Article pubs.acs.org/EF
Improvement of Acid Hydrolysis Procedures for the Composition Analysis of Herbaceous Biomass Matthew B. Whitfield,† Mari S. Chinn,*,† and Matthew W. Veal‡ †
Department of Biological and Agricultural Engineering, North Carolina State University, Campus Box 7625, Raleigh, North Carolina 27695-7625, United States ‡ Bayer CropScience, 2 T W Alexander Drive, Research Triangle Park, North Carolina 27709, United States S Supporting Information *
ABSTRACT: The accurate characterization of biomass is critical for development of bioenergy feedstocks and their utilization. Most analytical approaches involve acid hydrolysis of the polysaccharides in biomass, leaving most of the lignin as insoluble residue. A limitation of this approach is that the same conditions used to hydrolyze polysaccharides also degrade the liberated monosaccharides. The NREL-compiled procedures account for this effect with “Sugar Recovery Standards”, in which a solution of the expected monosaccharides is prepared and subjected to the dilute-hydrolysis portion of the procedure; however, this tends to overestimate monosaccharide degradation and introduce bias between polysaccharides of different lability. The following recommended method modifications are intended to reduce these errors: (1) quantification of immediate degradation products of monosaccharides and their stoichiometric addition to the monosaccharide yield; (2) the adjustment of this combined yield with sugar recovery standards; and (3) preautoclave analysis of hydrolyzates to improve the estimation of monosaccharide concentration profiles for adjustment calculations.
1. INTRODUCTION Accurate quantification of the components in biomass is critical for the development of bioenergy and bioproduct feedstocks and the processes necessary for their utilization. Conversion of feedstocks for fermentation, in particular, requires the quantification of both soluble saccharides, which are typically directly fermentable, as well as polysaccharides, which can be degraded into fermentable saccharides. Because the major polysaccharides (starch, cellulose, and hemicellulose) are not easily soluble, they are typically quantified indirectly via their degradation products.1 Many such methods are derived from the Klason lignin method,2 standard versions of which have been compiled by the National Renewable Energy Laboratory (NREL)1,3−5 and ASTM.6−11 The previously solvent-extracted biomass is typically treated with concentrated sulfuric acid to disrupt the linkages among the lignin, cellulose, and hemicellulose; disrupt the superstructure of the cellulose; and partially degrade the lignin, in order to facilitate the hydrolysis of the polysaccharides to soluble oligomers. The resulting solution is then diluted with water and heated to degrade the oligosaccharides. The resulting monosaccharides (typically quantified by HPLC) are taken to represent the polysaccharides in the biomass: glucose is assumed to derive from cellulose, and the pentose sugars (such as xylose and arabinose) are assumed to derive from hemicellulose. A limitation of this approach is that these conditions also degrade the resulting monosaccharides. For the most part, this process (proceeding from cross-linked polysaccharide, to labile polysaccharide, to oligosaccharides, to monosaccharides, to monosaccharide degradation products, to further degradation products) occurs approximately as a series of irreversible, pseudo-first-order reactions.12 Specifically, hexoses (e.g., © XXXX American Chemical Society
glucose) degrade to 5-hydroxymethylfurfural (5-HMF) (Figure 1) and pentoses (e.g., xylose and arabinose) degrade to 2furaldehyde (Figure 2); in turn, 5-HMF degrades to levulinic acid and formic acid, while 2-furaldehyde degrades to formic acid.13,14 Therefore, the resulting monosaccharide concentration will depend not only on the initial quantity of polysaccharide, but the extent to which the monosaccharides themselves degrade. That extent depends, in part, on the concentration profile of the monosaccharides over the course of the hydrolysis. This profile is extremely difficult to predict (largely because of the complexity of the degradation kinetics of the lignin-cellulose linkages), and impractical to determine empirically on a routine basis. The NREL-compiled procedures prescribe the use of “Sugar Recovery Standards” (SRS) to model monosaccharide losses during hydrolysis.4 The SRS, containing monosaccharides in their expected final concentrations, is subjected to the diluteacid hydrolysis portion of the procedure, and used to adjust the monosaccharide concentrations in the samples. This is intended to account for the monosaccharide degradation, and, particularly, for any variations in the procedure that might affect yield. There are several potential issues with this approach: 1.) The monosaccharides in the SRS are exposed to the dilute-acid hydrolysis conditions for the entire hydrolysis period. In actual biomass samples, the liberated monosaccharides are only available for degradation after oligosaccharide cleavage. More degradation thus Received: June 7, 2016 Revised: August 26, 2016
