + filter + sample and record. Subtract weight of filter cup and filter
paper to get dry sample weight. Record dry weight of washed sample to the nearest
0.01 grams.
12.8 Perform total solids on the as received material as described in Lap-001. The % total

Issue Date:
Supersedes:
solids on the washed sample is calculated from dry weight of washed sample in step
12.7.
12.9 Discard or store sample as necessary.
13. Calculations
13.1 Calculate the percent insoluble solids and fraction insoluble solids for each sample on
a percent dry weight basis. Total solids by LAP-001, on the as received and the washed
sample will be necessary for each procedure. However, in Procedure B (Filtration) the
percent total solids of the washed material is calculated based on the whole sample
weight.
Procedure A (Centrifugation)
Weight of washed sample = (Weight of bottle, cap, & washed sample) – (Weight of bottle and cap)
Dry Weight of Washed sample = (Weight of washed sample) (% Total solids of washed sample)
100
% Insoluble Solids = Dry weight of washed sample X 100

Weight of sample as received
Dry Weight of Sample As Received = (Weight of Sample As Received) X (% Total Solids As Received)
100
% Fraction Insoluble Solids = Dry Weight of Washed sample X 100

Dry weight of sample as received
13.2 Percent insoluble solids and percent fraction insoluble solids can be calculated as
described in Procedure A provided the percent total solids are calculated as
demonstrated below. The percent total solids of the washed sample are based on the
entire sample that is washed and dried in the filter cup.
Procedure B (Filtration)
Weight of washed sample = (Weight of filter cup, filter, & washed sample) – (Weight of filter cup & filter)
% Total Solids on As Received Sample = As described in LAP 001

Issue Date:
Supersedes:
% Total Solids on Washed Sample = Dry Weight of the Washed Sample X 100
Wet Weight of Washed Sample
14. Report
15.1 Analysis in one laboratory of a method verification standard showed a sample recovery
of 95.6% (IS) and 100.48 (FIS) with a coefficient of variation of 1.28% (IS) and 1.52%
(FIS) for Procedure B (Filtration). In Procedure A (Centrifugation) the recovery values
were 96.49% (IS) and 100.09 (FIS) with a coefficient of variation of 1.56% (IS) and
1.78% (FIS). Estimation of standard deviation from duplicate numbers was 2.46 (IS)
and .03(FIS) for Procedure A(centrifugation) and 0.09(IS) and 0.006)(FIS) for
Procedure B(filtration).
15.2 Statistical analysis for several different types of biomass material gave an estimation
of standard deviation of 0.59(IS) for Procedure A and 0.10(IS) Procedure B. Statistical
analysis comparing both procedures indicated no significant difference between the two
methods, assuming a 95% confidence level.
16. Quality Control
16.1 Reported significant figures: Report all data to two significant figures.
16.2 Replicates: Run all samples in duplicate or triplicate if time permits.
16.3 Blank: Not applicable.
16.4 Relative percent difference criteria: % RPD should not exceed 6%.
16.5 Method verification standard: A method verification standard should be run with each
sample set. Solka Floc can be processed as the MVS by both procedures. Use 10-15g
sample size when selecting Solka Floc as the MVS.

Issue Date:
Supersedes:
16.6 Calibration verification standard: Not Applicable.
16.7 Sample size: A minimum of 25-50 grams should be processed in these procedures. It
is possible scale up the process depending on equipment and time constraints.
16.8 Sample storage Samples shall be stored in the refrigerator.
16.9 Standard storage: Not Applicable.
16.10 Standard preparation: Not Applicable.
16.11 Definition of a sample set: Any number of samples analyzed together and recorded
together within the limitation of instrumentation and time requirements.
16.12 Control charts: A control chart should be kept for the method verification standard.
Procedure #018 Page 9 of **

Issue Date:
Supersedes:
Ethanol Project

Procedure
Determination of Insoluble Solids of Pretreated Biomass
Procedure Title: Material
Author: Fannie Posey Eddy, Jody Okafor, Candic Date: 9/23/98

Roberson
ISSUE DATE:9/18 SUPERSEDES:

