ARTICLE pubs.acs.org/IECR
Improved Xylan Hydrolysis of Corn Stover by Deacetylation with High Solids Dilute Acid Pretreatment Xiaowen Chen,* Joseph Shekiro, Rick Elander, and Melvin Tucker National Bioenergy Center, National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden, Colorado 80401, United States ABSTRACT: To produce ethanol cost-effectively from herbaceous feedstocks such as corn stover, efficient xylan hydrolysis with monomeric xylose yields approaching 90% are necessary. Dilute acid pretreatment is well established as one of the pretreatment technologies for xylan hydrolysis; however, the accumulation of salts from neutralization, the production of toxic byproducts, and the release of acetic acid can inhibit enzymatic saccharification and fermentation, resulting in depressed ethanol yields. Successful removal of acetyl groups from native corn stover by alkali de-esterification could potentially increase monomeric xylose yields from pretreatment and enzymatic hydrolysis, improve cellulose digestibility, and reduce the cytotoxicity of the fermentation broth. Results presented in this article show that alkaline extraction removed significant amounts of acetyl groups from corn stover, improved xylan hydrolysis in high solids dilute acid pretreatment by more than 50%, and improved xylan and glucan hydrolysis in low solids enzymatic hydrolysis by 15% and 30% over control samples. In whole slurry enzymatic hydrolysis, a 30% improvement in cellulose digestibility was found over the control.
’ INTRODUCTION Over the past 150 years, fossil fuels catalyzed the development of modern society by providing an inexpensive, reliable energy source. However, as the political and environmental issues posed by the procurement, production, and combustion of these fuels receive more attention, the development of a more sustainable energy economy has become pressing. To make the transition, the development of a biorefinery capable of producing liquid transportation fuels from biomass is critical. Cellulosic ethanol produced from corn stover is one of the most promising near term solutions. Corn stover is primarily composed of cellulose, hemicelluloses (primarily xylan), and lignin, in addition to small amounts of extractives and ashes. Typical corn stover composition can be assumed to be approximately 37% cellulose, 21% xylan, 18% lignin, and 3% acetyl groups on a dry weight basis; however, significant variation can occur as a function of plant species, growing season, and soil type.1 Structurally, hemicellulose surrounds and supports chains of cellulose fibers, while acetyl groups are esterified directly to the xylan backbone in positions O-2 or O-3,2 leading to an acetate/xylose molar ratio of ∼2:5. Conventionally, the biochemical production of ethanol from corn stover involves three fundamental stages: pretreatment, saccharification, and fermentation. Dilute acid pretreatment of biomass is a promising technology by which soluble monomeric and oligomeric xylose is produced from xylan through the partial deacetylation and depolymerization of hemicellulose.3 Many previous studies have shown a direct relationship between cellulose digestion and xylan removal.46 Next, a fraction of the remaining insoluble xylan and newly exposed cellulose is hydrolyzed into xylose and glucose through enzymatic saccharification. In the final stage, the sugar broth is fermented into ethanol using yeast or other genetically engineered bacteria. It has been reported in the literature that removal of acetyl groups during pretreatment has a significant effect on enzymatic r 2011 American Chemical Society
saccharification. Grohmann et al.4 showed that removal of 75% of the acetyl groups from xylan prior to pretreatment increased xylan digestibility by 57 times over native xylan. Deacetylation also increased the digestibility of the residual cellulose fraction by a factor of 23 due to improved enzyme accessibility.4 Agger et al.2 demonstrated a doubling of the xylose released from the insoluble fraction of pretreated corn bran by adding acetyl xylan esterase (AXE) to their monocomponent enzyme mixture. The AXE is an enzyme that catalyzes the deacetylation reaction of xylan and xylo-oligomer. Selig et al.7 showed that acetyl groups bound to the xylan backbone hindered enzyme access to the β-1,4 glycosidic linkages and that deacetylation provided more sites for enzymatic attack. Acetic acid liberated during pretreatment and enzymatic hydrolysis also has strong inhibitory effects on bacterial growth and ethanol fermentation. High solids (at 45% solids) dilute acid pretreatment at 150 °C, 0.5% acid for 20 min produces 4.9 g/L acetic acid. Pretreatment with higher severity at 160 °C, 2% acid for 5 min produces 11.8 g/L acetic acid. Takahashi et al.8 observed that acetate concentrations between 2.0 and 15 g/L significantly impacted the growth and fermentation performance of E. coli KO11. The presence of acetate not only extended the lag phase but also decreased ethanol production and cell growth rates. Others have shown that, of the common byproduct of biomass pretreatment, acetate was the most toxic to Zymomonas mobiliz CP4(pZB5) xylose fermentation.3,9 Despite the substantial evidence on the inhibitory effects of acetic acid, few efforts have been made to evaluate the effect of deacetylation on high solids dilute acid steam explosion (HSDASE) pretreatment and subsequent enzymatic digestion. In this study, Received: July 11, 2011 Accepted: November 19, 2011 Revised: October 24, 2011 Published: November 19, 2011 70
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we compare the overall carbohydrate yields for native and deacetylated corn stover in HSDASE pretreatment and enzymatic saccharification.
