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Energy & Fuels 2007, 21, 3684–3688
More Accurate Determination of Acid-Labile Carbohydrates in Lignocellulose by Modified Quantitative Saccharification Geoffery Moxley and Y.-H. Percival Zhang* Biological Systems Engineering Department, Virginia Tech, Blacksburg, Virginia 24061 ReceiVed July 9, 2007. ReVised Manuscript ReceiVed September 1, 2007
Production of biofuels and biobased products from lignocellulosic materials is an emerging industry. Quantitative saccharification (QS) is a widely used method for determining carbohydrate composition in lignocellulosic materials. The NREL methods (versions 1996 and 2006) involve a primary hydrolysis in 72% w/w sulfuric acid at 30 °C, converting polysaccharides into oligosaccharides, and a secondary hydrolysis in 4% w/w sulfuric acid at 121 °C, converting all oligosaccharides to monomeric sugars. Because some polysaccharides are degraded during the harsh hydrolysis processes, a sugar control set of monomeric sugars was run in parallel, and the correction coefficients of monomeric sugars were used to compensate the polysaccharide degradation. This assumption may be invalid because polysaccharide and monosaccharide sugars have different degradation rates, especially for acid-labile carbohydrates. Here we propose a modified quantitative saccharification involving a primary hydrolysis (72% sulfuric acid, 30 °C, 1 h), followed by a secondary hydrolysis (4% sulfuric acid, 121 °C, 1 h, for glucose, galactose, and mannose) and a parallel secondary hydrolysis (1% sulfuric acid, 121 °C, 1 h, for xylose and arabinose). The weaker secondary hydrolysis can decrease degradation of acid-labile xylose by ∼4.4-fold. The data of acid-labile hemicellulose carbohydrates for all of the five tested lignocellulosic materials from herbs to hardwood to softwood suggest that the current QS protocol results in a 4.2%–9.1% overestimation of xylan contents. Such statistically significant overestimation is attributed to theoretical errors from the methods’ assumption.
Introduction Lignocellulose is the most abundant renewable biological resource produced by photosynthesis. Lignocellulose, a natural composite, consists of three main polymeric components: cellulose, hemicellulose, and lignin, as well as other minor components, such as extractives, pectin, or protein.1,2 Cellulose is a linear polymer consisting of anhydroglucose connected by β-1,4-glycosidic bonds; hemicellulose is a polymeric carbohydrate with some branching, composed of pentose and hexose units. The complete separation of hemicellulose and cellulose with full carbohydrate recovery is still a challenging task because of the complicated linkage among cellulose, hemicellulose, and lignin.2 Knowledge of carbohydrate composition is important for the paper and pulping industries, agriculture and forest product industries, emerging biofuels and biobased products industries, and the new carbohydrate economy.1–8 * Corresponding author: Tel 540-231-7414; Fax 540-231-7414; e-mail
[email protected]. (1) Zhang, Y.-H. P.; Lynd, L. R. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems. Biotechnol. Bioeng. 2004, 88, 797–824. (2) Fengel, D.; Wegener, G. Wood: Chemistry, Ultrastructure, Reactions; Walter de Gruyter & Co.: Berlin, 1984. (3) Wyman, C. E.; Dale, B. E.; Elander, R. T.; Holtzapple, M.; Ladisch, M. R.; Lee, Y. Y. Coordinated development of leading biomass pretreatment technologies. Biores. Technol. 2005, 96, 1959–1966. (4) Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.; Cairney, J.; Eckert, C. A.; Frederick, W. J., Jr.; Hallett, J. P.; Leak, D. J.; Liotta, C. L.; Mielenz, J. R.; Murphy, R.; Templer, R.; Tschaplinski, T. The path forward for biofuels and biomaterials. Science 2006, 311, 484– 489. (5) Zhang, Y.-H. P.; Himmel, M.; Mielenz, J. R. Outlook for cellulase improvement: Screening and selection strategies. Biotechnol. AdV. 2006, 24, 452–481.