Determination of Insoluble Solids in Pretreated Biomass
1. Introduction
1.1 Pretreated biomass samples are composed of water-soluble and water insoluble
components. These two fractions are analyzed separately. Analytical results are then
mathematically recombined in the computation of the mass balance on the pretreatment
process. To separate the water soluble portion from the sample a thorough water
extraction is performed leaving a portion of solid material called the insoluble solids
fraction of the sample. This method describes two reliable procedures for determining
both the percent insoluble solids and percent fraction insoluble solids in a sample of
hydrolyzate slurry from pretreated biomass.
2. Scope
2.1 This procedure is intended to determine the percentage of water insoluble solids in a
pretreated biomass sample after all soluble components have been extracted with
aggressive water washing.
2.2 All analyses shall be performed according to guidelines established by the Ethanol
Quality Insurance Plan.
3. References
3.1 NREL Ethanol Project Laboratory Analytical Procedure #012, "Standard Test Method
for Moisture, Total Solids, and Total Dissolved Solids in Biomass Slurry and Liquid
Process Samples.
4. Terminology
4.1 Pretreated Biomass-Biomass which has been chemically and/or thermally altered to
change the structural composition.
4.2 Hydrolyzate slurry-The liquid and solid material in a sample resulting from biomass
pretreatment.

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4.3 Hydrolyzate Liquor- Liquid portion of hydrolyzate slurry.
4.4 Washed Solids-Water insoluble portion of hydrolyzate slurry.
4.5 Pressate-Liquid product from pretreated biomass pressed via centripetal force, manual
or hydraulic pressure.
4.6 Filtrate-Hydrolyzate liquid product from hydrolyzate slurry which has been placed in
a Buchner funnel and vacuum filtered.
4.7 Oven Dried Weight-The moisture-free weight of a biomass sample as determined by

Lap-001, Standard Method for Determination of Total Solids in Biomass.
4.8 Insoluble Solids (IS)-The oven dried weight of water insoluble solids divided by the
weight of whole hydrolyzate slurry sample (as received).
4.9 Fraction Insoluble Solids (FIS)-The oven dried weight of water insoluble solids
divided by the oven dried weight of the whole hydrolyzate slurry.
5.1 The percent insoluble solids and the percent fraction insoluble solids are used to
combine the liquid and solid compositions of the pretreated biomass in the mass
balance determination.
6. Interference
6.1 Technique is critical to minimizing the amount of material lost during the wash steps.
Care should be taken when separating the liquid from the solids or results will not meet
standard quality assurance requirements.
6.2 Hydrolyzate slurries separate quickly. Special attention is recommended when taking
samples .To obtain a representative sample thoroughly mix prior to taking a sample.
7. Apparatus Procedure A (Centrifugation)

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7.2 Convection ovens with temperature control to 45 °C ± 3 and 105 °C ± 3.
7.3 Analytical balance readable to 0.01g.
7.4 Desiccator.
7.5 Centrifuge refrigerated to 4°C and rotor specified to hold 300 ml. capacity bottles and
rated at least 9,000 rpm.
7.6 Aluminum foil weighing dish.
7.7 Centrifugation bottles with wide opening, caps with seals and 300 ml
capacity/reservoir.
7.8 Biochemical analyzer (YSI) for measurement of glucose (optional).
7.9 Eppendorf microcentrifuge tubes and transfer pipettes (optional).
8. Apparatus Procedure B (Filtration)
8.1 Whatman GF/D 2.7um glass microfiber FilterCup (1600R823) with FilterCup stem
(1600R900). An alternate is Buchner funnels (two-part, polypropylene) with GF/D
glass microfiber filter paper.
8.2 Glass microfiber filter paper sized for the Buchner funnel chosen.
8.3 1000 ml vacuum flask.
8.4 Filtration set-up including vacuum source and vacuum adapters for Buchner funnels.
8.5 Items 1-4 as described in section 7.
8.6 Teflon coated spatulas.

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9.2 Electronic pipette and disposable pipettes in the range of 25 ml.
9.3 2,000 ml flask or beaker.
9.4 YSI dextrose standards 2.0 g/L, 9.0 g/L (optional).
9.5 Water 18 megaohm deionized.
11. Procedure A (Centrifugation)
11.1 Record weight of centrifuge bottle and cap to nearest the 0.01g on an analytical balance
and tare.
11.2 Add 25-50 grams of sample. Record weight of sample as received to the nearest 0.01g.
11.3 Add 175-200 grams of deionized water. Shake vigorously for 60 seconds. (Note: If
collecting liquor for further analysis, do so prior to this step).
11.4 Cap and spin in centrifuge at 4°C for 20 minutes at 9,000 rpm.
11.5 Carefully remove bottles from centrifuge to minimize disturbance of sample pellet.
11.6 Remove liquid portion by decanting without disturbing sample pellet. Use electronic
or manual pipetter to remove small amounts of liquid until small traces are left.
11.7 Repeat steps 10.3-10.6 three more times or until sample pH is at least 4.5-7.0. (For
some applications it may be necessary to test the amount of glucose present by
biochemistry analyser in determining if another wash is necessary. Glucose remaining
in the last wash should not exceed more that 0.5 g/L.)
11.8 Record weight of bottle, cap and sample to the nearest 0.01g. Then subtract weight of
bottle and cap from this recorded weight to caluculate and record the weight of washed