corn stover, an aliquot of NaOH-extracted and washed corn stover feedstock was immersed in 120 L of 0.5% H2SO4 at 60 °C for 2 h. For the control, dilute acid-only impregnated sample (S3), 2 kg (93%) of native corn stover was immersed in 120 L of 0.5% H2SO4 at 60 °C for 2 h. Following each respective acid impregnation, the feedstock was suspended in the basket and drained of excess liquid (to approximately 20% solids), loaded into the mold of a hydraulic dewatering press, and pressed to ∼43% solids. Steam Explosion Reactor. A 4-L steam explosion reactor (NREL Digester) was used for all pretreatment experiments. The NREL Digester was preheated to pretreatment temperature, loaded with 500.0 g of dewatered feedstock (40%45% solids), and quickly reheated (∼5 to 10 s) via direct steam injection to reaction temperature. Pretreatments were carried out at 150 °C with 20 min residence times. Low Solids Enzymatic Hydrolysis. Low solids enzymatic hydrolysis was performed according to an NREL Laboratory Analytical Procedure (LAP) in 120 mL screw-capped Erlenmeyer flasks. Hydrolysis was carried out using exhaustively washed pretreated solids using 20 mg protein per g cellulose loading (12FPU) of Genencor GC220 (120 FPU/mL, 2:1 CPU: FPU), 1% cellulose loading, and 50 g total weight, at 50 °C and 130 rpm. Saccharification samples were taken at 24, 48 (optional), 72 (optional), 96 (optional), and 168 h and analyzed by HPLC for glucose and xylose content. High Solids Whole Slurry Enzymatic Hydrolysis. The unwashed whole slurries produced in HSDASE were also subjected to enzymatic hydrolysis at 15% total solids. Samples were prepared as described in Table 2. Pretreated slurry (125 g) was first diluted with 75% of the calculated water required to achieve a 15% solids loading. Slurry then was adjusted to a pH of 5.0 by the slow addition of 28% ammonium hydroxide (NH4OH) to the continually stirred slurry. If the pH rose above 5.0 during neutralization, 10 N sulfuric acid (H2SO4) was used to correct the pH to 5.0. The neutralized slurry was then diluted to 15% solids with deionized water, and 60 g samples of prepared slurry were loaded into 125-mL polypropylene roller bottles with 20 mg protein (Genencor GC220) per g cellulose. Samples were incubated for 112 h at 50 °C, and bottles were rolled at 4 rpm. Analysis. Pretreatment and enzymatic saccharification liquors were analyzed using HPLC according to NREL LAPs.12,13 Solid residues were analyzed according to NREL LAPs.1214 Chemical analysis uncertainties have been addressed by Templeton et al.15
’ MATERIALS AND METHODS Feedstock. Whole corn stover (Pioneer variety 33A14) was harvested in 2002, tub ground at the farm in Wray, Colorado, and further milled at NREL through a Mitts & Merrill rotary knife mill (model 10 12) to pass a 1/4-in screen. NaOH Extraction. Deacetylation is the de-esterification of acetylated xylan catalyzed by either acid or alkali. Song et al.10 extracted all acetyl groups from spruce wood by soaking the wood in a bath of 70 °C NaOH solution at pH 12 for 1 h. For this study, deacetylation of corn stover was performed by alkali extraction in a Recirculating Atmospheric Pressure Impregnation (RAPI) system as described by Weiss et al.11 Approximately 10 kg of dry (∼93 wt %) 1/4-in milled corn stover was loaded into Hastelloy C-276 wire mesh (20 mesh screen) baskets and immersed in the recirculating bath containing 120 L of 0.4 wt % NaOH (0.1 M) solution at 60 or 80 °C for 3 h. The initial pH was approximately 12. After extraction, the excess alkali solution was drained, and a sample