Pretreatment of Corn Stover for Sugar Production with Combined

ARTICLE pubs.acs.org/EF

Pretreatment of Corn Stover for Sugar Production with Combined Alkaline Reagents Ximing Zhang, Jiele Xu, and Jay J. Cheng* Department of Biological and Agricultural Engineering, Campus Box 7625, North Carolina State University, Raleigh, North Carolina 27695-7625, United States ABSTRACT: Corn stover pretreatment using a combination of sodium hydroxide (NaOH) and calcium oxide (CaO) at room temperature was investigated for improved cost-effectiveness of biomass-to-sugar conversion in this study. The effects of NaOH loading, CaO loading, and residence time on enzymatic hydrolysis were studied, and the total reducing sugar yield in the enzymatic hydrolysis was used to evaluate the pretreatment conditions. Compared with NaOH pretreatment, pretreatment with the combination of NaOH and CaO resulted in a similar sugar production rate but at a potentially lower cost. The addition of CaO not only increased the alkalinity, which favored biomass digestibility improvement, but also contributed to better biomass preservation in the pretreatment. On the basis of the sugar production rate and cost-benefit considerations, the two recommended pretreatment conditions were 3 h, 0.05 g NaOH g 1 raw biomass, 0.1 g CaO g 1 raw biomass and 6 h, 0.05 g NaOH g 1 raw biomass, 0.05 g CaO g 1 raw biomass, at which the total reducing sugar yields were 529.0 and 538.9 mg g 1 raw biomass, respectively, with overall carbohydrate conversions of 75.6 and 77.0%, respectively.

1. INTRODUCTION Fuel ethanol is considered one of the best alternative/supplementary fuels to gasoline to reduce United States dependence on foreign oil. It not only can be produced on a renewable basis from a variety of biomass feedstocks but also contributes to the reduction of automobile tailpipe emissions by promoting more complete fuel combustion. Corn grain is currently the predominant feedstock for the production of fuel ethanol in the U.S. In 2010, the U.S. ethanol industry produced 49.2 billion liters of fuel ethanol from 118.1 million tons of corn grain, with an increase of 23% over the previous year.1 However, as a result of the limitations in the agricultural land and resources needed for food and feed production, corn conversion for fuel ethanol production negatively affects the food/feed provision.2 The growing concerns over the sustainability of corn ethanol have stimulated explorations for nonedible biomass feedstocks. Corn stover refers to corn stalks, leaves, and cobs that remain in corn fields after the grain harvest, and it is considered a potential biomass feedstock for ethanol production because of its great amount and easy accessibility. Corn stover has a carbohydrate content of about 60% of the dry matter.3 It is expected that, if universal notill production of corn is adopted in the U.S., over 100 million tons of corn stover could be collected annually without causing erosion to exceed the tolerable soil loss.4 As with all other lignocellulosic feedstocks, corn stover is more challenging for conversion to ethanol than corn grain because of the complex structure of the lignocellulosic materials. Lignocellulose consists of three major components: cellulose, hemicellulose, and lignin. They form a recalcitrant structure that resists the access of hydrolytic enzymes to carbohydrates, mainly cellulose and hemicellulose, for fermentable sugar production. An effective pretreatment step, therefore, is required to reduce this recalcitrance by altering the chemical and structural features of the biomass. Sodium hydroxide (NaOH) pretreatment at high r 2011 American Chemical Society

