A national laboratory of the U.S. Department of Energy

Office of Energy Efficiency & Renewable Energy

National Renewable Energy Laboratory 

Innovation for Our Energy Future 

Determination of Protein 

Content in Biomass 

Laboratory Analytical Procedure (LAP) 

Issue Date: 05/23/2008 

B. Hames, C. Scarlata, and A. Sluiter 

Technical Report 

NREL/TP-510-42625 

Revised May 2008 

NREL is operated by Midwest Research Institute ● Battelle Contract No. DE-AC36-99-GO10337 
Determination of Protein 

Content in Biomass 

Laboratory Analytical Procedure (LAP) 

Issue Date: 05/23/2008 

B. Hames, C. Scarlata, and A. Sluiter 

Technical Report 

NREL/TP-510-42625 

Revised May 2008

National Renewable Energy Laboratory

1617 Cole Boulevard, Golden, Colorado 80401-3393 

303-275-3000 • www.nrel.gov 

Operated for the U.S. Department of Energy 

Office of Energy Efficiency and Renewable Energy 

by Midwest Research Institute • Battelle 

Contract No. DE-AC36-99-GO10337
DISCLAIMER 

These Standard Biomass Analytical Methods (“Methods”) are provided by the National 

Renewable Energy Laboratory (“NREL”), which is operated by the Midwest Research Institute 

(“MRI”) for the Department Of Energy. 

Access to and use of these Methods shall impose the following obligations on the user. The 

user is granted the right, without any fee or cost, to use, copy, modify, alter, enhance and 

distribute these Methods for any purpose whatsoever, except commercial sales, provided that 

this entire notice appears in all copies of the Methods. Further, the user agrees to credit 

NREL/MRI in any publications that result from the use of these Methods. The names 

NREL/MRI, however, may not be used in any advertising or publicity to endorse or promote any 

products or commercial entity unless specific written permission is obtained from NREL/MRI. 

The user also understands that NREL/MRI is not obligated to provide the user with any support, 

consulting, training or assistance of any kind with regard to the use of these Methods or to 

provide the user with any updates, revisions or new versions. 

THESE METHODS ARE PROVIDED BY NREL/MRI "AS IS" AND ANY EXPRESS OR IMPLIED 

WARRANTIES, INCLUDING BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF 

MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN 

NO EVENT SHALL NREL/MRI BE LIABLE FOR ANY SPECIAL, INDIRECT OR 

CONSEQUENTIAL DAMAGES OR ANY DAMAGES WHATSOEVER, INCLUDING BUT NOT 

LIMITED TO CLAIMS ASSOCIATED WITH THE LOSS OF DATA OR PROFITS, WHICH MAY 

RESULT FROM AN ACTION IN CONTRACT, NEGLIGENCE OR OTHER TORTIOUS CLAIM 

THAT ARISES OUT OF OR IN CONNECTION WITH THE ACCESS, USE OR PERFORMANCE 

OF THESE METHODS. 
Procedure Title: Determination of Protein Content in Biomass 

Laboratory Analytical Procedure 

1. Introduction 

1.1 Biomass used as a feedstock for conversion to fuels and chemicals may contain 

protein and other nitrogen containing materials. These constituents are measured as 

part of a comprehensive biomass analysis. Protein in biomass is difficult to measure 

directly. In many cases the nitrogen content of the biomass sample is measured by 

combustion or Kjeldahl methods and the protein content is estimated using an 

appropriate Nitrogen Factor (NF). There are published methods that recommend 

using an NF of 6.25 for all types of biomass except wheat grains where an NF of 

5.70 is recommended. Use of these published nitrogen factors may lead to errors 

when applied to biomass feedstock or process intermediates. This LAP includes a 

procedure for calculating an appropriate NF for most biomass types. 

1.2 The procedure for the determination of Nitrogen Factors is substantially similar to 

that described in Mossé (1990). 

1.3 This procedure covers the determination of nitrogen-to-protein conversion factors 

that are used to estimate the amount of protein in a biomass sample. 