A
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amounts of monosaccharides. Where the experimental treatment is one that might affect polysaccharide accessibility (e.g., pretreatment severity), the resulting change in the polysaccharide degradation rate will result in a change in monosaccharide concentrations. Because the SRS cannot account for such differences among samples, any actual effects of the treatments on polysaccharide content will be confounded with effects on polysaccharide analytical recoveries. 5.) The current NREL LAP recommends performing the SRS analysis in a volume of 10 mL instead of the 84 mL volume of the sample. In the relevant ASTM standard, the SRS approach involves subjecting monosaccharides to the concentrated-acid hydrolysis as well as the dilute-acid.10 While this does account for the effects of the concentrated-acid hydrolysis on monosaccharides, it almost certainly compounds the overestimation of monosaccharide losses, given that only small quantities of monosaccharides are actually liberated during the concentrated-acid hydrolysis of typical biomass (as we confirm; see SI). Some attention has been given to the quantification of the degradation products of monosaccharide that are formed during the hydrolysis procedure. Kaar et al.16 pioneered the use of sealed reaction vessels in the degradation of polysaccharides in woody biomass in order to include the analysis of monosaccharide degradation products (which are volatile under reaction conditions). A recent study17 subjected solutions containing a range of monosaccharide concentrations to the hydrolysis conditions and proposed that the ratio of degradation product thereby formed to the monosaccharide lost in the process be used to derive correction factors to estimate the monosaccharide losses from the degradationproduct concentrations. However, as with the SRS procedure, this approach does not account for the kinetics of the factors leading to the monosaccharide release. The ratio between the degradation extent of a given monosaccharide and the concentrations of its immediate degradation products (IDPs) (e.g., (glucose initial − glucose final)/HMF final) will increase over time (if there is any subsequent degradation of the IDPs, and assuming that the degradation steps both occur as irreversible reactions of similar order). The application of this type of correction factor will therefore result in the overestimation of the degradation that occurs in the actual samples, leading to a consistent overestimation of initial polysaccharide amounts. The confounding of this effect with the different labilities of hemicellulose and cellulose (Point 3) and with polysaccharides quantified from samples subjected to different experimental treatments (Point 4) will also occur with this approach. Another approach, developed by Moxley and Zhang,18 is to subject the samples to two different dilution levels in order to improve the hemicellulose quantification by reducing the pentose degradation. Practically speaking, after the concentrated acid hydrolysis step, the sample is diluted to a 4% sulfuric acid concentration as described by the NREL method; an aliquot of that dilution is then further diluted 4-fold to generate a sample in 1% sulfuric acid. The two dilutions are then subjected to the heating step together. Cellulose is then quantified from the 4% dilution and hemicellulose from the 1% dilution, under the reasoning that the 1% acid is concentrated enough to hydrolyze hemicellulose, but not cellulose. This
Figure 1. Hydrolysis of cellulose to glucose via oligosaccharides, followed by degradation of glucose to 5-HMF, and 5-HMF to levulinic acid and formic acid.
occurs in the SRS than in the dilute-acid hydrolysis of samples.15 2.) Any degradation that occurs during the concentratedacid hydrolysis is not accounted for. 3.) The hemicelluloses are more labile than cellulose, and so will hydrolyze to a greater extent in the concentrated-acid hydrolysis and more quickly during the dilute-acid hydrolysis. The hemicellulose degradation is therefore closer to the monosaccharide degradation rate than cellulose is, which will lead to a bias in the hemicellulose/ cellulose ratio. 4.) Any differences in polysaccharide lability that may exist among the particular samples in a study will result in different degradation rates and/or extents. Samples with the same polysaccharide composition, but with differing polysaccharide characteristics, either inherent or introduced by the experimental treatments, will yield different B
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Figure 2. Hydrolysis of hemicellulose to xylose and arabinose, followed by degradation of both to 2-furaldehyde, and 2-furaldehyde to formic acid.
