Enhancing Enzymatic Hydrolysis of Maize Stover by Bayer Process

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Energy & Fuels 2009, 23, 2284–2289

Enhancing Enzymatic Hydrolysis of Maize Stover by Bayer Process Sand Pretreatment Michael Rodgers, Zhenhu Hu,* and Xinmin Zhan Department of CiVil Engineering, National UniVersity of Ireland, Galway, Ireland ReceiVed NoVember 28, 2008. ReVised Manuscript ReceiVed February 2, 2009

In a laboratory study, enzymatic hydrolysis of maize stover pretreated with Bayer process sand (BPS) which is a waste with high alkalinity generated in aluminum production was compared with that of maize stover pretreated with sodium hydroxide. The effects of BPS loading, enzyme loading, pretreatment temperature, and pretreatment duration were investigated. After pretreatment at a BPS loading of 0.093 g NaOH equiv/g maize stover, a temperature of 35 °C for 24 h, and at a cellulase enzyme loading of 15 FPU/g glucan, 95.6% of the total amount of glucan and xylan contained in the pretreated maize stover was enzymatically hydrolyzed. In combination of the pretreatment and enzymatic hydrolysis stages, 93.2% of the glucan and 94.5% of the xylan were converted into glucose and xylose, respectively, similar to those obtained by means of sodium hydroxide pretreatment. Since the BPS solution contained 40-50 mg/L aluminum ion (Al3+), the effect of Al3+ concentration on enzymatic hydrolysis of the maize stover pretreated with NaOH was examined. When Al3+ concentration was lower than 90 mg/L, it did not affect enzymatic hydrolysis. These results suggest that BPS should replace sodium hydroxide for the pretreatment of maize stover.

1. Introduction The use of fossil fuels has caused serious problems, such as greenhouse gas (GHG) emissions, energy security, and environmental pollution.1 Petroleum is the largest single energy source used in the world, and two-thirds of the world’s petroleum is consumed by the transportation sector, which is the largest emitter of greenhouse gases.2 The recent soaring price of petroleum has become a big challenge to economic and energy security. Biofuels have the potential to reduce the dependence of the transportation sector on petroleum1 and are beneficial in maintaining fuel price stability and energy security.3 Bioethanol has become one of the most used biofuels, and its use has rapidly increased by 2.5 times from 2001 to 2007.4 However, the current feedstock used for bioethanol production is mainly grains (like corn) and sugar cane, which competes for resources with food and animal feed production. Therefore, exploiting nonfood sources for bioethanol production is very necessary for the sustainable development of biofuels. Lignocellulosic biomass, such as agriculture and forest wastes, is among the most abundant renewable resources of energy in the world.5 Cellulose and hemicellulose in the lignocellulosic biomass can be used for ethanol production.3 The potential bioethanol yield in the USA from lignocellulosic biomass is 2.27 × 1011 L/y, which could replace 30% of the current transportation fuel consumption.6 The study of Schmer et al. * To whom correspondence should be addressed. Phone: +353 91 493085. Fax: +353 91 494057. E-mail: [email protected]. (1) Farrell, A. E.; Plevin, R. J.; Turner, B. T.; Jones, A. D.; O’Hare, M.; Kammen, D. M. Science 2006, 311, 506–508. (2) Wyman, C. E.; Dale, B. E.; Elander, R. T.; Holtzapple, M.; Ladisch, M. R.; Lee, Y. Y. Bioresour. Technol. 2005, 96, 1959–1966. (3) Otero, J. M.; Panagiotou, G.; Olsson, L. In Biofuels; Springer-Verlag Berlin: Berlin, 2007; Vol. 108, pp 1-40. (4) Mabee, W. E. In Biofuels; Springer-Verlag Berlin: Berlin, 2007; Vol. 108, pp 329-357. (5) Schubert, C. Nat. Biotechnol. 2006, 24, 777–784. (6) Service, R. F. Science 2007, 315 (5818), 1488.