temperatures (>100 °C) has been extensively studied to facilitate the enzymatic hydrolysis of lignocellulosic biomass, and it has been proven effective on a variety of feedstocks.5 8 At alkaline conditions, the lignin, which stiffens and surrounds the fibers of polysaccharides, is effectively removed; the hemicellulose is partially degraded; and the recalcitrance of lignocellulose is considerably reduced through solvation and saponification reactions.5,9 Research also shows that one of the biggest advantages of NaOH pretreatment over other pretreatment technologies, including acid pretreatment, steam explosion, ammonia fiber explosion (AFEX), and organosolv pretreatment, is that NaOH pretreatment can be effectively conducted even at mild temperatures.5,6 However, at mild temperatures, substantially higher chemical loadings and extended residence times are required for effective pretreatments,4 which would inevitably compromise the cost-effectiveness of the process and affect the application perspective of this technology. Our previous study showed that adding cheap lime (Ca(OH)2) in the NaOH pretreatment of switchgrass helped to reduce the requirement for the more costly NaOH without negatively affecting the pretreatment efficiency.10 Although Ca(OH)2 dissolves slightly in water and was not very effective in treating lignocellulose at room temperature,11 it not only replaces a significant portion of NaOH alkalinity at a lower cost but also helps to maintain a high pH throughout the pretreatment by gradually dissolving into the liquid phase to make up for the alkalinity consumed during the pretreatment. In addition, a high solid loss that normally occurs during NaOH pretreatment can be prevented as a result of the presence of calcium ions.10 These positive ions (each carries two positive charges) provide Received: July 29, 2011 Revised: August 29, 2011 Published: August 30, 2011 4796

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Table 1. Experimental Conditions for the Pretreatment of Corn Stover with Combined NaOH and CaO temp (°C) 21

water loading (mL g 10

1

raw biomass)

NaOH loading (g g

1

raw biomass)

CaO loading (g g

0.025, 0.05, 0.1

protection to biomass against solubilization under NaOH attack by forming linkages between negatively charged biomass components.11 On the basis of the previous work on switchgrass, the pretreatment with combined alkaline reagents was applied on corn stover in this study to investigate its effectiveness in improving biomass conversion. Because calcium oxide (CaO) has even more desirable properties, it was used in place of Ca(OH)2 in this study. CaO, commonly referred to as quicklime, is not only much cheaper in the chemical market12 but also able to provide more alkalinity than Ca(OH)2 per unit weight. Moreover, the heat energy CaO produces when forming the hydrate Ca(OH)2 in water is favorable for reaction acceleration during the pretreatment. The effects of NaOH loading, CaO loading, and residence time applied in the pretreatment on the sugar production in the subsequent enzymatic hydrolysis and the compositional change of biomass during the pretreatment were investigated in this study. The results were compared with those of NaOH pretreatment to evaluate the comparative advantages of this technology in the pretreatment of corn stover.

residence time (h)

0, 0.05, 0.1

3, 6, 9

percent solid recovery (%) CaO loading (g g

1

raw biomass)

NaOH residence

loading

time (h)

(%, w/v)

3

6

9

2. EXPERIMENTAL SECTION October, 2009, from a farm belonging to Novozymes North America, Inc. in Franklinton, North Carolina. The corn variety is AgVenture Variety R9534VBW. The stover was air-dried and ground using a Thomas Wiley laboratory mill (model no. 4) fitted with a 1 mm screen. The prepared biomass was collected in plastic bags, sealed, and stored at room temperature. The chemical composition of the raw biomass was as follows: glucan 36.2%, xylan 20.1%, galactan 1.45%, arabinan 3.00%, lignin 21.2%, ash 1.56%, and others 16.5%. 2.2. Pretreatment. Corn stover was pretreated at 21 °C (room temperature) in serum bottles. Table 1 shows the pretreatment conditions explored. Three grams of dry biomass, 30 mL of deionized (DI) water, and desired amounts of NaOH and CaO were mixed in a 150 mL serum bottle. All serum bottles were sealed and crimped before pretreatment to prevent water evaporation. After pretreatment, the pretreated biomass was recovered by filtration and 300 mL of DI water was used to remove excess alkali and dissolved byproducts that might be inhibitory to the subsequent enzymatic hydrolysis. Preliminary experiments showed that the wash intensity of 100 mL DI water g 1 raw biomass was sufficient for biomass washing, while further increasing the wash intensity up to 200 mL DI water g 1 raw biomass resulted in reductions in sugar yield, as a result of the increased carbohydrate loss during biomass washing. About 1 g of pretreated biomass (on a dry basis) was dried at 70 °C to a constant weight for composition analysis, and the rest was stored in a sealed plastic bag at 4 °C for enzymatic hydrolysis. To prevent the impact of microbial growth, the pretreated biomass was subjected to enzymatic hydrolysis within 24 h after pretreatment. 2.3. Enzymatic Hydrolysis. Enzymatic hydrolysis was carried out in 50 mL plastic tubes in a controlled reciprocal shaking water bath (model C76, New Brunswick Scientific, Edison, NJ) at 50 °C and 150 rpm for 72 h. A total of 0.5 g pretreated biomass (on a dry basis) was mixed with the desired volume of 0.05 M sodium citrate buffer to reach a total volume of 15 mL and a pH of about 4.8. Cellic CTec (aggressive cellulases and a high level of β-glucosidase) at an enzyme loading of 40 FPU (filter paper unit) g 1 dry biomass and Cellic HTec (endoxy-