approach does improve the hemicellulose/cellulose bias (Point 3) generated by the standard method. It also reduces the reliance of the hemicellulose analysis on the SRS, and so partially addresses Point 1 with respect to hemicellulose. However, this approach does not address the bias among samples of differing lability. Given that it is not possible to predict polysaccharide hydrolysis rates with a useful level of accuracy, practically quantify monosaccharide concentrations during hydrolysis, or fully quantify all the monosaccharide degradation products, the quantification of polysaccharides by acid hydrolysis is at least partially an empirical method. However, we demonstrate here
several modifications to the existing methods in the interests of better assessments of both the total fermentable-sugar production potential of biomass and the relative effectiveness of different biomass processing parameters. Specifically, these modifications entail the quantification of the degradation products of monosaccharides and their stoichiometric addition to the monosaccharide yield; the adjustment of this combined yield using SRS; and improvements in the estimation of monosaccharide concentration profiles during hydrolysis. We demonstrate the usefulness of these method modifications using separated pith and rind tissue from sweet sorghum stalks. This allows the direct comparison of the relative efficacy of C
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levulinic acid, 5-HMF, and 2-furaldehyde. This column is analogous to the Phenomenex RHM and Aminex HPX-87H columns. 2.4. Sugar Recovery Standards. A solution of glucose, xylose, and arabinose was prepared at concentrations similar to those anticipated in the biomass hydrolyzates based on typical compositions of the type of biomass analyzed. For this work the concentrations were approximated as 1 mg/mL for glucose and xylose and 0.5 mg/mL for arabinose. For each batch of biomass samples autoclaved (∼12 samples per batch), an 84 mL aliquot of this solution was added to a serum bottle, followed by 3 mL of 12 M sulfuric acid, for a final volume of 86.73 mL, and then processed as part of the batch of samples. 2.5. Individual Analyte Recovery Determination. A set of preparations, each containing one of the monosaccharides (glucose, xylose, and arabinose) or IDPs (5-HMF and 2-furaldehyde) of interest, was subjected to the dilute-acid hydrolysis described in Section 2.2 (in the same manner as the recovery standards analysis in Section 2.4). The glucose and xylose preparations were at approximately 100 and 120 mg/mL, respectively, arabinose at approximately 14 mg/mL, and 5-HMF and 2-furaldehyde at 22 and 24 mg/mL, respectively. These preparations were performed at higher concentrations to better observe the carbohydrate degradation from these individual monosaccharides and degradation products and to present confirmation of the reactions. A 1 μL aliquot of each sample was analyzed under the HPLC conditions described in Section 2.3. 2.6. Sugar Recovery Standard Volume Effects. Twelve SRS were prepared as in Section 2.4 (by adding 3 mL of 12 M sulfuric acid to 84 mL of monosaccharide solution). Six more were prepared by adding 348 μL of 12 M sulfuric acid to 10 mL of the same monosaccharide solution (as specified in the NREL LAP). Another six were prepared by adding 357 μL of 12 M sulfuric acid to 10 mL of monosaccharide solution (to generate an acid strength equivalent to 3 mL of acid added to 84 mL of solution); this generates a final volume of 10.325 mL. Six more were therefore prepared using 10.325 mL aliquots of a SRS prepared according to Section 2.4 (to account for any effect of the SRS preparation procedure). The resulting 18 approximately 10 mL standards were processed in the same serum bottles as the biomass samples (125 mL). In a separate analysis, the effect of bottle (or headspace) volume was examined in addition to the effect of liquid volume. Six SRS (86.73 mL) were prepared as in Section 2.4. Two additional standards were prepared in this manner and combined to provide sufficient volume for the remaining analyses without introducing any unnecessary changes in preparation procedure. From this, 10.325 mL each was placed in each of six 125 mL serum bottles (actual volume 158 mL) and six 10 mL serum bottles (actual volume 12 mL). (See Table 1 for summary.)
