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The following are laboratory analytical procedures (LAPs). 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.
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) (Excel 360 KB)
- Corn stover (corn stover feedstock) (Excel 532 KB)
- Biomass hydrolyzate (liquid fraction produced from dilute-acid pretreatment) (Excel 316 KB)
- Corn stover intermediates (solid fraction produced from dilute-acid pretreatment of corn stover, may also be used for wood intermediates) (Excel 327 KB)
- Corn fiber, single extraction method (Excel 410 KB)
- Corn fiber, two step sequential extraction method (Excel 566 KB)
- Nitrogen to protein factor calculator (Excel 25 KB).
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.
The following documents are available as Adobe Acrobat PDFs. Download Adobe Reader.
Determination of Acid Soluble Lignin Concentration Curve by UV-Vis Spectroscopy
(PDF 369 KB)
This procedure describes the isolation of lignin from an acidic solution and measurement of a maximum peak and absorptivity constant specific to that sample or biomass type. These values can usually be applied to other samples of the same matrix.
Determination of Structural Carbohydrates and Lignin in Biomass
(PDF 379 KB)
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. A measure of acetyl content is necessary for biomass containing hemicellulose with a xylan backbone, but not biomass containing a mannan backbone. Acetate is measured by HPLC.
Determination of Extractives in Biomass
(PDF 367 KB)
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 and 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
(PDF 319 KB)
This procedure describes methods for drying, 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 (PDF 419 KB)
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
(PDF 326 KB)
This test method covers the determination of ash, expressed as the percentage of residue remaining after dry oxidation (oxidation at 550 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
(PDF 383 KB)
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 Starch in Biomass Samples by High-Pressure Liquid Chromatography (HPLC)
(PDF 341 KB)
This method describes the procedure for measuring starch content in solid biomass samples, such as corn fiber. Starch is broken down into glucose using enzymic procedures, and measured by high-pressure liquid chromatography (HPLC). This method is based on the Megazyme Total Starch Assay Procedure (amyloglucosidase / a-amylase method).
Determination of Protein Content in Biomass
(PDF 318 KB)
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
(PDF 307 KB)
This procedure describes the rules for rounding numbers for the reporting of analytical data.
Determination of Insoluble Solids in Pretreated Biomass Material
(PDF 375 KB)
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
(PDF 346 KB)
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
(PDF 361 KB)
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
(PDF 409 KB)
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.
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