shows that switchgrass-based ethanol has the potential to reduce GHGs by 94% when compared with petroleum, whereas cornbased ethanol can only reduce GHGs by 30%.7 Therefore, the production of ethanol from lignocellulosic biomass can not only avoid competition with food and animal feed resources but also reduce GHG emissions.1 Bioethanol production from lignocellulosic biomass generally comprises the following four main steps: (i) pretreatment of lignocellulosic biomass, (ii) enzymatic hydrolysis of cellulose and hemicellulose, (iii) fermentation of hydrolyzed sugars, and (iv) production of ethanol from the fermentation broth.3 The key challenge in the commercial ethanol production from lignocellulosic biomass is to reduce operational costs so that cellulosic ethanol would be competitive in price with petroleum.2 Because of the recalcitrance of the lignocellulosic structure, it is important to develop technologies to reduce this recalcitrance prior to enzymatic hydrolysis. There are two approaches to reduce cellulosic recalcitrance: (i) pretreatment of lignocellulosic biomass so as to break down the lignocellulosic structure8 and (ii) selection or modification of plants with lower lignin contents.9,10 Physical, biological, and chemical pretreatment methods have been used to improve the hydrolysis and fermentation of lignocellulosic biomass. Physical pretreatment like ball milling and high-energy radiation is too expensive.8 Biological pretreatment by using lignin-degrading microorganisms is environmentally friendly and energy-saving but requires a long process duration; in addition, the cellulose and hemicellulose are partly consumed during the process.11 Chemical alkali pretreatment at ambient temperatures is simple and time(7) Schmer, M. R.; Vogel, K. P.; Mitchell, R. B.; Perrin, R. K. Proc. Natl. Acad. Sci. USA 2008, 105, 464–469. (8) Galbe, M.; Zacchi, G. In Biofuels; Springer-Verlag Berlin: Berlin, 2007; Vol. 108, pp 41-65. (9) Chen, F.; Dixon, R. A. Nat. Biotechnol. 2007, 25, 759–761. (10) Chen, F.; Dixon, R. A. In Vitro Cell. DeV-An. 2008, 44, S28-S29. (11) Shi, J.; Chinn, M. S.; Sharma-Shivappa, R. R. Bioresour. Technol. 2008, 99, 6556–6564.

10.1021/ef801032x CCC: $40.75  2009 American Chemical Society Published on Web 03/17/2009

Hydrolysis of Maize StoVer

saving and appears to have strong commercial potential.12 Lowlignin cellulosic plants could also be selected or genetically modified for use.9 Sodium hydroxide is a typical alkali used in alkaline pretreatment,13,14 and other alkaline chemicals have also been used for pretreatment.12 The Bayer process sand (BPS), containing a high content of alkalinity, is a waste generated in aluminum production. If the BPS can be used to efficiently break down the lignocellulosic structure, it will significantly reduce the pretreatment cost of the cellulosic ethanol production process and increase the added-value of the BPS as well. However, BPS contains high concentrations of aluminum oxide, the efficiency of BPS in pretreatment and its effects on successive enzymatic hydrolysis and fermentation need to be examined. The aims of this laboratory study were: (i) to examine the efficacy of using BPS to pretreat mature maize stover for enzymatic hydrolysis and (ii) to quantify the solubilization of maize stover in the pretreatment stage and the conversion of polysaccharides in enzymatic hydrolysis of the pretreated maize stover. This was achieved by carrying out pretreatment and enzymatic hydrolysis experiments at different conditions of BPS concentration, temperature, and experimental duration. 2. Materials and Methods 2.1. Materials. Fresh maize was obtained from the Teagasc Farm, Athenry, Ireland. The maize was harvested at the medium fruit stage when the maize was mature, but not completely lignified. After harvesting, the cob was removed. The remaining stover was dried at 50 °C, comminuted in a blender, ground to powder in a mill (IKA Grinder, Germany), and passed through a 1.00 mm sieve. The prepared maize stover was then stored at an ambient temperature of about 20 °C before use. 2.2. Pretreatment of the Ground Maize Stover. 2.2.1. Preparation of BPS Solution. The raw BPS was collected from a bauxite processing company and stored in a sealed container in the laboratory at an ambient temperature of about 20 °C. BPS contains 1-4% sodium oxide, 25-35% aluminum oxide, 35-50% iron oxide, 2-6% titanium oxide, 3% calcium oxide, and 2-32% quartz oxide. Before the pretreatment experiments, 1 kg of the BPS was mixed with 2 L of tap water. After settlement for 30 min, the supernatant of the BPS mixed liquor was collected and stored in a reagent bottle for use. The aluminum ion (Al3+) concentration in the supernatant ranged from 40-50 mg/L. The alkaline concentration of the BPS solution (NaOH equivalent) was determined by titration with 1 mol/L of HCl. 2.2.2. Alkaline Pretreatment of Maize StoVer. The pretreatment was carried out in 125 mL Erlenmeyer flasks. Each flask was loaded with 3.0 g of the ground maize stover (dry matter) and 18 mL of BPS solution. Different alkaline concentrations, pretreatment temperatures, and durations were used in the test experiment, to establish their effects on the hydrolysis processes. After complete mixing of maize stover and the BPS solution, the slurry was heated for a specified test duration. The pretreated slurry was then separated into liquid and solid fractions by centrifuging, and the solids were washed with four times their volume of deionized (DI) water (the volume of DI water was 4 times as much as the slurry volume). Both the liquid and solid fractions were collected for sugar analysis, and the solid fraction was used for the successive enzymatic hydrolysis. Each test was replicated three times, and the average is (12) Kim, S.; Holtzapple, M. T. Bioresour. Technol. 2005, 96, 1994– 2006. (13) Varga, E.; Szengyel, Z.; Reczey, K. Appl. Biochem. Biotechnol. 2002, 98, 73–87. (14) de Vrije, T.; de Haas, G. G.; Tan, G. B.; Keijsers, E. R. P.; Claassen, P. A. M. Int. J. Hydrogen Energy 2002, 27, 1381–1390.