these procedures on tissue types that differ in composition and fiber structure.19,20
2. EXPERIMENTAL SECTION 2.1. Analytical Materials. Sorghum samples from four sweet and two forage cultivars (Dale, Topper, Theis, Sugar T, ES5140, and ES5201, respectively) were collected from the Williamsdale Biofuels Field Laboratory near Wallace, NC (34.765 N, 78.100 W). Stalk samples were manually separated into pith and rind fractions and dried at 45 °C. The dried samples were ground in a Wiley mill (Model 4, Thomas Scientific, Swedesboro, NJ) to pass a 2 mm mesh screen. The ground samples were subjected to a Soxhlet extraction as previously described.21 Glucose, xylose, arabinose, 5-HMF, 2-furaldehyde, and levulinic acid were obtained from Sigma-Aldrich (St. Louis, MO). 2.2. Acid Hydrolysis. The acid hydrolysis procedures largely followed the NREL and ASTM methods, with modifications to the concentrated-acid hydrolysis, sampling points, acid neutralization, SRS volumes, and adjustment calculations (see Figure 3 for summary).
Figure 3. Steps in typical lignocellulosic analysis methods. Shaded blocks are modified in this study. Briefly, 0.3 ± 0.01 g of biomass (weighed to ± 0.0001 g) was placed in a 125 mL serum bottle, to which was added 3 mL of 12 M sulfuric acid (Ricca, Arlington, TX) using a bottletop dispenser (Dispensette III, Brandtech Scientific, Essex, CT). The mixture was incubated for 1 h at 30 °C with regular agitation (concentrated-acid hydrolysis). Agitation was performed by placing a magnetic stir bar in the bottle and placing the bottle over the magnetic drive of a Boekel reciprocating water bath (Feasterville, PA). (See Supporting Information for details.) Deionized water (84 mL) was added using a bottletop dispenser (EMD Polyfix, EMD Chemicals, Gibbytown, NJ), the resulting suspension was vortexed to mix, and 1 mL was removed to determine initial monosaccharide values by HPLC (presample). The sealed bottle was placed in an autoclave for a 1 h cycle at 121 °C (dilute-acid hydrolysis). After cooling, the samples were vacuum-filtered through prefired, tared filtering crucibles; the crucible and sample were weighed after drying overnight at 105 °C. 2.3. HPLC Analysis. HPLC samples were neutralized according to the NREL LAP with 0.057 g calcium carbonate per mL of sample. The use of a constant ratio of calcium carbonate was necessary because the neutralization affects the volume of the sample (through the precipitation of calcium sulfate and the generation of reaction water) and it was desired to normalize any volume changes. The neutralized samples were then centrifuged (10 min, 20 800g) and filtered through 0.2 μm nylon filters (Whatman, Maidstone, UK). A 10 μL aliquot of each sample (with the exception of Section 2.5, below) was analyzed using a Phenomenex (Torrance, CA) Rezex ROA column (300 mm × 7.8 mm) at 50 °C in 50 min runs, with 0.6 mL/min 5 mM sulfuric acid in HPLC water (Sigma-Aldrich) as the eluent, and quantified by refractive index detection (RID) for glucose, xylose, arabinose,
Table 1. SRS Analyses for Determination of Effects of Preparation Method, SRS Volume, and Bottle Volume quantity
SRS volume
bottle sizea
12
86.73 mL
125 mL
6
∼10 mL
125 mL
6
∼10 mL
125 mL
6
∼10 mL
125 mL
6
86.73 mL
125 mL
6
∼10 mL
125 mL
6
∼10 mL
10 mL
description matches hydrolyzate sample volume and bottle size NREL LAP defined approach (347 μL acid +10 mL water) scaled from sample acid/water ratio (357 μL acid +10 mL water) subsampled from an 86.73 mL preparation matches hydrolyzate sample volume and bottle size NREL defined volume in sample bottle size NREL defined volume in reduced bottle size
a
Bottle size has a nominal volume of 125 or 10 mL and actual volume of 158 and 12 mL, respectively.