Quantitative saccharification (QS) is a widely used method for determining carbohydrate composition in lignocellulosic materials. As shown in Figure 1, QS involves a primary hydrolysis, which converts polysaccharides (Pi) to oligosaccharides (Oi) using high sulfuric acid at low temperatures, and a secondary hydrolysis, which converts oligosaccharides (Oi) to monomeric sugars (Mi) using dilute acid at high temperatures. During the above processes, some carbohydrates degrade. Therefore, it is crucial to ensure complete conversion of polysaccharides to monosaccharides and avoid any significant degradation of monomeric carbohydrates. The researchers at the National Renewable Energy Laboratory (NREL) published the first HPLC-based QS (M1) in 1996,9 based on Saeman’s method.10 They used 3.00 mL of 72% w/w sulfuric acid for 2 h at 30 °C as the primary hydrolysis and diluted the hydrolysate to 4% w/w sulfuric acid for 1 h at 121 °C as the secondary hydrolysis. The monomeric sugars after neutralization were measured by HPLC with a carbohydrate assay column. Since some polysaccharides were degraded during the whole process, the degradation degrees of five monomeric sugars (glucose, xylose, arabinose, galactose, and mannose) were (6) Lynd, L. R.; Weimer, P. J.; van Zyl, W. H.; Pretorius, I. S. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. ReV. 2002, 66, 506–577. (7) Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Fractionating recalcitrant lignocellulose at modest reaction conditions. Biotechnol. Bioeng. 2007, 97, 214–223. (8) Zhang, Y.-H. P.; Evans, B. R.; Mielenz, J. R.; Hopkins, R. C.; Adams, M. W. W. High-yield hydrogen production from starch and water by a synthetic enzymatic pathway. PLOS ONE 2007, 2, e456. (9) Ruiz, R.; Ehrman, T. Determination of carbohydrates in biomass by high performace liquid chromatography. NREL: LAP-002, 1996. (10) Saeman, J. F.; Bubl, J. L.; Harris, E. E. Quantitative saccharification of wood and cellulose. Ind. Eng. Chem. Anal. Ed. 1945, 17, 35–37.
10.1021/ef7003893 CCC: $37.00 2007 American Chemical Society Published on Web 10/18/2007
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Figure 1. Simplified flowcharts of three quantitative saccharification methods for lignocellulose. M1, NREL 1996; M2, NREL 2006; M3, the newly developed method. The primary hydrolysis is conducted using high sulfuric acid (72% w/w), 30 °C, and the reaction time indicated in the figure. The secondary hydrolysis is conducted using dilute acid (4% or 1%) at 121 °C for 1 h. b, polysaccharides; ), oligosaccharides; O, monomeric sugars; 0, degraded sugars. Note: the primary hydrolysis for 2 h can generate some amount of monosaccharides (e.g., xylose and/or arabinose), but the primary hydrolysis for 1 h can produce a very small amount of monosaccharides from polysaccharides with a negligible degradation of monomeric sugars in the primary hydrolysis.
estimated for correcting the degradation degrees of the polysaccharides. In 2006, the researchers at NREL released an updated protocol (M2) with some modifications.11 They suggested (i) to shorten the primary hydrolysis time from 2 to 1 h in order to decrease sugar degradation and save measurement time and (ii) to measure the correction coefficients of the controls—monomeric sugars only in the secondary hydrolysis because only a small fraction of polysaccharides were converted to monomeric sugars and a negligible amount of monomeric sugars was degraded in the primary hydrolysis. But it is notable that the degradation degrees of polysaccharides are always smaller than those of monomeric sugars.12–14 When the degradation differences between polysaccharides and monomeric sugars are significantly large, polymeric carbohydrate composition determined by QS would be overestimated. Currently, there is an urgent need to develop rapid, highthroughput analytical instrumental methods for the rapid measurement of carbohydrate composition in lignocellulose, where QS is used as a calibration method.15,16 Small deviations (11) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. Determination of structural carbohydrates and lignin in biomass. In Laboratory Analytic Procedure LAP-002, 2006 (http://devafdc. nrel.gov/pdfs/9572.pdf). (12) Lloyd, T.; Wyman, C. E. Application of a depolymerization model for predicting thermochemical hydrolysis of hemicellulose. Appl. Biochem. Biotechnol. 2003, 105-108, 53–67. (13) Lloyd, T. A.; Wyman, C. E. Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids. Biores. Technol. 2005, 96, 1967–1977. (14) Li, X.; Converse, A. O.; Wyman, C. E. Characterization of molecular weight distribution of oligomers from autocatalyzed batch hydrolysis of xylan. Appl. Biochem. Biotechnol. 2003, 105/108, 515–522. (15) Schultz, T. P.; Templeton, M. C.; McGinnis, G. D. Rapid determination of lignocellulose by diffuse reflectance Fourier Transform Infrared spectrometry. Anal. Chem. 1985, 57, 2867–2869. (16) Bjarnestad, S.; Dahlman, O. Chemical compositions of hardwood and softwood pulps employing photoacoustic Fourier transform infrared spectroscopy in combination with partial least-squares analysis. Anal. Chem. 2002, 74, 5851–5858.