Energy & Fuels, Vol. 23, 2009 2285 presented here. The effects of different Al3+ concentrations on the hydrolysis were also investigated to establish limitations on the use of BPS. In the alkaline loading tests (at 35 °C for 24 h), the BPS solution was diluted to 0.20, 0.25, 0.30, 0.35, and 0.40 mol NaOH equiv/L with DI water giving the corresponding alkaline loading of 0.053, 0.066, 0.080, 0.093, and 1.007 g NaOH equiv/g untreated maize stover. When the alkaline loading was less than 0.053 g NaOH equiv/g untreated maize stover, the pH value of the mixed slurry of maize stover and BPS solution was lower than 10 and lignin did not appear to be removed. Tests at 20, 35, and 50 °C were carried out in an incubator for 24 h, at 100 °C in an oven for 2 h, and at 121 °C in an autoclave for 30 min. The effects of pretreatment durations of 0, 5, 10, 15, 20, and 24 h were examined when the temperature was 35 °C, and the NaOH equivalent of the BPS solution was 0.093 g/g maize stover. 2.3. Enzymatic Hydrolysis of Alkaline Pretreated Maize Stover. The enzymatic hydrolysis experiment was carried out in 125 mL Erlenmeyer flasks (AGB, Ireland) with each flask containing a 100 mL mixture of buffer solution (pH 4.8) and 2% (g/mL %) maize stover obtained from the pretreatment process. The buffer solution was 50 mM acetate buffer containing 20 mg/L tetracycline and 20 mg/L cycloheximide. Celluclast 1.5 L, Novozym 188, and xylanase (Sigma, Ireland) were used in the enzymatic hydrolysis. The Celluclast 1.5 L contained 87.4 FPU/mL of cellulase, the Novozym 188 contained 480 U/mL of β-glucosidase, and the xylanase (from Thermomyces lanuginosus) concentration was 68 000 U/g. The cellulase loading was 15 FPU/g glucan except at the test of enzyme loadings in which the celllulase loading ranged from 5 to 30 FPU/g glucan. The enzyme cocktail was made by mixing equal volumes of the Celluclast 1.5 L and the Novozym 188, with the xylanase loading of 1360 U/g maize stover. The enzymatic hydrolysis was carried out at 50 °C in a shaker incubator at a speed of 150 rpm for 120 h. During the hydrolysis experiment, samples of 1.0 mL were taken out of the flasks at the predetermined intervals, diluted twice with 2% H2SO4, centrifuged at 17968 g for 20 min, and then filtered through 0.45 µm filters for sugar analysis. 2.4. Effect of Aluminum Ion Concentrations. To investigate the influence of Al3+ concentrations on enzymatic hydrolysis, maize stover was first pretreated at 35 °C for 24 h with NaOH at the alkali loading of 0.093 g NaOH/g maize stover. After the pretreatment, the slurry was separated into liquid and solid fractions as described above. Enzymatic hydrolysis of the pretreated solids was carried out at an enzyme loading of 15 FPU/g glucan with the addition of aluminum sulfate in concentrations of 0, 30, 60, 90, 120, and 150 mg Al3+/L. 2.5. Analysis. The sugar composition of the solid and liquid fractions before and after pretreatment was analyzed according to the NREL Laboratory Analytical Procedures (LAP) 001 and 002.15,16 Acid-insoluble lignin was determined using the Klason lignin procedure published as NREL LAP 003.17 The filter paper activity of cellulase and the β-glucosidase were determined using the standard IUPAC procedures.18 The glucose and xylose contents were measured using an Agilent 1200 HPLC System with a 1200 series refractive index detector (Agilent Technologies, USA). An Aminex HPX-87H column (Bio-Rad Laboratories, USA) was used for the sugar analysis with 0.1% (v/ v) H2SO4 solution as the mobile phase. The flow rate was controlled at 0.6 mL/min, the column temperature was 65 °C, and the detector temperature was 40 °C. The structure of the maize stover before (15) Ruiz, R.; Ehrman, T. Dilute acid hydrolysis procedure for determination of total sugars in the liquid fraction process samples; National Renewable Energy Laboratory (NREL): Golden, CO, 1996. (16) Ruiz, R.; Ehrman, T. Determination of carbohydrates in biomass by performance liquid chromatography; National Renewable Energy Laboratory (NREL): Golden, CO, 1996. (17) Templeton, D.; Ehrman, T. Determination of acid-Insoluble lignin in biomass; National Renewable Energy Laboratory: Golden, CO, 1995. (18) Ghose, T. K. Pure Appl. Chem. 1987, 59 (2), 257–268.