D
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Energy & Fuels Table 2. Recoveries in Individual Analyte Degradations Under Dilute-Acid Hydrolysis Regimea glucose xylose arabinose 5-HMF 2-furaldehyde a
monosaccharide
2-levulinic acid
5-HMF
2-furaldehyde
sum
0.941 0.905 0.824 ND ND
0.00690 ND ND 0.692 ND
0.00791 ND ND 0.250 ND
ND 0.0586 ND ND 0.959
0.956 0.964 0.824 0.942 0.959
All values are on a mol per initial mol of analyte basis. Analytes not detected are marked ND.
2.7. Autoclave performance validation. In order to assess the effect of the autoclave step on the performance of the dilute-acid hydrolysis, 24 SRS were prepared as in Section 2.4. Three batches were separately autoclaved, with each batch of bottles physically arranged in a 2 × 4 grid.
[Hexose]̂ = [Glucose] +
and after (indicated by f) autoclaving. The recovery fraction is then used to estimate a loss based on this approximation, and the loss is added to the final monosaccharide equivalent. wCellulose =
⎛ 180.16 ⎞ ⎛ 180.16 ⎞ ⎜ ⎟[HMF] + ⎜ ⎟[LA] ⎝ 126.11 ⎠ ⎝ 116.11 ⎠ (1)
̂ = [Xylose] + [Arabinose] + [Pentose]
wHemicellulose =
⎛ 150.13 ⎞ ⎜ ⎟[Furaldehyde] ⎝ 96.08 ⎠ (2)
[Hexose]̂ f [Hexose]̂ f = [Hexose]i [Glucose]i
(3)
RPentose =
̂ f ̂ f [Pentose] [Pentose] = [Pentose]i [Xylose]i + [Arabinose]i
(4)
2.8.2. Sugar Recovery Standard Yield. The SRS yields (R) are calculated by dividing the monosaccharide equivalents (eq 1 and 2) in the autoclaved SRS (indicated by f) by the concentrations at which the SRS solution was prepared (indicated by i) (that is, the concentration prior to acid addition). R thus accounts for the degradation of the analytes, as well as the changes in sample volume that result from the addition of sulfuric acid and the subsequent neutralization with calcium carbonate. [Hexose]̂ f,adj =
⎞ [Hexose]̂ i + [Hexose]̂ f ⎛ 1 − 1⎟ ⎜ 2 ⎝ RHexose ⎠ + [Hexose]̂ f
̂ f,adj = [Pentose]
(5)
⎞ ̂ i + [Pentose] ̂ f⎛ 1 [Pentose] − 1⎟ ⎜ 2 ⎝ RPentose ⎠ ̂ f + [Pentose]
̂ f,adj ⎛ 132.11 ⎞ [Pentose] ⎜ ⎟ × 84 mL msample ⎝ 150.13 ⎠
(7)
(8)
2.8.4. Polysaccharide Mass Fractions. The respective mass fractions (w) of the polysaccharides in the initial sample are derived (similarly to the NREL LAP) by dividing the final monosaccharide equivalents by the mass of sample used in the analysis, and then multiplying both by an approximation of the mass change of the saccharide resulting from the hydrolysis reaction (i.e., the ratio of the approximate molar weight of a typical monomeric unit from cellulose and hemicellulose (162.14 and 132.11) to the molar weight of the corresponding monosaccharide of glucose and pentose (180.16 and 150.13)) and by the initial volume of water (84 mL). The SRS yields in eqs 3 and 4 are calculated using the initial water volume (that is, 84 mL, not the combined water and acid volume of 86.73 mL). Therefore, because the recovery accounts for the effect of acid dilution, the weight of sample is also calculated without the addition of acid (that is, using 84 mL). The same value must be used for both calculations: for the SRS to be a meaningful predictor of the samples, it must be assumed that any incidental volume changes (e.g., from vapor equilibration in the autoclave, or vaporization during vacuum filtration) are the same in both the SRS and the samples. (It would also be possible to use the initial SRS concentration after acid addition as the denominator of eq 3 and 4, in which case 86.73 mL should be used in place of 84 mL in eq 7 and 8; the results will be the same.) In summary, eqs 1 and 2 are used to calculate the monosaccharide equivalents in the biomass samples and recovery standards. (“Monosaccharide equivalents” are the stoichiometric sums of the surviving monosaccharides and their degradation products.) Eqs 3 and 4 are used to calculate the monosaccharide recovery yields in the sugar recovery standards, using the monosaccharide