of carbohydrate composition measured by QS could greatly affect these developing methods, resulting in methodological errors. In this technical note, we investigated the effects of hydrolysis conditions on the degradation of monomeric sugars and the hydrolysis of oligosaccharides (cellodextrins and oligo-xylosaccharides). We propose a slightly modified QS protocol based on the NREL 2006 version. This can more accurately determine acid-labile carbohydrate composition in lignocellulose. Our results clearly suggest that the previous QS protocol results in a statistically significant overestimation of acid-labile xylan composition for five different lignocellulosic materials, and such deviation is attributed to theoretical errors from the method’s invalid assumption. Methods and Materials Chemicals and Materials. All chemicals were reagent grade and purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Atlanta, GA) unless otherwise noted. Cellodextrins were prepared from microcrystalline cellulose treated by mixed acid hydrolysis; the cellodextrin components were separated by chromatography as described before.17 Xylooligosaccharides were hydrolyzed by hot water treatment from Sigma oat spelt xylans,14 or birchwood xylooligosaccharides were purchased from Megazyme International Ireland Ltd. (Ireland). Corn stover, switchgrass, wheat straw, and hybrid poplar were graciously provided by James McMillan at NREL (Golden, CO). Douglas fir was provided from Dr. Jack Saddler at the University of British Columbia (Vancouver, Canada). All nearly dry lignocellulosic materials were knife-milled (laboratory model 4, Arthur H. Thomas Co., Philadelphia, PA) and screened. The lignocellulose particles smaller than the 40 mesh screen and larger than the 60 mesh screen (250–420 µm) were used (17) Zhang, Y.-H. P.; Lynd, L. R. Cellodextrin preparation by mixedacid hydrolysis and chromatographic separation. Anal. Biochem. 2003, 322, 225–232.
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Figure 2. Comparison of monomeric sugar degradation among hydrolysis conditions in three conditions. C1, representing method 1, includes the primary hydrolysis using 72% sulfuric acid and the secondary hydrolysis using 4% sulfuric acid; C2, representing method 2, was only secondary hydrolysis in 4% sulfuric acid; and C3 was only secondary hydrolysis in 1% sulfuric acid.