2. Scope 

2.1 This procedure is appropriate for most types of biomass, including extractives-free 

biomass (biomass that has been extracted using LAP “Determination of Extractives 

in Biomass”) and biomass before extraction. 

2.2 This procedure is suitable for biomass feedstocks, process solids, and process 

liquids. 

2.3 Some types of biomass feedstocks, such as bark-free wood chips, contain little or no 

protein. For these samples, a protein measurement is not a necessary part of a 

comprehensive compositional analysis. 

2.4 All analyses shall be performed according to the guidelines established in an 

appropriate laboratory specific Quality Assurance Plan (QAP). 

2.5 Results are reported as a weight percent of the oven-dried biomass (105oC). 

3. Terminology 

3.1 Oven dry weight (ODW)- the weight of biomass mathematically corrected for the 

amount of moisture present in the sample at the time of weighing 

3.2 Nitrogen Factor – A conversion factor used to estimate protein content from 

measured nitrogen content. 

3.3 Non-protein nitrogen – The difference between the measured nitrogen content and 

the nitrogen content that can be accounted for in protein. This includes, but is not 

limited to, nitrogen found in nitrates, nitrites, chlorophyll, nucleic acids and free 

amino acids. 

 1
4. Significance and Use 

4.1 This procedure is used, possibly in conjunction with other procedures, to determine 

the amount of protein and other nitrogen containing materials in a solid biomass 

sample. 

4.2 This procedure is used at several stages in a comprehensive compositional analysis 

of a biomass sample. 

5. Interferences 

5.1 In this procedure the protein content of a biomass sample is estimated using the 

results of other determinations (nitrogen content and amino acid profile). The 

precision and accuracy of those methods are reflected in the results. 

5.2 Biomass samples containing less than 0.2 weight percent protein may fall below the 

validated range of analysis for some standard methods. AOAC 990.03 for crude 

protein by combustion states that the methods is applicable to solids samples 

containing 0.2% – 20% nitrogen 

5.3 Protein in biomass may be structurally modified during pretreatment and 

saccharification. Standard amino acid determinations may not quantify these 

portions of the protein. Assumptions made in this method concerning the 

relationship between nitrogen content and protein content may no longer be valid. 

6. Apparatus 

6.1 This procedure requires a measurement of sample nitrogen content. This 

measurement can be a combustion measurement performed according to AOAC 

990.03 or a Kjeldahl measurement performed according to AOAC 984.13 

6.2 This procedure requires multiple amino acid profiles as described by Mosse. These 

are modified versions of amino acid profiles procedures similar to AOAC 982.30 

E(a,b,c), CHP 45.3.05 (1995) or an equivalent method. 

7. Reagents and materials 

7.1 Reagents 

7.1.1 none 

7.2 Materials 

7.2.1 This procedure requires a measurement of sample nitrogen content. This 

measurement can be a combustion measurement performed according to AOAC 

990.03 or a Kjeldahl measurement performed according to AOAC 984.13. 

7.2.2 This procedure requires multiple amino acid profiles as described by Mosse. 

These are modified versions of amino acid profiles procedures similar to AOAC 

982.30 E(a,b,c), CHP 45.3.05 (1995) or an equivalent method. 

7.2.3 Parts of this procedure requires Nitrogen Factor Calculator worksheet, “NF 

calculator.xls”, available at 

http://www.nrel.gov/biomass/analytical_procedures.html. 

 2
8. ES&H Considerations and Hazards 

8.1 Follow all applicable NREL chemical handling procedures 

9. Sampling, Test Specimens and Test Units 

9.1 Many CHN analyzers have a sample size limit of 100 mg or less. Further size 

reduction may be necessary to assure that sample being analyzed is representative 

of the larger biomass sample. 

10. Procedure 

10.1 Calculate an appropriate Nitrogen Factor. 

10.1.1 Obtain a complete amino acid profile for each sample. 

10.1.1.1 Complete amino acid profile may be obtained according to AOAC 

982.30 E(a,b,c), CHP 45.3.05 (1995) 

10.1.1.2 A complete amino acid profile may also be obtained according to Mossé, 

J., “Nitrogen to Protein Conversion Factor for 10 Cereals and 6 Legumes 

or oilseeds – a Reappraisal of Its Definition and Determination – 

Variation According to Species and to Seed Protein Content”, 1990, 

Journal of Agriculture and Food Chemistry Vol. 38(1),18-24. 