equivalents in the SRS after autoclaving (calculated from eqs 1 and 2) and the initial monosaccharide content. Eqs 5 and 6 are used to calculate the final, SRS-adjusted, monosaccharide equivalents in the biomass samples, by calculating the monosaccharide equivalents present in the final biomass sample (term 2 in eqs 5 and 6) and adjusting them by an estimate of the losses in monosaccharide equivalents during autoclaving. The estimate of those losses is determined by applying the SRS yield (eqs 3 and 4) to an estimate of the average monosaccharide equivalent content during the autoclave step (term 1 in eqs 5 and 6). 2.9. Statistical Analysis. SRS volume, tissue-type (pith, rind), and cultivar fixed effects were modeled in PROC GLM in SAS 9.3. The variation in the results of the polysaccharide yield calculations was analyzed by Levene’s test using the approach described by Littell.22
2.8. Calculations. 2.8.1. Monosaccharide Concentration Equivalents (Adjustment A). [Hexose]̂ in eq 1 is defined as the degradation̂ product-adjusted, hexose concentration-equivalent, with [Pentose] mutatis mutandis for pentose in eq 2. The monosaccharide equivalents are the sums of the molar concentrations of the monosaccharides and their degradation products, or, as given here, the sum of the mass concentrations of the monosaccharides and their degradation products, adjusted by their molecular weights to the mass of monosaccharide consumed in their generation. In this regime, 5-HMF (MW 126.11) and levulinic acid (MW 116.11) are each assumed to be derived from an equimolar quantity of hexose (MW 180.16), and so are adjusted by their molecular weight ratios to what would be their equivalent hexose mass. Xylose and arabinose (MW 150.13) are quantified together, because they both degrade to 2-furaldehyde (MW 96.08) (which is also adjusted to its equivalent pentose mass).
RHexose =
[Hexose]̂ f,adj ⎛ 162.14 ⎞ ⎜ ⎟ × 84 mL msample ⎝ 180.16 ⎠
(6)
3. RESULTS 3.1. Individual-Analyte Degradations Under DiluteAcid Hydrolysis. The degradation profiles observed from the individual-analyte recovery determinations in Section 2.5
2.8.3. Adjusted Sample Monosaccharide Yields (Adjustment B). To adjust the final monosaccharide equivalents, the monosaccharide concentrations during the dilute-acid hydrolysis are approximated from the mean of the concentrations measured before (indicated by i) E
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Energy & Fuels Table 3. Sugar Recovery Standard Yields After Dilute-Acid Hydrolysis by Volumea standard volume (mL)
glucose
xylose
arabinose
5-HMF
2-levulinic Acid
2-furaldehyde
10 84 p-value
0.9623 ± 0.0036 0.9592 ± 0.0041 0.1430
0.8666 ± 0.0018 0.854 ± 0.0020 < .0001
0.9313 ± 0.0037 0.9195 ± 0.0043 < .0001
0.01036 ± 0.00025 0.01022 ± 0.00028 0.3280
0.01601 ± 0.00055 0.01845 ± 0.00063 < .0001
0.09682 ± 0.0037 0.1011 ± 0.0039 0.0073
a
Monosaccharide values are g/g initial; 5-HMF and levulinic acid values are g/g initial glucose; and 2-furaldehyde values are g/g initial pentose (means followed by 95% confidence interval).
Table 4. Sugar Recovery Standard Yields After Dilute-Acid Hydrolysis by Headspacea headspace (mL)
glucose
xylose
arabinose
5-HMF
2-levulinic Acid
2-furaldehyde
2 74 148 p-value
0.9524 ± 0.0041 0.9565 ± 0.004 0.9581 ± 0.004 0.0357
0.8524 ± 0.0023 0.8506 ± 0.0023 0.8583 ± 0.0023 0.0018
0.9068 ± 0.0038 0.9075 ± 0.0037 0.9142 ± 0.0037 0.0081
0.007371 ± 0.00022 0.00748 ± 0.00022 0.007388 ± 0.00022 0.6966
0.01099 ± 0.00027 0.01208 ± 0.00026 0.01055 ± 0.00026 < .0001
0.04947 ± 0.00174 0.05398 ± 0.00159 0.04879 ± 0.00159 < .0001
a
Monosaccharide values are g/g initial; 5-HMF and levulinic acid values are g/g initial glucose; and 2-furaldehyde values are g/g initial pentose (means followed by 95% confidence interval). Headspace volumes are based on actual volume capacity of the 10 mL (12 mL) and 125 mL (158 mL) bottles.