2286 Energy & Fuels, Vol. 23, 2009 and after pretreatment was observed with a Hitachi S-4700 scanning electron microscope at the accelerating voltage of 15 kV (Hitachi Ltd., Japan). The solid yield is defined as the percentage of the solid fraction recovered after pretreatment to the initial solid added. The total sugar is defined as the summation of glucose and xylose. The enzymatic hydrolysis efficiency is defined as the ratio of the sugars released after enzymatic hydrolysis to the sugars contained in the raw material. The overall conversion efficiency of glucan and xylan to glucose and xylose from the pretreatment to the enzymatic hydrolysis stages was calculated using the following equation (The conversion coefficients of glucan and xylan to glucose and xylose are 1.1 and 1.06, respectively.):19

conversion (%) ) {glucose released [g] + 1.053cellobiose released [g] + xylose released [g]} / {1.1glucan added [g] + 1.06xylan added [g]} × 100 (1) 3. Results and Discussion 3.1. Maize Stover Characteristics. The glucan, xylan, lignin, and ash contents in the untreated maize stover (on the basis of dry matter) were 38.6, 20.5, 7.8, and 5.1%, respectively. The contents of glucan and xylan in the maize stover were similar to those in completely lignified corn stover, but the lignin content was significantly lower than that in lignified corn stover, which is usually about 15-25%.13,19 The protein content of the maize stover was 7.5%, while that of lignified corn stover is generally less than 4%.19 This indicates that the maize stover residue, after enzymatic hydrolysis and fermentation, may contain a high level of protein, which can be recovered for animal feed; this will increase the added-value of maize stover. 3.2. Effect of BPS Loading. The pretreatment of maize stover with BPS was carried out at 35 °C for 24 h with BPS loadings ranging from 0 to 0.107 g NaOH equiv/g maize stover (dry matter). In the pretreatment without BPS addition, 14.8% of glucan and 12.5% of xylan were solubilized after 24 h of soaking, but there was no apparent lignin removal (Table 1) and the lignin content of the remaining solid residue increased to 9.2%. With the increase in the BPS loading, the lignin content of the pretreated biomass decreased from 7.5% at a BPS loading of 0.053 g NaOH equiv/g maize stover to 2.8% at 0.107 g NaOH equiv/g maize stover (Table 1). Because of the solubilization of the solids during the pretreatment, the glucan content in the solid residues increased by about 10% regardless of the BPS loading