for determining structural carbohydrate contents. The right-size lignocellulose particles were dried in a convection oven at 105 ( 3 °C for 4 h and then removed from the oven and cooled to room temperature in a desiccator. Sugar Assays. Monomeric sugars were measured by a Shimadzu HPLC (Kyoto, Japan) with a Bio-Rad Aminex HPX-87P column (Richmond, CA) operated at 80 °C with a mobile phase at a rate of 0.6 mL of distilled water/min.7 Oligosaccharides (cellodextrins and oligo-xylosacchardies) were measured by a Waters HPLC with a Bio-Rad HPX-87A column with a flow rate of 0.4 mL/min distilled water at 80 °C, as described previously.14,17 QS Protocols. QS method 1, developed by NREL in 1996,9 was adopted by ASTM as the standard test method, E1758-95. QS method 2 was updated by NREL in 2006.11 Method 3 that we propose was developed based on QS method 2 with some modifications. The whole procedure is described below. The samples were dried at a convection drying oven at 105 °C for 4 h or until a constant weight was achieved, and then they were cooled to room temperature in a desiccator containing dryer. 300 ( 10.0 mg of the completely dry lignocellulose sample was weighed and put into a test tube. 3 ( 0.001 mL of 72% (w/w) sulfuric acid was added to the tubes that were placed in an ice-cold water bath. The solid particles were well-mixed using a glass stir rod. The tubes were placed in a water bath set at 30 ( 1 °C for 60 ( 1 min. Proper mixing by shaking at 120 rpm or frequent manual mixing was essential to ensure even acid-to-particle contact and uniform hydrolysis. After the high acid hydrolysis, the tubes were place in an ice-cold water bath to quench the reaction. The hydrolysate in the tubes was transferred to the 125 mL serum bottles using 84.00 mL of distilled water (i.e., acid concentration was diluted to 4% sulfuric acid). After mixing well, exactly 1.000 mL of 4% hydrolysate was transferred to another pressure anaerobic tube (Bellco Biotech Co., Vineland, NJ) and supplemented by 3.000 mL of distilled water (i.e., 1% sulfuric acid). All serum vials and pressure anaerobic tubes were sealed by butyl rubber septum type stoppers and aluminum seals. After autoclave (121 °C) for 1 h in the microprocessor-controlled autoclave with slow-exhaust mode, the hydrolysate samples were cooled to room temperature slowly. About 1 mL of the supernatant of the hydrolysate was withdrawn and neutralized to pH ∼6 by adding calcium carbonate. The neutralized samples were transferred to 1.5 mL microcentrifuge tubes and then centrifuged at 13 000 rpm for 5 min. The supernatant was transferred to another microcentrifuge tube and frozen overnight. After thawing, the samples were vortexed well and then centrifuged at 13 000 rpm for 5 min. The supernatant was placed in a HPLC vial for sugar assays. Alternatively, the neutralized hydrolysate was filtered through a 0.2 µm filter, and the filtrate was used for HPLC assay. The concentrations of glucose, mannose,
Moxley and Zhang and galactose were measured on the basis of results from the 4% sulfuric acid hydrolysate; the concentrations of xylose and arabinose were measured on the basis of results from the 1% sulfuric acid hydrolysate. The parallel autoclaving for both of the lignocellulose hydrolysate samples and monomeric sugars controls can minimize the influences from variations in autoclaving. The data presented in Figure 2 and Table 1 were obtained for the microprocessordriven autoclaves at Virginia Tech. Estimation of Correction Coefficients. The first monomeric sugar set containing the known amount of glucose, galactose, and mannose (close to the respective sugar composition of lignocellulosic materials) was dissolved in 87 mL of 4% w/w sulfuric acid. The second acid-labile monomeric sugar set containing the known amounts of xylose and arabinose, whose amounts are close to those of the real lignocellulosic samples, was dissolved in 328 mL of 1% w/w sulfuric acid, which was diluted from 4% sulfuric acid. Half-volume of the two sugar sets was sealed in the tubes and then autoclaved for an hour; the other half-volume was used for the known concentration of the sugars. After neutralization using calcium carbonate, the correction coefficients of monomeric sugars RMi were calculated as RMi )