was retained for pH, acetate, and sugar concentration analysis. The extracted corn stover feedstock was separated into two portions. Approximately 20 kg (∼15 wt % wet solids) was pressed to ∼40% solids using a hydraulic dewatering press and used for dilute alkali pretreatments in the steam explosion reactor (see S1 in Table 1). The remaining 40 kg of deacetylated corn stover was washed four times. For each wash, the basket of deacetylated stover was immersed in the RAPI filled with 120 L of warm (∼60 °C) water for 1 h with recirculation and allowed to drain between washes. After four washes, the pH of the washed liquor measured ∼8.0. Approximately 40 kg (15 wt % solids content) of washed corn stover was recovered and divided into three parts. One 10-kg aliquot was pressed to ∼40% solids using a hydraulic dewatering press for steam-only pretreatment (refer to S2 in Table 1). The other two aliquots (15 kg each) were impregnated with dilute acid as described below (S4 and S5). Table 1 lists the preparation conditions for each sample. Acid Impregnation. Acid impregnation was carried out using the same 200-L RAPI system. To impregnate alkali-extracted
Table 1. Sample ID and Corresponding Preparation H2SO4 acid
pretreatment
impregnation
conditions
0.4 wt % NaOH sample ID
extraction
washed
S1
60 °C, 3 h
no
no
150 °C, 20 min
S2
60 °C, 3 h
yes
no
150 °C, 20 min
S3
No
yes
0.5%, 60 °C, 2 h
150 °C, 20 min
S4
60 °C, 3 h
yes
0.5%, 60 °C, 2 h
150 °C, 20 min
S5
80 °C, 3 h
yes
0.5%, 60 °C, 2 h
150 °C, 20 min
’ RESULTS AND DISCUSSION Deacetylation of Corn Stover. Table 3 shows the compositional analysis of native, dilute acid impregnated, alkali extracted, and the combination of alkali extracted and dilute acid impregnated (CAEDAI) corn stover feedstocks. After CAEDAI, approximate glucan and xylan fractions increased from 34 to 42 wt % and from 22 to 25 wt %, respectively.
Table 2. Whole Slurry Enzymatic Hydrolysis Sample Preparation sample amount of initial total DI-water for initial pH volume of added NH4OH ID
slurry (g)
solids (%)
dilution (g)
of slurry
(29%32%) (mL)
pH after adding the amount of NH4OH
volume of H2SO4 (10N) total amount of slurry to pH 5.0 (mL)
(15%) prepared (g)
S3
87.1
21.5
35.6
2.03
1.5
5.0
0
124.1
S4
108.69
17.3
14.5
2.08
1
6.0
0.3
124.6
S5
87.4
21.4
35.7
2.03
1
8.4
0.5
124.9
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Table 3. Chemical Compositional Analysis of Untreated, Dilute Acid Impregnated, Alkali Extracted, and Combination Alkali Extracted and Acid Impregnated Corn Stover Feedstocks sample
lignin (%)
glucan (%)
xylan (%)
acetyl (%)
ash (%)
total mass balance (%)
untreated corn stover
12.29
34.00
21.95
2.87
6.09
99.1
0.5% H2SO4 at 60 °C NaOH extracted at 60 °C
18.06 15.56
42.29 42.76
24.83 25.36
2.94 0.95
3.48 3.49
96.71 94.14
NaOH extracted at 80 °C
16.59
41.46
23.03
0.72
2.46
89.58
NaOH extracted at 60 °C and washed and resoaked
15.77
41.71
25.12
1.1
5.02
96.01
with 0.5% H2SO4 at 60 °C
Table 4. Chemical Analysis by HPLC of Extraction Liquors from Alkali-Extracted Corn Stover Feedstock sample
lignina
alkali extracted at 60 °C
1.15 g/L (12.1%)c
0.24 g/L (0.8%)
0.57 g/L (2.9%)
2.25 g/L (72.5%)
2.45 g/L (78.9%)
alkali extracted at 80 °C
2.23 g/L (23.4%)
0.55 g/L (1.9%)
0.90 g/L(4.7%)
3.12 g/L (100.5%)
4.11 g/L (132.4%)
glucan
xylan
free acetate
total acetateb
a Spectrophotometric determination of soluble lignin. b Acetate is present in two forms in the NaOH extraction liquor: (1) soluble free acetate/acetic acid that is already cleaved from the xylan backbone and (2) acetyl groups still bound to xylo-oligomer.