and the xylan contents in the residues increased slightly. In comparison, Varga et al.13 found in their study on corn stover, that when NaOH loading increased to 0.1 g NaOH/g corn stover, nearly 80% of xylan was solubilized during the pretreatment. Because Cellulase 1.5 L and Novezym 188 only contained small amounts of xylanase, xylanase was added to improve the hydrolysis of xylan. The pretreated maize stover was subjected to enzymatic hydrolysis at an enzyme loading of 15 FPU/g glucan. For the maize stover without pretreatment, 54.7% of glucan and 58.7% of xylan were hydrolyzed to monosaccharides (Figure 1) after 120 h of incubation at 50 °C, whereas corresponding values for untreated corn stover or switchgrass were only 5-15%,13,20 suggesting that the low lignin content in the raw material should result in improved enzymatic hydrolysis without pretreatment, which is consistent with the saccharification of lignin-modified alfalfa.9 The enzymatic hydrolysis efficiency increased with increase in the BPS loading, and 95.6% of the total amount of glucan and xylan was (19) Wyman, C. E.; Dale, B. E.; Elander, R. T.; Holtzapple, M.; Ladisch, M. R.; Lee, Y. Y. Bioresour. Technol. 2005, 96, 2026–2032. (20) Hu, Z. H.; Wen, Z. Y. Biochem. Eng. J. 2008, 38, 369–378.

Rodgers et al. Table 1. Components of the Solid Residues and the Solid Yields after Pretreatment and the Overall Conversion Efficiency of Polysaccharides in Raw Maize Stover under Various Pretreatment Conditions component (%)d glucan

xylan

lignin

overall sugar conversion solid yield (%) efficiency (%)e

0 0.053 0.066 0.080 0.093 0.107

BPS loading (g NaOH equiv/g maize)a 38.5 (0.9)f 21.0 (0.8) 9.2 (0.3) 85.5 (1.7) 41.7 (1.4) 22.8 (1.3) 7.5 (0.4) 78.7 (1.9) 42.5 (0.8) 22.3 (0.7) 5.5 (0.2) 77.8 (1.6) 42.3 (1.3) 22.1 (1.2) 4.2 (0.2) 76.3 (2.1) 42.8 (1.1) 21.5 (1.2) 3.2 (0.3) 76.3 (2.3) 42.4 (1.5) 21.5 (1.4) 2.8 (0.2) 75.5 (1.8)

20 35 50 100 121

40.6 (1.2)c 41.4 (1.7) 42.5 (2.1) 41.3 (1.8) 41.3 (2.2)

pretreatment temperature (°C)b 20.6 (0.4) 3.7 (0.2) 80.4 (2.1) 20.7 (0.6) 3.4 (0.2) 76.8 (2.5) 21.1 (0.4) 3.3 (0.1) 76.5 (1.8) 20.1 (0.5) 2.8 (0.2) 75.1 (1.4) 19.9 (0.7) 2.6 (0.3) 73.0 (1.6)

82.7 (1.7) 94.2 (1.9) 94.6 (1.6) 89.5 (1.2) 88.0 (1.4)

0 5 10 15 20 24

39.2 (0.8)c 40.9 (1.1) 41.4 (0.9) 42.8 (1.2) 42.6 (1.4) 42.3 (1.3)

pretreatment duration (h)c 21.3 (0.3) 9.2 (0.2) 83.0 (2.2) 21.4 (0.5) 6.4 (0.3) 80.9 (2.3) 20.8 (0.5) 5.8 (0.3) 77.5 (1.8) 21.4 (0.4) 4.2 (0.2) 76.0 (1.5) 21.6 (0.6) 3.8 (0.2) 75.0 (1.7) 21.0 (0.5) 3.4 (0.3) 75.7 (1.9)