raw biomass)

Table 2. Solid Recoveries after Pretreatments with the Combination of NaOH and CaO at Room Temperature

a

2.1. Biomass Preparation. The corn stover was collected in early

1

0 a

0.05

0.1

0.25

82.3 (1.65)

83.6 (0.52)

88.7 (1.11)

0.5

79.3 (0.77)

82.5 (2.47)

85.7 (1.11)

1

73.4 (0.64)

79.5 (1.25)

85.7 (2.05)

0.25

81.7 (0.51)

82.0 (1.30)

85.7 (1.40)

0.5

80.1 (1.47)

83.5 (1.27)

88.5 (0.47)

1

73.1 (1.57)

76.4 (1.41)

85.2 (2.58)

0.25 0.5

81.0 (0.92) 79.0 (1.54)

81.5 (0.42) 79.8 (0.02)

84.8 (1.80) 85.4 (1.88)

1

71.2 (1.37)

76.9 (1.95)

83.3 (0.63)

The number in parentheses is standard deviation of triplicate samples.

lanase) at an enzyme loading of 75 FXU (fungal xylanase unit) g 1 dry biomass were added to the slurry, making a final solid loading of 3.3% for hydrolysis. Both enzymes were obtained from Novozymes North America, Inc. (Franklinton, NC). The activities of Cellic CTec and Cellic HTec were respectively 115.6 FPU mL 1 (with a βglucosidase activity of 281.8 CBU mL 1) and 1090 FXU mL 1. After the addition of enzymes, sodium azide (0.3%, w/v) was added to inhibit microbial growth during hydrolysis. After the hydrolysis time elapsed, the hydrolyzate was centrifuged at 8  103g, and then, the supernatant was collected and stored at 20 °C for sugar analysis at a later time. 2.4. Analytical Methods. The total solids, ash, structural carbohydrates, and lignin of raw and pretreated biomass were determined using Laboratory Analytical Procedures (LAP) established by National Renewable Energy Laboratory (NREL).13 15 The total reducing sugars in the hydrolyzate were measured by the DNS (3,5-dinitrosalicylic acid) method, using glucose as the standard.16,17 The major monomeric sugars present in the hydrolyzate, which include glucose and xylose, were determined using high performance liquid chromatography (HPLC) (Dionex UltiMate 3000, Dionex Corporation, Sunnyvale, CA). The HPLC system was equipped with a Bio-Rad Aminex HPX-87H column (300 mm  7.8 mm), a Bio-Rad Micro-Guard column, a thermostatted autosampler, an isocratic analytical pump, and a refractive index detector. The analytical column was operated at 65 °C with 0.005 M H2SO4 as the mobile phase at a flow rate of 0.6 mL min 1. The sugar profiles of raw and pretreated biomass were determined by measuring the monomeric sugars derived from the composition analysis using ion chromatography (IC) (Dionex ICS-5000, Dionex Corporation, Sunnyvale, CA). The IC instrument was equipped with a pulsed electrochemical detector. The column used was a CarboPac PA1 (4 mm  250 mm) column operated at 18 °C with 0.018 M potassium hydroxide as the mobile phase at a flow rate of 0.9 mL min 1. All treatments in this study were conducted in triplicate. The GLM (general linear model) procedure with Tukey adjustment in SAS 9.1 software (SAS Institute Inc., Cary, NC) was used for all data analysis. 4797

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Figure 1. Yields of (a) total reducing sugars, (b) glucose, and (c) xylose in the enzymatic hydrolysis of corn stover pretreated using the combination of NaOH and CaO at room temperature (the tick label of the horizontal axis: A + B represents A g NaOH g 1 raw biomass + B g CaO g 1 raw biomass).