HPLC CM i known CM i
(1)
known is the known concentration of monomeric sugar in which CMi HPLC is the concentration of (Mi) without autoclave (mg/mL) and CMi monomeric sugar after autoclave measured by HPLC (mg/mL). Polymeric Carbohydrate Calculation. The contents of polysaccharides (Pi, %) in lignocellulosic materials are calculated using the correction coefficients of polysaccharides (RPi)
Pi )
CiV MWPi ⁄ wtfeedstock × 100% RPi MWMi
(2)
in which i denotes sugar types for polysaccharides or monomeric sugars: G, glucose or glucan; M, mannose or mannan; Gal, galactose or galactan; X, xylose or xylan; A, arabinose or arabinan, respectively; Ci is the monomeric sugar concentration measured by HPLC (mg/mL); V is the volume of the hydrolysate (mL), 87 or 348 for 4% sulfuric acid or 1% sulfuric acid hydrolysate, respectively; RPi is the correction coefficient for polysaccharide i (Pi); MWPi is the molecular weight for hexose and pentose polysaccharides (162.14 or 132.11 g/mol, respectively); MWMi is the molecular weight of monomeric hexose or pentose (180.16 or 150.13 g/mol, respectively); and wtfeedstock is the weight of the feedstock sample (mg). Because RPi is not available, RMi, the correction coefficient for monomeric sugars (Mi), was used to approximately represent RPi for determining Pi in eq 2. When the differences between polymeric (RPi) and monomeric sugars (RMi) are very close, such approximations are operative, and no statistics significant difference can be obtained. Lignin and Ash Determination. About 86 mL of the 4% sulfuric acid hydrolysate in the serum vials was filtrated through a known weight medium-porosity crucible with a nominal maximum pore size of 10–15 µm (W1) by vacuum. The solid was rinsed by 50 mL of fresh deionized water. The crucible containing the filtrated solids (acid-insoluble ash + acid-insoluble lignins) were dried in an oven at 105 ( 3 °C for 12 h or longer until a constant weight was achieved. The crucibles were cooled to room temperature in a desiccator and then weighed as W2. After that, the crucibles were placed in a muffle furnace at 575 ( 25 °C for 24 h, were cooled in the desiccator, and then were weighed as W3. The amount of acid-insoluble lignin equals (W2 – W3), and the amount of ash equals (W3 – W1). The absorbency of the filtrate at 240 nm was measured for determining acid-soluble lignin.11 Notes of Environment, Safety, and Health. Sulfuric acid is corrosive and should be handled with care. Be cautious when handling hot pressure bottles and tubes after autoclaving. When
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Table 1. Carbohydrate Compositions of Five Lignocellulosic Materials by Quantitative Saccharification by the NREL 1996 Protocol (Method 1, C1), the NREL 2006 Protocol (Method 2, C2), and the Newly Proposed Protocol (Data Italicized; C3, for Xylan and Arabinose; C2 for Glucan, Galactan, and Mannan)a condition
a
no.
glucan (mg/g)
xylan (mg/g)
galactan (mg/g)
mannan (mg/g)
arabinan (mg/g)
10.4 ( 0.7 10.6 ( 0.5 10.2 ( 0.5
7.7 ( 0.3 7.6 ( 0.6 7.5 ( 0.7
37.6 ( 0.6 35.0 ( 0.7 34.1 ( 0.5
1 2 3
5 5 5
336.1 ( 1.2 334.3 ( 1.3 NAb
Switchgrass 218.4 ( 1.3 215.0 ( 1.7 206.3 ( 1.6
1 2 3
5 5 5
393.1 ( 1.8 389.0 ( 1.9 NA
Corn Stover 234.4 ( 1.1 230.6 ( 1.1 212.6 ( 1.6
20.4 ( 1.0 18.8 ( 0.9 17.8 ( 0.9
8.9 ( 0.9 9.6 ( 0.9 8.6 ( 1.3
26.0 ( 1.8 23.6 ( 1.4 23.1 ( 1.6
1 2 3
5 5 5
393.3 ( 1.9 388.5 ( 2.1 NA
Wheat Straw 222.0 ( 1.8 10.0 ( 0.6 215.0 ( 1.3 9.8 ( 0.7 198.5 ( 1.5 10.3 ( 0.9
7.1 ( 0.8 7.9 ( 0.8 7.2 ( 0.5
34.2 ( 0.9 29.2 ( 0.8 28.5 ( 1.0
1 2 3
5 5 5
491.9 ( 1.9 490.7 ( 1.8 NA
Hybrid Poplar 152.3 ( 0.8 12.0 ( 0.9 149.1 ( 1.3 10.5 ( 0.7 137.9 ( 1.6 10.8 ( 1.1
9.1 ( 1.2 9.4 ( 1.3 9.5 ( 1.4
14.9 ( 1.2 14.5 ( 1.3 14.2 ( 1.0
1 2 3
5 5 5
476.5 ( 1.6 471.6 ( 1.4 NA
127.9 ( 1.0 125.2 ( 1.9 120.7 ( 1.4
19.0 ( 1.3 18.2 ( 1.1 17.8 ( 1.1
Douglas Fir 99.1 ( 3.0 94.4 ( 1.4 86.5 ( 1.5
28.9 ( 0.9 27.7 ( 1.6 28.4 ( 1.4
The raw experimental data are available as Supporting Information. b NA ) not available.