60.3 (1.6) 64.0 (1.4) 63.3 (1.2) 86.7 (1.8) 92.3 (1.3) 93.2 (1.5)

62.4 (1.6) 63.7 (1.4) 80.0 (1.3) 88.2 (1.6) 95.4 (1.5) 95.4 (1.6)

a Pretreatment at 35 °C for 24 h. b Pretreatment at BPS loading of 0.093 g NaOH equiv/g maize stover for 24 h. c Pretreatment at 35 °C and 0.093 g NaOH equiv/g maize stover for 24 h. d The contents are based on the solid residues after pretreatment. e The overall sugar conversion efficiency is the percentage of monosaccharides released from both the pretreatment and enzymatic hydrolysis stages to the total sugars contained in raw maize stover. f Standard deviations are shown within parentheses.

Figure 1. Effect of BPS loading on enzymatic hydrolysis of maize stover pretreated at 35 °C for 24 h with an enzyme loading of 15 FPU/g glucan.

converted into monosaccharides at a loading of 0.093 g NaOH equiv/g maize stover. Above this loading, there was no further increase in saccharification with increase in the BPS loading. The high conversion efficiency of the pretreated maize stover suggests that a BPS loading of 0.093 g NaOH equiv/g maize stoversat 35 °C for 24 hsshould replace NaOH as a suitable chemical to pretreat lignocellulosic biomass. 3.3. Effect of Enzyme Loading. In the enzyme loading experiment, the maize stover was pretreated at 35 °C for 24 h with a BPS solution that had an equivalent alkaline loading of 0.093 g NaOH equiv/g maize stover. Nearly 80% of the enzymatic hydrolysis efficiency was achieved at an enzyme loading as low as 5 FPU/g glucan (Figure 2). When the enzyme loading was 15 FPU/g glucan, 93.7% of the total amount of glucan and xylan was hydrolyzed into monosaccharides, and a maximum of 97.3% was achieved at 20 FPU/g glucan. The experiment suggests that an enzyme loading of 15-20 FPU/g glucan should be suitable for hydrolysis of maize stover. In a previous study, Varga et al.13 found that the enzymatic conver-

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Figure 2. Effect of enzyme loading on enzymatic hydrolysis of maize stover pretreated at 0.093 g NaOH equiv of BPS solution/g maize stover and 35 °C for 24 h.

Figure 4. Effect of pretreatment duration on enzymatic hydrolysis of maize stover pretreated at 0.093 g NaOH equiv of BPS solution/g maize stover and 35 °C with an enzyme loading of 15 FPU/g glucan.

Figure 3. Effect of pretreatment temperature on enzymatic hydrolysis of maize stover pretreated at 0.093 g NaOH equiv of BPS solution/g maize stover for 24 h with an enzyme loading of 15 FPU/g glucan.

Figure 5. Effect of Al3+ concentration on enzymatic hydrolysis of maize stover pretreated at 0.093 g NaOH /g maize stover and 35 °C with an enzyme loading of 15 FPU/g glucan.