3. RESULTS AND DISCUSSION 3.1. Enzymatic Hydrolysis Improvement. When CaO was reacted with water to form Ca(OH)2 in the pretreatment slurry, the heat energy generated was not sufficient to considerably increase the pretreatment temperature. At the highest CaO loading of 0.1 g g 1 raw biomass, the slurry temperature increased by less than 1 °C and dropped back to room temperature within just a few minutes. It is, therefore, expected that the heating effect of CaO-to-Ca(OH)2 conversion can be neglected during corn stover pretreatment. Solid recovery is an important parameter to evaluate pretreatment performance, as it determines the total amount of biomass that can be potentially converted to sugars in enzymatic hydrolysis. Although the solubilization of biomass components

including valuable carbohydrates is inevitable under alkaline attack, using the combination of NaOH and CaO could substantially reduce its extent. Table 2 shows that the solid recovery significantly (P < 0.05) decreased with the increase of NaOH loading, while the change of residence time was not significantly (P > 0.05) associated with solid recovery. When CaO was used with NaOH, the solid recoveries were much higher. At room temperature, the solid recovery with the CaO addition ranged 76.4 88.7%, which was considerably higher than that without CaO addition (71.2 82.3%). In addition, with the increase of CaO loading, the solid recovery increased accordingly. This indicates that adding CaO, although it causes an increase of alkalinity in the system, provided some extent of biomass preservation. This is consistent with the observation in our previous study on the lime (Ca(OH)2) pretreatment of switchgrass.11 4798

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Figure 2. Total reducing sugar yields in the enzymatic hydrolysis of corn stover pretreated at recommended residence times and NaOH loadings with different CaO loadings.

Figure 3. Changes of pH during enzymatic hydrolysis of the corn stover pretreated using 0.1 g NaOH g 1 raw biomass for 9 h at different CaO loadings.

The formation of calcium linkages within the biomass structure provided a plausible explanation for this phenomenon, which can also explain the increased calcium content in the lime pretreated poplar wood in a previous study by Chang et al.18 More research, however, is required to obtain a mechanistic understanding. After pretreatment, the recovered biomass was subjected to enzymatic hydrolysis for sugar production. The total reducing sugar yield was used to evaluate the overall pretreatment performance. Because glucose and xylose are the dominant monomeric sugars in the hydrolyzate and can well represent the conversions of cellulose and hemicellulose, respectively, their yields were also determined to understand the impacts of pretreatment conditions on different carbohydrate components. Figure 1a shows that the total reducing sugar yield generally increased with the increase of NaOH loading. However, with CaO addition, the application of the highest NaOH loading of 0.1 g g 1 raw biomass did not necessarily result in the maximum sugar productions. This is because more solubilization of the

hydrolyzed carbohydrates occurred during the alkaline pretreatment with the higher NaOH concentration. Adding CaO successfully increased sugar production, which demonstrates the effectiveness of supplementing CaO in NaOH pretreatment. However, applying the highest CaO loading of 0.1 g g 1 raw biomass did not necessarily favor sugar production, and the sugar production even decreased with the increase of CaO loading from 0.05 to 0.1 g g 1 raw biomass at the NaOH loading of 0.1 g g 1 raw biomass. A plausible explanation is that, in an already intense pretreatment environment created by a high NaOH load of 0.1 g g 1 raw biomass, an excessive alkalinity addition would cause more carbohydrate solubilization in spite of the improved biomass preservation by more available calcium ions. Similarly, although extending residence time should favor the digestibility improvement of biomass, it was not always positively associated with the total reducing sugar yield as a result of the increased biomass solubilization at longer residence times. On the basis of the total reducing sugar yield and the potential pretreatment cost, the conditions 3 h, 0.05 g NaOH g 1 raw biomass, 0.1 g CaO g 1 raw biomass and 6 h, 0.05 g NaOH g 1 raw biomass, 0.05 g CaO g 1 raw biomass were selected as the recommended conditions for corn stover pretreatment at room temperature. At these conditions, the total reducing sugar yields in enzymatic hydrolysis were respectively 529.0 and 538.9 mg g 1 raw biomass, with overall carbohydrate conversions of 75.6 and 77.0%, respectively. Further economic analysis weighing the impacts of residence time and CaO loading on the cost of ethanol production is required before the best pretreatment conditions for practical application can be determined. The effects of pretreatment conditions on both glucose and xylose yields were similar to those on total reducing sugar yield (Figure 1b and c). The glucose and xylose yields of the biomass pretreated at the first recommended conditions of 3 h, 0.05 g NaOH g 1 raw biomass, 0.1 g CaO g 1 raw biomass were 320.1 and 137.4 mg g 1 raw biomass, respectively, with overall glucan and xylan conversions of 79.5 and 60.2%, respectively. The glucose and xylose yields of the biomass pretreated at the second recommended pretreatment conditions of 6 h, 0.05 g NaOH g 1 raw biomass, 0.05 g CaO g 1 raw biomass were 331.6 and 4799