Table 5. Acetate Distribution in the Whole Pretreatment Process (Calculation Based on Solid Compositional Analysis)
a
acetate left in extraction
acetate in residue
acetate in acid
acetate mass
sample
liquor (%)a
solids (%)
hydrolysate (%)
closure (%)
control, 20 min NaOH extracted at 60 °C, 20 min
∼0 73.0
16.7 8.6
67.0 28.0
83.7 109.6
NaOH extracted at 80 °C, 20 min
80.0
4.7
24.5
109.2
This is calculated from acetyl groups in the alkali extracted/dilute acid soaked corn stover shown in Table 3.
The increases are attributed to the removal of acetyl groups, ash, soluble sugars, and other water-soluble extractives, which accounts for approximately 20% of the total weight loss during deacetylation/acid impregnation. While acid impregnation alone did not remove significant amounts of acetyl groups, as illustrated in Tables 4 and 5, alkali extraction at 80 °C successfully reduced the acetyl groups present in the solids by more than 75%. Alkali extraction decreased the acetyl content of native corn stover from 2.9 to 1.0 wt % and 0.7 wt % for 60 and 80 °C extractions, respectively. The postextraction liquors remaining in the recirculating bath were analyzed by HPLC, and the results are reported in Table 4. Assuming that no water evaporated during the 3 h extraction and that the sample was well mixed, the acetyl group yield is calculated to be 79% at 60 °C and 132% at 80 °C. The high acetate yield at 80 °C is likely attributed to standard error in solids compositional analysis (( 1.5 wt %) and water loss due to evaporation at the 80 °C extraction temperature. Small amounts of sugar are extracted under these conditions; less than 4% of the total sugar (glucose + xylose) was extracted at 60 °C, and less than 7% total sugars was extracted at 80 °C. A substantial amount of lignin (∼23%) is extracted at 80 °C, compared to only ∼12% at 60 °C. The extracted acetate calculated using data from HPLC analysis of the 60 °C extraction liquor is ∼79%, in close agreement with the ∼73% calculated from the solids compositional analysis. Effects of Deacetylation on High Solids Dilute-Acid Steam Explosion Pretreatment. Pretreatment experiments were carried out using feedstock prepared according to the conditions listed in Table 1. Improved monomeric and total xylose yields were found using alkali-extracted, dilute acid pretreatment
conditions (S4 and S5) compared to yields from the dilute acidonly pretreated control (S3), as reported in Figure 1. The soluble monomeric xylose yields increased from ∼52% in the dilute acid control (S3) to ∼76% for the deacetylated and HSDASE treated feedstocks (S4 and S5), while the total xylan solubilized increased from ∼77% for S3 to ∼86% for S4 and S5. Residual xylan in the pretreated solids correspondingly decreased from 21.9% in S3 to 17.5% in S4 and 17.0% in S5. No significant difference was apparent between samples deacetylated at 60 and 80 °C. Alkali extraction did not affect xylan degradation; in all dilute acid pretreatments, ∼1.8% of the xylan was converted to furfural. The unwashed alkali-extracted sample (S1) released a comparatively low amount of xylo-oligomers (7.7%) and only a trace amount of monomeric xylose (0.6%). Without the presence of added catalyst or liberated acetic acid, the alkali-extracted and washed sample (S2) achieved even lower yields, only releasing 2.1% xylo-oligomers and 0.2% xylose monomers. Figure 1 also shows the xylan mass balance for dilute acid pretreatment. Overall, xylan component mass balance closures vary between ∼98% and ∼106%. The improvement in xylose monomer yields reported for pretreated deacetylated samples is likely attributed to deacetylation of the xylan backbone, resulting in decreased recalcitrance through the formation of simpler, more easily hydrolyzed xylo-oligomers with fewer side branches during the initial stages of pretreatment. As a result, these oligomers are hydrolyzed to monomers during pretreatment. Reports in the literature describe such variations in reaction rates of xylan hydrolysis during dilute acid pretreatment.1618 Approximately 60%70% of xylan is hydrolyzed in a characteristic 72
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Figure 1. Xylan mass balance closures of S1S5 pretreated at 150 °C and 20 min.