sion efficiency of corn stover pretreated with 1, 5, and 10% NaOH was only 70-80% at 42 FPU/g glucan. 3.4. Effect of Pretreatment Temperature. Temperature is an important factor in the pretreatment of lignocellulosic biomass. High pretreatment temperatures can cause: (i) increased energy input; (ii) increased complexity of the process operation; and (iii) generation of possible inhibitors to enzymatic hydrolysis and fermentation.8 In this study, the effects of pretreatment temperatures from 20 to 121 °C were investigated at the BPS loading of 0.093 g NaOH equiv/g maize stover for 24 h. After pretreatment, the lignin contents decreased to 2.6-3.7% from the original 7.8% (Table 1). With increase in pretreatment temperature, the glucan content of the solid residue slightly increased and the xylan content slightly decreased. When the pretreatment temperature increased from 20 to 121 °C, the solid yield decreased from 80.4% to 73.0%. At an enzyme loading of 15 FPU/g glucan, 76.6% of glucan and 83.7% of xylan were hydrolyzed into monosaccharides for the maize stover pretreated at 20 °C (Figure 3). When the pretreatment temperature increased to 35 °C, 93.8% of glucan and 95.8% of xylan were hydrolyzed. At the pretreatment temperature of 121 °C, the yield of xylose slightly decreased, which may be due to the degradation of xylan at high temperatures. These experimental results suggest that the temperature of 35 °C should be suitable for maize stover pretreatment. At 35 °C, there was a maximum polysaccharide conversion of 94.2% (Table 1) from the raw maize stover in comparison with the 70% conversion from miscanthus pretreated with 12% NaOH at 70 °C.14 The higher yield in this study was probably due to using the less-lignified cellulosic maize stover as feedstock. 3.5. Effect of Pretreatment Duration. In the pretreatment duration experiment carried out at 35 °C and 0.093 g NaOH equiv/g maize stover for a period of 0-24 h, there was a slight increase in the glucan content, little change in the xylan content,

Table 2. Comparison of Pretreatment of Maize Stover with NaOH and BPS at 35°C and 0.093 g NaOH or NaOH equiv/g Maize Stover for 24 h component (%)a

b

pretreatment

glucan

xylan

lignin

solid yield

NaOH BPS

42.6 (2.3)b 41.8 (2.0)

21.4 (0.5) 20.7 (0.6)

3.4 (0.3) 3.2 (0.2)

75.9 (2.4) 76.4 (2.7)

a The contents are based on the solid residues after pretreatment. Standard deviations are shown within parenthesis.

and a decrease in the lignin content (from 9.2% to 3.4%) (Table 1). This occurred mainly in the first 15 h of the pretreatment experiment. When the pretreatment duration extended from 0 to 10 h, the enzymatic hydrolysis efficiency of glucan and xylan to monosaccharides only slightly rose from 55.4 to 60.3%, although 42.3% of lignin had been removed (Figure 4). This indicates that factors, other than the lignin content, can also affect enzymatic hydrolysis of polysaccharides, such as cellulose crystallinity.21 There was an increase in enzymatic hydrolysis of the total amount of glucan and xylan from 60.3% at 10 h to 96.4% at 24 h, so 24 h was a suitable pretreatment duration (Figure 4). Compared with the pretreatment of corn stover with slake lime, which required 4 weeks to reach 92% glucan conversion,12 the pretreatment of maize stover with the BPS solution had a better conversion efficiency at 24-h pretreatment duration (Table 1). 3.6. Effect of Al3+ Concentration and Comparison between BPS and NaOH Pretreatments. The BPS is a waste generated in aluminum production and it contains a high content of aluminum oxide. Aluminum ion (Al3+) concentration up to (21) Yoshida, M.; Liu, Y.; Uchida, S.; Kawarada, K.; Ukagami, Y.; Ichinose, H.; Kaneko, S.; Fukuda, K. Biosci. Biotech. Biochem. 2008, 72, 805–810.

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Figure 6. SEM images: (A) raw maize stover (×1000); (B) raw maize stover (×10 000); (C) maize stover pretreated with BPS at 0.093 g NaOH equiv/g maize stover and 35 °C for 24 h (×1000); and (D) maize stover pretreated with BPS at 0.093 g NaOH equiv/g maize stover and 35 °C for 24 h (×10 000).

Figure 7. Mass balance analysis of conversion of maize stover to monosaccharides. The pretreatment conditions were 0.093 g NaOH equiv of BPS solution/g maize stover and 35 °C for 24 h, and the enzymatic loading in the hydrolysis stage was 15 FPU/g glucan.