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sample.
11.9 Perform total solids on the as received and washed material as described in Lap-001.
11.10 Store remaining sample in refrigerator or freezer as appropriate to maintain sample

integrity.
12. Procedure B (Filtration)
12.1 Record weight of buchner funnel filter cup with glass filter paper and record to the
nearest 0.01g on analytical balance and tare.
12.2 Add 25-50 grams of sample. (Record weight of sample as received).
12.3 Gradually pour 200-250 ml of deionized water. Stir gently to mix sample. Allow
vacuum filtration to proceed slowly.
12.4 Use pH sticks to test the pH of the filtrate and sample.Repeat steps 12.2-12.3 three
more times or until sample pH is at least 4.5-7.0. (For some applications it may be
necessary to test the amount of glucose present by biochemistry analyser in determining
if another wash is necessary. Glucose remaining in the last wash should not exceed
more than 0.5 g/L.)
12.5 Let process sit under vacuum for 5 minutes to remove excess water. Remove filter cup
and place on paper towel to allow water accumulation on filter cup bottom to escape.
12.6 Weigh filter cup with glass fiber filter paper and sample. Record weight. Subtract the
weight of the filter cup and filter to get the wet weight of the washed sample. Then
place in 105°C oven for overnight.. (If alternate Buchner funnel is used check with
manufactuer for temperature limitations. Use 45°C as oven temperature if necessary
and dry to a constant weight.)
12.7 Remove filter cup with sample from oven and place in desiccator for five minutes.
Weigh filter cup + filter + sample and record. Subtract weight of filter cup and filter
paper to get dry sample weight. Record dry weight of washed sample to the nearest
0.01 grams.
12.8 Perform total solids on the as received material as described in Lap-001. The % total

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solids on the washed sample is calculated from dry weight of washed sample in step
12.7.
12.9 Discard or store sample as necessary.
13. Calculations
13.1 Calculate the percent insoluble solids and fraction insoluble solids for each sample on
a percent dry weight basis. Total solids by LAP-001, on the as received and the washed
sample will be necessary for each procedure. However, in Procedure B (Filtration) the
percent total solids of the washed material is calculated based on the whole sample
weight.
Procedure A (Centrifugation)
Dry Weight of Washed sample = (Weight of washed sample) (% Total solids of washed sample)
100
% Insoluble Solids = Dry weight of washed sample X 100

Weight of sample as received
Dry Weight of Sample As Received = (Weight of Sample As Received) X (% Total Solids As Received)
100
% Fraction Insoluble Solids = Dry Weight of Washed sample X 100

Dry weight of sample as received
13.2 Percent insoluble solids and percent fraction insoluble solids can be calculated as
described in Procedure A provided the percent total solids are calculated as
demonstrated below. The percent total solids of the washed sample are based on the
entire sample that is washed and dried in the filter cup.
Procedure B (Filtration)
Weight of washed sample = (Weight of filter cup, filter, & washed sample) – (Weight of filter cup & filter)
% Total Solids on As Received Sample = As described in LAP 001

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% Total Solids on Washed Sample = Dry Weight of the Washed Sample X 100
Wet Weight of Washed Sample
14. Report
15.1 Analysis in one laboratory of a method verification standard showed a sample recovery
of 95.6% (IS) and 100.48 (FIS) with a coefficient of variation of 1.28% (IS) and 1.52%
(FIS) for Procedure B (Filtration). In Procedure A (Centrifugation) the recovery values
were 96.49% (IS) and 100.09 (FIS) with a coefficient of variation of 1.56% (IS) and
1.78% (FIS). Estimation of standard deviation from duplicate numbers was 2.46 (IS)
and .03(FIS) for Procedure A(centrifugation) and 0.09(IS) and 0.006)(FIS) for
Procedure B(filtration).
15.2 Statistical analysis for several different types of biomass material gave an estimation
of standard deviation of 0.59(IS) for Procedure A and 0.10(IS) Procedure B. Statistical
analysis comparing both procedures indicated no significant difference between the two
methods, assuming a 95% confidence level.
16. Quality Control
16.1 Reported significant figures: Report all data to two significant figures.
16.2 Replicates: Run all samples in duplicate or triplicate if time permits.
16.3 Blank: Not applicable.
16.4 Relative percent difference criteria: % RPD should not exceed 6%.
16.5 Method verification standard: A method verification standard should be run with each
sample set. Solka Floc can be processed as the MVS by both procedures. Use 10-15g
sample size when selecting Solka Floc as the MVS.