Figure 2. Solubilization of acetate vs xylose for native and deacetylated corn stover.
fast first-order reaction. Obtaining higher soluble xylan yields in pretreatment requires going to higher severity conditions where the xylan hydrolysis initially proceeds at a fast rate for increased yields and eventually proceeds at a reduced rate.19 It is postulated that the less reactive fraction is embedded within or linked to lignin via lignincarbohydrate bonds20 such as ester bonds between hydroxide groups on lignin and acetyl groups branching off the xylan backbone.21 Thus, deacetylation prior to pretreatment may increase the fraction of xylan available to react at the faster rate. Figure 2 depicts the relationship between concentrations of monomeric xylose and acetate released in hydrolysate liquors from 127 HSDASE pretreatments using native corn stover. The samples generated as part of this study are also shown. Increasing pretreatment severity results in a nonlinear positive relationship between solubilized monomeric xylose and acetate as shown in Figure 2. Using low severity pretreatment conditions of 150 °C, 0.5 wt % H2SO4, and 90 s to 20 min residence times,
hydrolysis of 1 kg of stover released ∼0.5 mol of xylose and 0.1 mol of acetate, resulting in a xylose to acetate molar ratio of 5:1. As pretreatment severity was increased, this ratio increased to approximately 4:1 for moderate severity pretreatment and reached nearly 2:1 in the most severe pretreatments of 170 °C, 1 wt % H2SO4, and 30 to 60 min residence times. This trend indicates that the degree of acetylation correlates to the recalcitrance of xylan; less acetylated xylo-oligomers are easily reduced to monomers, while hydrolysis of xylo-oligomers having a higher degree of acetylation requires increased severity. The deacetylated samples, shown in red, support this hypothesis. The solubilized xylose to acetate ratio was ∼9:1 for samples deacetylated at 60 °C and ∼10:1 for samples deacetylated at 80 °C. For a feedstock with a lower degree of acetylation, less acetate was released during pretreatment, and more xylose was released with lower pretreatment severity. Table 5 reports the acetate distribution between the amounts measured in alkali and dilute acid RAPI liquors, the pretreated 73
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Table 6. Compositional Analysis of Washed Pretreated Solids Residues sample ID
conditions
% lignin
% glucan
% xylan
% acetyl
S1
alkali treated
12.3
50.2
25.4
0.0
S2
stream treated
16.4
43.5
26.0
1.4
S3
acid impregnated
25.1
58.6
8.4
1.2
S4
deacetylated, acid impregnated
23.8
59.1
7.0
0.7
S5
deacetylated (80 °C), acid impregnated
22.8
59.7
6.8
0.4
Deacetylation of corn stover feedstock improved the total xylan yield by ∼10% and improved monomeric xylose yield by 13%25%. Extraction with dilute sodium hydroxide effectively removed 70%80% of acetyl groups from the native corn stover feedstock. The molar ratios of acetyl to xylose in the residual xylan are significantly reduced by alkali extraction. Effect of Deacetylation in Low Solids Enzymatic Hydrolysis. Dilute acid and deacetylated/dilute acid pretreated corn stover slurries were washed extensively and subjected to low solids (∼2 wt %) enzymatic saccharification using Genencor GC220. The yields of xylose and glucose were calculated based on the xylose and glucose amount in treated solids. Figure 3a shows the monomeric xylose yields found over the 168 h time course of enzymatic saccharifications. The final monomeric xylose yield of the dilute acid native corn stover pretreated control (S3) reached ∼48%, whereas the xylose yields of the deacetylated corn stover dilute acid pretreated residues reached 55% (S4) and 58% (S5). During the time course of the saccharifications, the deacetylated dilute acid pretreated residue samples showed improved xylan hydrolysis compared to the dilute acid impregnated control. Low solids enzymatic saccharification of the alkali-extracted and steam pretreated residue samples achieved lower yields, likely due to lower pretreatment effectiveness and a large amount of intact hemicellulose. In 24 h, 10% of the available xylan was hydrolyzed in both S1 and S2. However, during the later residence time (24168 h), S1 displayed a faster conversion rate of xylan to xylose and ultimately achieved a xylose yield ∼10% higher than that of S2, likely due to any residual alkaline in the unwashed sample (S1) acting as a catalyst in pretreatment. In low solids enzymatic hydrolysis, the loading of GC220 was based on the mass of cellulose contained in the solids. While all samples contained the same level of cellulose, xylan content varied by sample, as illustrated in Table 6. The dilute acid impregnated samples (S3S5) had similar xylan content, but the alkali-only samples had significantly higher xylan content. As a result, the xylanase loading was approximately 4 times lower for the alkali-only samples. Figure 3b shows monomeric glucose yields resulting from low solids enzymatic hydrolysis of washed pretreated residues. Deacetylated and acid pretreated samples (S4 and S5) showed improved reaction rates and final yields over the dilute acid-only pretreatment control (S3). The glucose yield from enzymatic digestion of the dilute acid control residue achieved ∼72% yield in 168 h, versus S4 and S5 where final yields of 79% and 84%, respectively, were found. Deacetylated sample S1 reached ∼60% conversion after 168 h digestion, ∼20% higher than S2. While improvements in cellulose digestibility may not be attributed directly to deacetylation, improved xylan hydrolysis is known to increase cellulose accessibility, thus increasing saccharification yields. The removal of 4% more lignin in S1 (see Table 6) is another possible cause of the improved saccharification yields, by
Figure 3. Effect of deacetylation on monomeric xylose and glucose yields during low solids enzymatic hydrolysis.
solid residues, and the hydrolysate liquors. After alkali extraction, 73%80% of acetyl groups are removed in the form of sodium acetate in the extraction liquor. Roughly 20%30% are recovered as acetic acid in the dilute acid hydrolysis liquor, leaving less than 10% esterified to the xylan backbone in the insoluble solid residues. Solids compositional analysis data of washed pretreated solids for S1 through S5 are reported in Table 6. The dilute acid pretreated control, labeled S3, contains ∼1.2% acetate, giving a molar ratio of acetyl to xylan in the sozlids of 1:2. Deacetylation at 60 °C (S4) prior to acid impregnation and pretreatment shows a residual acetyl concentration of 0.7%, giving an acetyl-to-xylan ratio of approximately 1:3. Deacetylation of corn stover at 80 °C (S5) results in a lower acetyl content of ∼0.4% and lower acetylto-xylan ratio of 1:5. 74
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Figure 4. Effect of deacetylation on feedstock reactivity.
Figure 5. Deacetylation enhances glucan conversion during whole slurry enzymatic hydrolysis.
creating additional pores and increasing available surface area and exposure of cellulose fibers. Deacetylation effectively improved enzymatic saccharification yields for both xylose and glucose likely because of decreased steric hindrance of deacetylated xylose units in the xylan backbone and increased exposure of cellulose fibers. Alkali pretreated
corn stover reached 60% glucose and 40% xylose yields at a constant cellulase loading. For dilute acid pretreated corn stover, reduction of the acetyl/xylose ratio from 1:2 to 1:5 resulted in a 10% gain in glucose yield and a 12% increase in xylose yield. Feedstock Reactivity. Feedstock reactivity is defined in the following equation:
feedstock reactivity ¼
hydrolyzed glucose and xylose in pretreatment and enzymatic hydrolysis total glucan and xylan in native corn stover
In the equation above, the numerator includes the portion of glucose and xylose monomers hydrolyzed during dilute acid pretreatment and the portion of glucose and xylose hydrolyzed during low solids enzymatic hydrolysis (1% cellulose loading, GC220 20 mg protein/g of cellulose). It is divided by the total glucan and xylan in native corn stover. Therefore, feedstock reactivity represents the highest glucose and xylose fraction that can be utilized for fermentation under certain pretreatment and enzymatic hydrolysis conditions. Here, we assume that glucose and xylose oligomers cannot be directly utilized by microbes in fermentation. Figure 4 shows the reactivity of Kramer 33A14 corn stover pretreated at 150 °C, 0.5% H2SO4 (8 mg H2SO4/g of biomass) for 20 min. The feedstock reactivity for the control sample (S3) is 68.4%. The two major contributors