placing crucibles in a furnace or removing them, use appropriate personal protective equipment, including heat-resistant gloves.
Results and Discussion First, we investigated the degradation degrees of monomeric sugars in 72% sulfuric acid at 30 °C and in 1–4% sulfuric acid at 121 °C (data not shown). The degradation order of monomeric sugars in high acid/low temperature or dilute acid/high temperature were xylose > arabinose > mannose > galactose > glucose, in a good agreement with previous results.18,19 These results suggest that the five-carbon sugars of xylose and arabinose are more acid-labile than are the other six-carbon sugars of glucose, galactose, and mannose. Second, we investigated the conditions of acid concentrations from 1 to 4% sulfuric acid and autoclave time on hydrolysis of a mixture of cellodextrins (cellooligosaccharides) or xylooligosaccharides with various degrees of polymerization (DP) (data not shown). Complete hydrolysis of cellobiose (DP ) 2) requires at least 2% sulfuric acid and a 60 min reaction time; longer cellodextrins, e.g., G10 (DP ) 10), required higher acid concentrations (4% sulfuric acid) for >40 min. But it was found that 1% sulfuric acid and 60 min at 121 °C were enough for complete cleavage of oligo-xylosaccharides with DP from 2 to greater than 20 to monomeric xylose unit (data not shown). Furthermore, longer reaction time or higher acid concentration significantly increased xylose degradation. The hydrolysis and degradation kinetics of much easily hydrolyzed hemicellulose and its derivatives, oligo-xylosaccharides, have been investigated recently.12,13,20 The above results suggest that we can measure monomeric sugar contents with higher accuracy by decreasing the disparity between RPi and RMi. The putative best strategy may be to (1) (18) Saeman, J. F. Kinetics of wood saccharification: hydrolysis of cellulose and decomposition of sugars in dilute acid at high temperature. Ind. Eng. Chem. Anal. Ed. 1945, 37, 44–52. (19) Albersheim, P.; Nevins, D. J.; English, P. D.; Karr, A. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohyd. Res. 1967, 5, 340–345. (20) Brennan, M. A.; Wyman, C. E. Initial evaluation of simple mass transfer models to describe hemicellulose hydrolysis in corn stover. Appl. Biochem. Biotechnol. 2004, 113/116, 965–976.