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16.6 Calibration verification standard: Not Applicable.
16.7 Sample size: A minimum of 25-50 grams should be processed in these procedures. It
is possible scale up the process depending on equipment and time constraints.
16.8 Sample storage Samples shall be stored in the refrigerator.
16.9 Standard storage: Not Applicable.
16.10 Standard preparation: Not Applicable.
16.11 Definition of a sample set: Any number of samples analyzed together and recorded
together within the limitation of instrumentation and time requirements.
16.12 Control charts: A control chart should be kept for the method verification standard.
Procedure #018 Page 9 of **

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Chemical Analysis and Testing

Laboratory Analytical Procedures

08-12-96 Determination of Carbohydrates in Biomass by High Performance

01-30-95 Determination of Acid-Insoluble Lignin in Biomass ................................................................... LAP-003

09-25-96 Determination of Acid-Soluble Lignin in Biomass ...................................................................... LAP-004

04-28-94 Standard Method for Ash in Biomass........................................................................................... LAP-005

08-12-96 Measurement of Cellulase Activities ............................................................................................ LAP-006

05-31-95 Preparation of Dilute-Acid Pretreated Biomass........................................................................... LAP-007

05-31-95 SSF Experimental Protocols: Lignocellulosic Biomass Hydrolysis

08-26-96 Enzymatic Saccharification of Lignocellulosic Biomass ........................................................... LAP-009

04-22-94 Standard Method for the Determination of Extractives in Biomass.......................................... LAP-010

05-05-94 Determination of Ethanol Concentration in Biomass to Ethanol

08-26-96 HPLC Analysis of Liquid Fractions of Process Samples for Monomeric

08-12-96 Dilute Acid Hydrolysis Procedure for Determination of Total Sugars in

09-25-96 HPLC Analysis of Liquid Fractions of Process Samples for Byproducts

09-25-96 Determination of Starch in Biomass Samples by Chemical Solubilization

07-31-98 Determination of O-Acyl Groups in Biomass by High Performance

09-23-98 Determination of Insoluble Solids of Pretreated Biomass Material .......................................... LAP-018

Chemical Analysis and Testing Task

Author: Tina Ehrman Date:

ISSUE DATE: 11/1/94 SUPERSEDES: 8/17/92

Laboratory Analytical Procedure #001

4. Significance and Use

Procedure #001 Page 2 of 7

5.1 Analytical balance, sensitive to 0.1 mg.

5.4 Aluminum foil weighing dishes.

6. ES&H Considerations and Hazards

7. Sampling, Test Specimens and Test Units

Procedure #001 Page 3 of 7

9. Infrared Moisture Analyzer Procedure

Procedure #001 Page 4 of 7

weight dried sample plus dish - weight dish

10.2 If desired, the percent moisture can also be calculated:

(weight dried sample plus dish - weight dish)

12. Precision and Bias

Procedure #001 Page 5 of 7

13. Quality Control

13.6 Calibration verification standard: Not applicable.

13.9 Standard storage: Not applicable.

Procedure #001 Page 6 of 7

Chemical Analysis and Testing Task

Author: Raymond Ruiz and Tina Ehrman Date: 2-14-96

Laboratory Analytical Procedure #002

Procedure #002 Page 2 of 11

5. Significance and Use

7.3 Guard columns, cartridges appropriate for the column used.

7.4 Analytical balance readable to 0.1 mg.

Procedure #002 Page 3 of 11

7.6 Autoclave capable of maintaining 121 ± 3°C.

7.7 Water bath set at 30 ± 3°C.

7.8 Desiccator containing anhydrous calcium sulfate.

8. Reagents and Materials

8.1.3 Calcium carbonate, ACS reagent grade.

8.1.4 Water, 18 megohm deionized.

8.2.1 Glass test tubes, 16x100 mm.

8.2.3 pH paper, suitable to cover the pH range of 4 to 7.

8.2.4 Disposable nylon syringe filters, 0.2 µm.

8.2.5 Disposable syringes, 3 mL.

8.2.6 Autosampler vials, with crimp top seals to fit.