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Figure 4. Lignin reductions of corn stover pretreated using the combination of NaOH and CaO at room temperature (the tick label of the horizontal axis: A + B represents A g NaOH g 1 raw biomass + B g CaO g 1 raw biomass).

Table 3. Material Balances for Raw Corn Stover and the Corn Stover Pretreated under the Recommended Conditions content (%) pretreated biomass 3 h, 0.05 g NaOH g

6 h, 0.05 g

1

NaOH g

raw biomass, raw component total solids

biomass 100

0.1 g CaO g

1

1

raw biomass, 0.05 g CaO g

raw biomass

raw biomass

85.7 (1.11)

83.5 (1.27)

glucan

36.2 (0.91)

34.0 (3.04)

35.4 (0.76)

xylan

20.1 (0.58)

17.3 (2.05)

17.7 (0.43)

galactan

1.45 (0.01)

1.23 (0.12)

arabinan

3.00 (0.08)

2.60 (0.12)

lignin ash others

21.2 (0.40) 1.56 (0.14) 16.5 (1.93)

15.1 (0.13) 0.85 (0.19) 14.6 (3.01)

1

1.35 (0.07) 2.99 (0.09) 15.8 (1.22) 0.87 (0.21) 9.39 (1.40)

143.7 mg g 1 raw biomass, respectively, with overall glucan and xylan conversions of 82.4 and 62.9%, respectively. The overall conversions of xylan were lower than those of glucan at all pretreatment conditions. This is because of the greater loss of xylan during pretreatment, as a result of hemicellulose solubilization. Hemicellose is an amorphous, heterogeneous, and branched compound with much lower molecular weight than cellulose, which makes it more susceptible to degradation and solubilization than semicrystalline cellulose under alkaline attack. As a result of the limited solubility of Ca(OH)2 in water, part of the Ca(OH)2 would remain as solids throughout the pretreatment period and be washed and wasted after biomass recovery. To reduce chemical waste, more accurate investigations are required to determine the best CaO loading. On the basis of the recommended pretreatment conditions (3 h, 0.05 g NaOH g 1 raw biomass, 0.1 g CaO g 1 raw biomass and 6 h, 0.05 g NaOH g 1 raw biomass, 0.05 g CaO g 1 raw biomass), CaO loadings of 0.075 0.125 and 0.025 0.075 g g 1 raw biomass were studied, respectively (Figure 2). The results show that the best CaO loadings were 0.1 and 0.05 g g 1 raw biomass, respectively, for