10.1.2 Obtain a measurement of the total nitrogen content of each biomass sample. 

If necessary, convert the values to a percent dry weight basis. 

10.1.3 Enter the amino acid and nitrogen weight percent values into the Nitrogen 

calculator spreadsheet. 

10.1.4 Calculate kA, kP, k1, k2 and avg(k1, k2) according to the equations shown in 

section 11 

10.1.5 The average of kA and kP is a customized NF for this sample. k1, k2 are the 

plausible upper and lower limits (or the plus/minus error) for the customized 

NF as in 11.4. 

11. Calculations 

11.1 The N-factor limits in the NF calculator are calculated from amino acid and 

nitrogen data. 

11.2 The upper limit is defined as: 

A ∑ ∑DE = k ii / 

Where: 

Ei = the grams of the ith Amino Acid per100 grams of sample (dry weight basis) 

Di = the grams nitrogen of the ith Amino Acid per100 grams of sample (dry weight 

basis) 

11.3 The lower limit is defined as: 

 3
NE = kP i ∑ /

Where: 

Ei = the grams of the ith Amino Acid per100 grams of sample (dry weight basis) 

N = the grams nitrogen per100 grams of dry sample 

11.4 A range of highest probability (k1, k2) is calculated as follows: 

)),((25.0),( 1 PA PA kkavg = k + kkavg

and 

)),((25.0),( 2 PA PA kkavg = k − kkavg

11.5 The appropriate N-Factor is avg(kA, kP). 

11.6 The protein content of this sample is calculated as follows: 

% %nitrogen = protein × NF

Where: 

NF = nitrogen factor = N-Factor 

12. Report Format 

12.1 Report protein as a weight percent of the oven-dried biomass. Report average 

and RPD 

13. Precision and Bias 

13.1 Determined by data quality objectives and laboratory specific Quality Assurance 

Plan. 

14. Quality Control 

14.1 Reported Significant Figures or decimal places: Determined by data quality 

objectives and laboratory specific Quality Assurance Plan, see LAP “Rounding and 

Significant Figures”. 

14.1.1 Replicates: Analyses should be performed in duplicate wherever possible 

14.1.2 Relative percent difference criteria: Determined by data quality objectives and 

laboratory specific Quality Assurance Plan 

14.2 Blank: not applicable 

14.3 Relative percent difference criteria: not applicable 

14.4 Calibration verification standard: not applicable 

14.5 Sample size: 0.5 grams 

14.6 Sample storage: not applicable 

14.7 Standard storage: not applicable 

 4
 5

14.8 Standard preparation: not applicable 

14.9 Definition of a batch: not applicable 

14.10 Control charts: not applicable 

15. Appendices 

15.1 Excel spreadsheet N-Factor calculator.xls 

16. References 

16.1 Mossé, J., “Nitrogen to Protein Conversion Factor for 10 Cereals and 6 Legumes or 

oilseeds – a Reappraisal of Its Definition and Determination – Variation According 

to Species and to Seed Protein Content”, 1990, Journal of Agriculture and Food 

Chemistry Vol. 38(1),18-24. 

16.2 Jones, D. "Factors for converting percentages of nitrogen in foods and feeds into 

percentages of proteins." U.S. Department of Agriculture, 1931, Circular(183): 21 

16.3 Mosse, J., J. C. Huet, et al. "The Amino-Acid Composition of Wheat-Grain as a 

Function of Nitrogen-Content." Journal of Cereal Science, 1985, 3(2): 115-130. 

16.4 Tkachuk, R. "Amino acid composition of wheat flours." Cereal Chemistry, 1966, 

43: 207-223. 

16.5 Tkachuk, R."Nitrogen-to-protein conversion factors for cereals and oilseed meals." 

Cereal Chemistry, 1969, 46: 419-423.