Standard Biomass Analytical Procedures
NREL has developed the following laboratory analytical procedures (LAPs) for standard biomass analysis. The American Society for Testing and Materials (ASTM) and the Technical Association of the Pulp and Paper Industry (TAPPI) may have adopted similar procedures. ASTM and TAPPI versions may be ordered from those organizations.
NREL Biomass Program researchers have also developed calculation workbooks (Excel spreadsheets) that automatically calculate desired compositional analysis and mass closure, based on the equations and measurement procedures in the pertinent LAPs. Workbooks are available for:
- Wood (hardwood or softwood)
- Corn stover (corn stover feedstock)
- Biomass hydrolyzate (liquid fraction produced from dilute-acid pretreatment)
- Corn stover intermediates (solid fraction produced from dilute-acid pretreatment of corn stover, may also be used for wood intermediates)
- Nitrogen-to-protein factor calculator.
These workbooks are designed for use in conjunction with the appropriate NREL LAPs. They were developed to assist with the calculations necessary in the LAPs. Each workbook combines the calculations for one or more LAPs, guiding the user to summative mass closure or summative analysis. The "Read Me" page of each workbook should be read prior to use, as it contains important instructions and legal information. It is up to the user to determine the most appropriate workbook to use, as each workbook is product specific. Comments and suggestions are welcome.
Summative Mass Closure — Laboratory Analytical Procedure Review and Integration: Feedstocks
Choosing the appropriate combinations of LAPs allows for the summative mass closure of biomass feedstocks. By combining the appropriate LAPs, the goal is to break the biomass feedstock sample down into constituents that sum to 100% by weight. There are several important points within the compositional analysis suite where decisions must be made to optimize the analysis. Some of these decisions are based on the type of biomass present, and some decisions must be made to obtain complete summative mass closure of all constituents. This report includes discussions to aid these decisions for individual LAPs. Following the LAP discussions is a flowchart of analyses and a walkthrough of the flowchart using an example feedstock, including some troubleshooting.
Summative Mass Closure – Laboratory Analytical Procedure Review and Integration: Pretreated Slurries
This summary Laboratory Analytical Procedure (LAP) includes a discussion of each LAP that is necessary to obtain complete compositional analysis of dilute acid-pretreated biomass slurries. LAPs developed at the National Renewable Energy Laboratory (NREL) have been optimized to provide compositional analysis for biomass feedstocks as well as for intermediary products of dilute acid pretreatment. This LAP reviews several important points within the compositional analysis suite where decisions must be made to optimize the analysis and achieve summative mass closure of process intermediates.
Determination of Structural Carbohydrates and Lignin in Biomass
This procedure for measuring carbohydrate and lignin content is suitable for samples that do not contain extractives. (See "Determination of Extractives in Biomass" for samples that do contain extractives.) This procedure uses a two-step acid hydrolysis to fractionate the biomass into forms that are more easily quantified. The lignin fractionates into acid insoluble material and acid soluble material. The acid insoluble material may also include ash and protein, which must be accounted for during gravimetric analysis. The acid soluble lignin is measured by UV-Vis spectroscopy. During hydrolysis the polymeric carbohydrates are hydrolyzed into the monomeric forms, which are soluble in the hydrolysis liquid. They are then measured by HPLC. Protein may also partition into the liquid fraction. Acetate is measured by HPLC.
Determination of Extractives in Biomass
This procedure covers the determination of soluble non-structural materials in a biomass sample. The results are reported, on a dry weight basis, as a weight percentage of the biomass. Extractives percentages are measured and used to convert compositions from an extractives-free basis to an as-received basis. Determining the amount of water extractable sucrose is also covered. This LAP should be used in conjunction with other assays to determine the total composition of biomass samples. This procedure should be performed prior to LAP "Determination of Structural Carbohydrates and Lignin in Biomass."
Preparation of Samples for Compositional Analysis
This procedure describes methods for sample drying and size reduction, obtaining samples with a uniform particle size, and representative sampling of biomass samples.
Determination of Total Solids in Biomass and Total Dissolved Solids in Liquid Process Samples
This procedure describes the methods used to determine the amount of solids or moisture present in a solid or slurry biomass sample. It also covers the determination of dissolved solids in a liquor sample. A traditional convection oven drying procedure is covered as well as solids determination using an automatic infrared moisture analyzer.
Determination of Ash in Biomass
This test method covers the determination of ash, expressed as the percentage of residue remaining after dry oxidation (oxidation at 550°C to 600°C). All results are reported relative to the 105°C oven-dried weight of the sample.
Determination of Sugars, Byproducts, and Degradation Products in Liquid Fraction Process Samples
The concentrations of monomeric sugars (soluble monosaccharides) and cellobiose, total sugars (monosaccharides and oligosaccharides), as well as carbohydrate degradation products and sugar alcohols can be determined using this procedure. Monomeric sugars are quantified by HPLC with refractive index detection. Oligomeric sugars are converted into the monomeric form using acid hydrolysis and quantified by HPLC with refractive index detection. Byproducts and degradation products are quantified by HPLC with refractive index detection.
Determination of Protein Content in Biomass
This procedure covers the determination of nitrogen-to-protein conversion factors that are used to estimate the amount of protein in a biomass sample.
Rounding and Significant Figures
This procedure describes the rules for rounding numbers for the reporting of analytical data.
Determination of Insoluble Solids in Pretreated Biomass Material
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.
Measurement of Cellulase Activities
This 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.
Enzymatic Saccharification of Lignocellulosic Biomass
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).
SSF Experimental Protocols: Lignocellulosic Biomass Hydrolysis and Fermentation
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.