pretreatments at 3 h and 0.05 g NaOH g 1 raw biomass and 6 h and 0.05 g NaOH g 1 raw biomass. Pretreatments involving Ca(OH)2/CaO usually raise concerns over their effects on the hydrolysis pH. It was noticed that, even after biomass washing, the residual Ca(OH)2 particles could still increase the pH of the subsequent enzymatic hydrolysis beyond the optimal range, thus reducing the sugar production.10 To investigate the effect of CaO-induced pH rise on the enzymatic hydrolysis of pretreated corn stover, the changes of hydrolysis pH were monitored. Figure 3 shows the pH change during the enzymatic hydrolysis of the corn stover pretreated using 0.1 g NaOH g 1 raw biomass for 9 h at different CaO loadings. Throughout the 72 h hydrolysis period, the values of pH at different CaO loadings were quite stable, except that a slight drop of pH was observed after 24 h in the samples without CaO addition, which is probably due to the release of organic acids from the biomass during hydrolysis. The results also show that there is a positive correlation between the CaO loading applied and the average pH. Although sodium citrate buffer (pH 4.8) was used for pH stabilization, the residual Ca(OH)2 could still significantly (P < 0.05) increase the hydrolysis pH. Nonetheless, the pH change was not expected to substantially affect sugar production as the Novozymes product data sheet indicated that, for practical application the optimal pH is around 5.0 for Cellic CTec and 4.5 6.0 for Cellic HTec. Although it is theoretically possible to remove all the residual Ca(OH)2 particles through intensive biomass washing, increasing wash intensity was not recommended because it would inevitably result in greater solid loss, which would compromise sugar production in hydrolysis. 3.2. Lignin Reduction. Lignin is a complex aromatic polymer present in plant cell walls, which confers structural support, impermeability, and resistance against microbial attachment and oxidative stress.19 Because lignin is a major physical barrier limiting enzyme access to cellulose and hemicellulose, its removal/degradation is of great importance to the improvement of enzymatic hydrolysis. Figure 4 shows that higher NaOH loadings generally resulted in greater lignin reductions. This is in agreement with the previous reports that NaOH is a strong base and can effectively remove lignin from lignocellulosic biomass.5 8 CaO addition caused reduced lignin removal, especially at higher NaOH loadings when lignin was under more intense alkaline attack. The observed lignin preservation caused by CaO addition 4800

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Table 4. Comparison between Pretreatments using NaOH + CaO and NaOH Alone pretreatment 3 h, 0.05 g NaOH g

1

raw biomass, 0.1 g CaO g

6 h, 0.05 g NaOH g

1

raw biomass, 0.05 g CaO g

9 h, 0.1 g NaOH g

1

raw biomass

1

solid

glucan

xylan

lignin

recovery (%)

conversion (%)

conversion (%)

reduction (%)

potential cost

raw biomass

85.7 (1.11)

79.5 (1.44)

60.2 (3.66)

27.8 (1.70)

low

1

83.5 (1.27)

82.4 (3.92)

62.9 (0.11)

31.4 (2.43)

low

71.2 (1.37)

80.2 (4.27)

62.4 (0.83)

48.0 (1.04)