differently sized bottles to determine whether the headspace above the sample would affect recoveries. As can be seen in Tables 3 and 4, both treatments did significantly affect saccharide and degradation-product yields. The fact that degradation-product yields increased while saccharide yields decreased implies that there is a change in the extent of the monosaccharide degradation. Although both factors contribute to this effect, it is unclear to what extent it is a direct result of the differences in volume, as opposed to the differences in headspace. Specifically, in the case of glucose, while the effect of volume within bottle size was not significant, the effect of bottle size within the smaller volume (10 mL) was significant. The effect on 5-HMF yields was smaller than for other analytes, corresponding to its position as an intermediate in glucose degradation. Even without a deep understanding of the mechanisms, it appears that a change in either the volume or headspace of a SRS can cause a bias in the yield of some analytes. Thus, preparing SRS in a manner that mimics as closely as possible the conditions of the biomass samples should improve their accuracy in modeling those samples. 3.3. Autoclave Performance Study. As Table 5 indicates, the only significant effect on monosaccharide and mono-
(Table 2) were generally those expected from the literature.23−27 A typical degradation profile observed from individual analyte recoveries, performed as described in Section 2.5, is shown in Table 2. The values were those generally expected from the literature: glucose yielded 5-HMF and no detectable 2-furaldehyde, while xylose yielded 2-furaldehyde and no 5-HMF. This implies that those degradation products can be added (on a molar basis) to their corresponding monosaccharides. Xylose was more labile than glucose, as expected; however, when their degradation products were taken into account, the recoveries for the two monosaccharides were similar. The arabinose preparation resulted in a recovery lower than the other sugars’, and contained no detected degradation products; this was most likely because a lower quantity of arabinose was used (∼14 mg/mL as opposed to ∼100 mg/mL) to better approximate the conditions in the biomass. 2-Furaldehyde was relatively stable under hydrolysis conditions while 5-HMF was not. The 5-HMF degradation results in equimolar amounts of levulinic acid and formic acid.25 However, to the extent that it does degrade, 2-furaldehyde also generates formic acid, so formic acid cannot be directly assigned to one monosaccharide. It should be possible to use the levulinic acid yield to determine the quantity of formic acid attributable to the hexose degradation, and assume the remainder derives from the pentose reaction chain. However, the quantities of formic acid assigned in this manner would be very small, and would include the errors not only of the formic acid quantification, but also of the levulinic acid quantification, as well as any unaccounted-for degradation of levulinic acid that may occur. The different ratios of levulinic acid to 5-HMF in the glucose (0.87) and 5-HMF preparations (2.8) illustrate the effect of the exposure time of intermediates on degradation extent. The 5HMF in the 5-HMF preparation was exposed to the hydrolysis conditions for the full time period, while the 5-HMF in the glucose preparation was exposed for a lesser time as a result of secondary reaction rates, and so did not degrade as extensively. 3.2. Sugar Recovery Standard Volume. In order to determine the effect of SRS volume on the extent of monosaccharide degradation during the dilute-acid hydrolysis, SRS were prepared both in the same volume as samples (86.73 mL, after acid addition) and in 10 mL volumes as specified in the NREL LAP. The 10 mL samples were autoclaved in two
Table 5. p-Values for the Effect of Autoclave Position and Autoclave Batch on the Recoveries of Monosaccharides and Monosaccharide Equivalents
column row batch
glucose
glucose equivalent
xylose
arabinose
pentose equivalent
0.2476 0.7335 0.6135
0.1945 0.7843 0.9975
0.5849 0.4329 0.0004
0.3035 0.4147 0.4428
0.2182 0.6412 0.4042
saccharide equivalent concentrations in the autoclave runs was the effect of batch on xylose recoveries; all position effects, and other batch effects, were not significant. The magnitude of the significant effect on Rxylose was small, with batch means of 86.8, 87.4, and 88.3%. There were no significant effects in the pentose equivalent yield when the 2-furaldehyde was included. Although the magnitude of effects such as these will be dependent upon the specific equipment used, this indicates that at least in this case, little recovery variability occurred from one run to another, and that what variability did occur was mitigated by the use of monosaccharide equivalents. As a result, F