are hydrolyzed xylose from pretreatment and glucose from enzymatic hydrolysis. Deacetylation improved xylose monomer yields by almost 20% during pretreatment, resulting in approximately 12%14% higher feedstock reactivities. The improvement also comes partly because the cellulose in deacetylated corn stover is more digestible in enzymatic hydrolysis. As shown in Figure 4, about 80% of the glucan and xylan in deacetylated samples S4 and S5 can be converted into fermentable sugars. Whole Slurry Enzymatic Hydrolysis. While the low solids enzymatic digestions display promising results, a process requiring the exhaustive washing of pretreated slurry is not likely to be commercially viable. To demonstrate the effectiveness of alkali extraction in a commercially relevant manner, whole slurry enzymatic saccharification was also preformed. When the dilute acid pretreated slurries with initial pH ∼2 were neutralized prior to enzyme addition, the deacetylated samples required significantly less NH4OH. As can be seen in Table 2, addition of ∼80 μL of 28% NH4OH was required to bring 1 g of S3 solids to a pH of 5.0, while the addition of ∼53 μL per gram solids brought
S4 and S5 to pH 6.0 and pH 8.4, respectively. This indicates that alkali extraction prior to dilute acid impregnation and pretreatment reduces ammonium hydroxide requirements for neutralization by 33% over dilute acid-only pretreatment samples. This could possibly reduce the costs of whole slurry enzymatic hydrolysis. Figure 5 shows the glucose yields for S3S5 after whole slurry digestion. After the 112 h time course, the control slurry (S3) reached a glucose yield of 47%, while the deacetylated slurries (S4 and S5) obtained conversions of 63% and 68%, respectively. The glucose yield is calculated based on the equations developed by Zhu.22 As expected, overall conversion yields were lower than yields from low-solids hydrolysis. The lower glucose yield is caused by several reasons. During high solids whole slurry enzymatic hydrolysis, the high xylose concentration in the background strongly inhibits the hydrolysis reaction by xylanase, and little xylan in the solids is further converted. Compared to the accessibility of cellulose in washed solids with high xylan conversion, the cellulose accessibility in the high solids whole slurry is reduced. Second, the xylo-oligomers in the liquor are strong inhibitors of cellulose hydrolysis by enzymes.23 However, alkaliextracted and dilute acid pretreated samples enhanced glucose yield improvements by more than 30% over the dilute acid-only control in whole slurry hydrolysis.
’ CONCLUSIONS Alkali extraction at 60 and 80 °C removed more than 70% of acetyl groups from native xylan and improved the acetyl-toxylose ratio from 2:5 in native corn stover to 1:2 and 1:5 at 60 and 80 °C, respectively. High solids dilute acid steam explosion pretreatment of corn stover feedstock deacetylated at either 60 or 80 °C showed ∼52% improvement in monomeric xylose 75
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yield over the dilute acid only control. Yield improvements were also found in low solids enzymatic saccharification, where deacetylated samples increased xylose and glucose hydrolysis yields by ∼15% and ∼30%, respectively, over the control. Feedstock reactivity indicates that deacetylation significantly improved the amount of fermentable sugars produced from pretreatment and enzymatic hydrolysis. Deacetylated samples also showed a 30% improvement in cellulose digestibility over the control in a process-relevant whole slurry scenario. Additionally, these samples required more than 33% less ammonium hydroxide for neutralization prior to enzyme addition. However, deacetylation also adds process costs through increases in chemical (NaOH) and water consumption and because of the required solidliquid separation after alkaline extraction. Therefore, to understand the impact of deacetylation on the total process cost, future work will be performed to incorporate the process into a techno-economic model.
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’ AUTHOR INFORMATION Corresponding Author
*Tel: 303-384-5676. Fax: 303-384-6877. E-mail: Xiaowen.Chen@ nrel.gov.
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dx.doi.org/10.1021/ie201493g |Ind. Eng. Chem. Res. 2012, 51, 70–76