high

raw biomass

is consistent with the reports that divalent calcium ions from Ca(OH)2 dissociation have a high affinity for lignin and can effectively cross-link lignin molecules.20 22 Residence time did not have a significant (P > 0.05) effect on lignin reduction. A plausible explanation is that lignin removal from corn stover occurred during the initial stage of pretreatment when the high initial pH restrained the dissociation of Ca(OH)2 to produce Ca2+ and, thus, the formation of calcium linkages among lignin molecules for their preservation. 3.3. Material Balances. Material balances were performed on raw biomass, and the biomass was pretreated using the recommended pretreatment conditions (3 h, 0.05 g NaOH g 1 raw biomass, 0.1 g CaO g 1 raw biomass and 6 h, 0.05 g NaOH g 1 raw biomass, 0.05 g CaO g 1 raw biomass). After pretreatment at the recommended conditions, the total dry weight of biomass was determined, and the biomass components, including glucan, xylan, galactan, arabinan, lignin, ash, and others, were determined and compared with those of the raw biomass. Table 3 shows that the pretreatment using the combination of NaOH and CaO resulted in satisfying carbohydrate preservation. After pretreatment at 3 h, 0.05 g NaOH g 1 raw biomass, 0.1 g CaO g 1 raw biomass and 6 h, 0.05 g NaOH g 1 raw biomass, 0.05 g CaO g 1 raw biomass, the glucan reductions were respectively 6.07 and 2.21%, while the xylan reductions were respectively 13.7 and 11.8%, which were higher than those of glucan as a result of the more susceptible structure of hemicellulose. Changes of galactan and arabinan were similar to that of xylan. It can be concluded that, although the total amount of carbohydrates in the biomass was not substantially changed after pretreatment, the biomass structure had been remarkably altered, which led to a great improvement of enzymatic hydrolysis. The impact of pretreatment on noncarbohydrate components was more significant. Lignin, ash, and other undefined components including protein, waxes, fats, resins, gums, and chlorophyll23,24 were all considerably removed. 3.4. Comparison with NaOH Pretreamtent. Because the total reducing sugar yield of the pretreatment using 0.1 g NaOH g 1 raw biomass for 9 h was comparable with those at recommended conditions, a comparison was conducted between the pretreatment using the combination of NaOH and CaO and that using NaOH alone. Table 4 shows that the pretreatment using 0.1 g NaOH g 1 raw biomass resulted in a much greater lignin reduction. A high overall carbohydrate conversion could also be achieved at the expense of a more intensive biomass solubilization. However, the potential cost of NaOH pretreatment could be higher than those of using the combination of NaOH and CaO. Assuming that the market price of NaOH is 4.5 times as much as that of CaO,12,25 to produce a similar amount of sugar for ethanol fermentation, the chemical cost of the pretreatment using 0.1 g NaOH g 1 raw biomass for 9 h is 38.5 and 63.6% higher than those of the pretreatments at 3 h, 0.05 g NaOH g 1 raw biomass, 0.1 g CaO g 1 raw biomass and 6 h, 0.05 g NaOH g 1 raw biomass, 0.05 g CaO g 1 raw biomass, respectively. Moreover,

the extended residence time required in NaOH pretreatment could result in higher capital investment and operational cost in practical applications. The preliminary comparison indicates that corn stover pretreatment using the combination of NaOH and CaO is potentially a more promising technology than NaOH pretreatment at room temperature. Nonetheless, a more comprehensive economic analysis is required before the final conclusion can be reached.

4. CONCLUSIONS Compared with NaOH pretreatment at room temperature, the pretreatment of corn stover using the combination of NaOH and CaO was proven effective in reducing the pretreatment cost without compromising the sugar yield in enzymatic hydrolysis. After pretreatment at recommended conditions of 3 h, 0.05 g NaOH g 1 raw biomass, 0.1 g CaO g 1 raw biomass and 6 h, 0.05 g NaOH g 1 raw biomass, 0.05 g CaO g 1 raw biomass, the overall conversions of glucan in raw biomass were respectively 79.5 and 82.4%, and those of xylan reached 60.2 and 62.9%, respectively. The application of CaO resulted in substantially reduced lignin reductions. Nonetheless, the sugar yield in hydrolysis was still high despite the presence of a large amount of lignin in the biomass. It can be concluded that, instead of total lignin reduction, the structural alternations of lignin and the breakup of lignin carbohydrate complex at alkaline conditions were responsible for the improved enzyme access to carbohydrates. However, the report shows that lignin limits enzyme access to carbohydrates not only through posing physical barrier but also by causing the unproductive binding of enzymes.26 It is necessary, therefore, to further investigate the impact of this pretreatment technology on enzyme loading in the future work. ’ AUTHOR INFORMATION Corresponding Author

*Telephone: +1-919-515-6733. Fax: +1-919-515-7760. E-mail: [email protected].

’ ACKNOWLEDGMENT The authors would like to thank Novozymes North America, Inc. for providing a corn stover and enzymes for this research. ’ REFERENCES (1) Renewable Fuels Association. Building Bridges to a More Sustainable Future: 2011 Ethanol Industry Outlook; Renewable Fuels Association: Washington, DC; St. Louis, MO; Omaha, NE, 2011. Available online: http://www.ethanolrfa.org/page/-/2011%20RFA%20Ethanol %20Industry%20Outlook.pdf?nocdn=1. (2) Sun, Y.; Cheng, J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 2002, 83, 1–11. (3) National Renewable Energy Laboratory. Biomass Feedstock Composition and Property Database; U.S. Department on Energy: 4801

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dx.doi.org/10.1021/ef201130d |Energy Fuels 2011, 25, 4796–4802