use 72% sulfuric acid at 30 °C for an hour, converting all polysaccharides to oligosaccharides; (2a) use 4% sulfuric acid at 121 °C for an hour, converting all cellodextrins to glucose; and (2b) use 1% sulfuric acid at 121 °C for an hour, converting all hemicellulose oligosaccharides to monomeric units: xylose, arabinose, mannose, and galactose. Figure 2 presents the monomeric sugar degradation at different conditions. C1, representing method 1, includes the primary hydrolysis using 72% sulfuric acid and the secondary hydrolysis using 4% sulfuric acid; C2, representing method 2, is the secondary hydrolysis only in 4% sulfuric acid; and C3, representing putative method 3, is the secondary hydrolysis only in 1% sulfuric acid. Regardless of sugar type, the majority of sugar degradation happened in the secondary hydrolysis. Method 2 is expected to obtain somewhat more accurate data than method 1 because its correction coefficients do not include the primary hydrolysis. Furthermore, the degradations of acid-labile sugars xylose and arabinose in C3 using a weaker acid was decreased by 4.4- and 4.0-fold from those of C2. That is, RMi values of xylose and arabinose increased from 0.860 to 0.968 and from 0.913 to 0.978, respectively. Table 1 presents the carbohydrate contents of switchgrass, corn stover, poplar, wheat straw, and Douglas fir measured by different protocols. Most sugar values have a decreasing trend from C1 to C2 to C3 because their RMi values have an increasing trend from C1 to C2 to C3 (in Figure 2). Obviously, the NREL 2006 version resulted in more accurate results for sugar contents, but there are no statistically significant differences in the data from method 2 and method 1 based on t test values (data not shown). Using a t test with R ) 0.01 level, the t-values of the xylan contents for switchgrass, corn stover, wheat straw, hybrid poplar, and Douglas fir by C2 and C3 are 3.98, 5.79, 3.72, 3.75, and 3.34, respectively. They are significantly higher than 2.67 (R ) 0.01). Therefore, statistical analysis clearly indicates that there are significant differences between acid labile xylans by C2 and C3. Such overestimations are 4.2% for switchgrass, 8.5% for corn stover, 8.3% for wheat straw, 8.1% for hybrid poplar, and 9.1% for Douglas fir. These results suggested that xylan contents based on method 2 were overestimated due to the invalid assumption that the monomeric sugar degradation degree
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is the same as that of the polysaccharide. Similarly, arabinan contents by C3 showed some improvement as compared to those by C2, but they did not present any statistical difference (data not shown). It is anticipated that some statistical difference could be shown when more duplicate assays/samples are conducted for decreasing the standard deviation of the assays. We have also tested several different autoclaves (two modern microprocessor-driven autoclaves at Virginia Tech and one at Dartmouth College) and a manual autoclave at Dartmouth College.21 The results from the proposed QS always showed statistically significant difference from those of the NREL protocol regardless of autoclave type and operation mode.21 The parallel autoclaving for both of the lignocellulose hydrolysate samples and monomeric sugar controls can minimize the influences from variations in autoclaving. Here we propose the slightly modified QS for determining carbohydrate composition in lignocellulosic materials. The protocol involves the primary hydrolysis (72% w/w sulfuric acid, 30 °C, 1 h), followed by the secondary hydrolysis (4% w/w sulfuric acid, 121 °C, 1 h, for glucose, galactose, and mannose) and a parallel secondary hydrolysis (1% w/w sulfuric acid, 121 °C, 1 h, for xylose and arabinose), as shown in Figure 1. We do not recommend measuring mannose and galactose content in the 1% sulfuric acid hydrolysate because ultralow sugar concentrations (high dilution of low-content hemicellulose (21) Zhang, Y.-H. P.; Lynd, L. R. An improved method for quantitative saccharification of lignocellulose. Presented at the 6th Symposium on Biotechnology for Fuels and Chemicals, Chattanooga Choo Choo, TN, 2004.
Moxley and Zhang
sugars—mannose and galactose) could be associated with large standard deviations. The modified QS protocol can provide more accurate hemicellulose contents than do NREL 1996 and 2006 protocols because it can significantly decrease the disparity in degradation between monomeric sugars and polysaccharides. Our data for five lignocellulosic materials—herbaceous (switch grass, corn stover, and wheat straw), hardwood (hybrid poplar), and softwood (Douglas fir)—suggest that there is a significant overestimation of acid-labile xylose contents by the NREL 2006 protocol. The recommended change adds a little complication to testing but can significantly improve the accuracy of acidlabile hemicellulose sugar composition. Finally, the researchers working on other lignocellulosic materials may check whether the overestimation of acid-labile carbohydrate composition occurs by comparing the results from the NREL 2006 protocol and the proposed modified QS. Acknowledgment. This work was made possible with the new faculty start-up support of Biological Systems Engineering Department of Virginia Tech and DOE ONRL-led BioEnergy Science Center (BESC). The authors are grateful to James McMillan, Richard Elander, and Jack Saddler for providing lignocellulosic materials and to Lee Lynd for useful discussions. Supporting Information Available: Raw experimental data. This material is available free of charge via the Internet at http://pubs.acs.org. EF7003893