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Energy & Fuels
also greater in the pith than in the rind. These differences between the hydrolysis yields of the pith and rind, if the result of the difference in the accessibility of the two types of cellulose, would confirm that (under the conditions of this analysis) more-accessible cellulose (as in pith) is hydrolyzed to monomer more quickly than less-accessible cellulose (as in rind), in which case we should expect cellulose accessibility to be confounded with cellulose quantity in these analyses, as discussed in point 4 of the Introduction. The difference between the xylose ratios of the two tissue types was not significant; this may be because the hemicellulose hydrolysis proceeds further in the concentrated-acid step, or it may be an indication that cellulose crystallinity per se, as opposed to lignocellulosic recalcitrance generally, is the major contributor to the effect in this case. 3.4.2. Application of Correction Factors. The degradation products in both the SRS and the samples were assigned to monosaccharides, and the resulting monosaccharide equivalent values were used to calculate final hydrolysis yields for cellulose and hemicellulose (Adjustment A) (eqs 1 and 2). In addition, the mean of the initial and final concentrations of each analyte over the dilute-acid hydrolysis was used to estimate monosaccharide losses (Adjustment B) (eqs 5 and 6). The results of these two adjustments, individually and combined, on both cellulose and hemicellulose yields in sorghum pith and rind samples are shown in Table 7. No differences were observed among the variances of the yields resulting from the standard method and the adjustments, as determined by Levene’s test (data not shown). The effect of the degradationproduct adjustment alone (Adjustment A) was significant for hemicellulose, but not for cellulose. This may be in part because this adjustment accounts for the degradation products generated during the concentrated-acid hydrolysis (which are detectable for hemicellulose, but not for cellulose), but also because xylose is more labile than glucose. The magnitude of this adjustment is small in this case; however, it does serve to reduce the reliance of the calculation on the SRS (by directly quantifying more of the polysaccharide constituents) and therefore reduce the magnitude of the SRS error. The adjustment of the concentration profile of the monosaccharides (Adjustment B) reduces both the cellulose and hemicellulose yields this is because this adjustment reduces the concentration basis for the recovery calculation. This has a larger effect on hemicellulose because the hemicellulose monosaccharides are more labile than the cellulose monosaccharides. The combined adjustment (A and B) reflects a recovery calculation that includes the degradation products and is based on the estimation of monosaccharide concentrations in solution over the course of the dilute-acid hydrolysis. Overall, this causes a reduction in the calculated cellulose yields and an increase in
it may be appropriate to perform a validation study of this kind ahead of a composition analysis study, and, if consistent, use the results of the validation study to generate what should be a more accurate estimate of the true recovery values than would be generated from a single SRS. Subsequently, an SRS would be analyzed with each batch of samples as described, but be used to verify that the system is in control (that is, that the batch recovery values are within a certain distance of the validation study means). This becomes important if autoclave reliability is questionable. 3.4. Monosaccharide Degradation Adjustment. 3.4.1. Effect of Cellulose Accessibility. In order to improve the estimation of monosaccharide degradation during the polysaccharide hydrolysis, sample hydrolyzates were analyzed for monosaccharide and major degradation product content both before and after autoclaving. This approach was applied to both the pith and rind fractions of sweet sorghum, which are known to differ in lignocellulosic content and structure, which presumably affects the accessibility of the polysaccharides for hydrolysis. The glucose and xylose liberated during the concentrated-acid hydrolysis as a fraction of glucose and xylose released in the entire procedure is presented in Table 6. A Table 6. Glucose and Xylose Released During ConcentratedAcid Hydrolysis and Degradation Products Generated During Dilute-Acid Hydrolysisa tissue type
glucose
5-HMF
2-levulinic acid
xylose
2furaldehyde
pith rind p-value
0.082 0.0693