40-50 mg/L in the solution may inhibit or inactivate enzymes.22 In investigating the effects of Al3+ on enzymatic hydrolysis, maize stover was pretreated using a NaOH solution at an alkali loading rate of 0.093 g NaOH/g maize stover at 35 °C for 24 h. The pretreatment of maize stover with the BPS solution was also carried out under the same condition for comparison. The lignin removal and the solid yield were very similar after the BPS and NaOH pretreatments (Table 2) indicating that BPS can be used as effectively, and more economically and sustainably as NaOH. The enzymatic hydrolysis efficiency of the NaOH pretreated maize stover (Figure 5) was very close to that of the BPS pretreated one under the same pretreatment condition. Enzymatic hydrolysis was not affected by Al3+ when its concentration was lower than 90 mg/L (Figure 5). However, when the Al3+ concentration increased to 120 mg/L, the hydrolysis efficiency decreased to 74.5%, which may be due to the destabilization of the celulase by Al3+ around 120 mg/L and, consequently, reduce the effective contacts of cellulose and cellulase.23 (22) Vanderberg, L. A.; Foreman, T. M.; Attrep, M.; Brainard, J. R.; Sauer, N. N. EnViron. Sci. Technol. 1999, 33, 1256–1262.

Because the concentration of Al3+ in the BPS solution was between 40-50 mg/L, the result above suggests that the Al3+ concentration should have no negative influence on enzymatic hydrolysis. 3.7. SEM Image Analysis. The structural change of the maize stover before and after the BPS pretreatment (0.093 g NaOH equiv/g maize stover at 35 °C for 24 h) was imaged with a scanning electron microscope (SEM) (Figure 6). As shown in Figure 6A (×1000), the fiber surface was rough and particles were observable. After pretreatment, the surface of the fibers became smooth, and the structural detail was more observable (Figure 6C (×1000)). In Figure 6B (×10 000), it appeared that the fiber surface was covered with thin films, which might be wax layers commonly found in herbaceous biomass.24 After pretreatment with the BPS solution, the covered layers partly disappeared (Figure 6D). The removal of the covered layers would benefit enzymatic hydrolysis by increasing (23) Fanklin, L.; Burton, F. L.; Stensel, H. D.; Tchobanoglous, G. Wastewater engineering: treatment and reuse, 4th ed.; McGraw-Hill Book Company: New York, 2003. (24) Yan, L. F.; Li, W.; Yang, J. L.; Zhu, Q. S. Macromol. Biosci. 2004, 4, 112–118.

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the diffusivity of cellulose molecules and accessibility of fibers to the cellulase.25 3.8. Mass Balance and Economic Analysis. The study above shows that pretreatment conditions suitable for enzymatic hydrolysis of maize stover were the following: (i) a pretreatment duration of 24 h, (ii) a pretreatment temperature of 35 °C, and (iii) a BPS loading of 0.093 g NaOH equiv/g maize stover. Under these pretreatment conditions and with an enzyme loading of 15 FPU/g glucan, a mass balance was analyzed with 100 g of maize stover dry matter (Figure 7). Other components of maize stover, such as proteins, acid-solubilized lignin, and other non-cell-wall components, were not measured in this study and were not considered in the mass balance. This was why the summations of the components in the flowchart in different stages were under 100%. In the pretreatment stage, pretreatment of maize stover with the BPS solution resulted in 68% lignin and 15.3% glucan removal and 21.1% xylan solubilization. In the enzymatic hydrolysis stage, 95.6% of the total amount of glucan and xylan of the pretreated biomass was hydrolyzed into monosaccharides. Theoretically, 42.5 g of glucose and 21.7 g of xylose can be yielded from 100 g of raw maize stover (dry matter). In this study, overall yields of glucose and xylose in the pretreatment and enzymatic hydrolysis stages were 39.6 and 20.5 g, corresponding to 93.2% and 94.5% of the maximum theoretical yields for glucose and xylose, respectively. This indicates that using BPS as a pretreatment alkali at very moderate pretreatment conditions, a high production efficiency of monosaccharides can be achieved with maize stover as the feedstock. On the basis of the flowchart, 1 ton of maize stover could produce 601 kg of glucose and xylose. The pretreatment of 1 (25) Wang, L. S.; Zhang, Y. Z.; Gao, P. J.; Shi, D. X.; Liu, H. W.; Gao, H. J. Biotechnol. Bioeng. 2006, 93, 443–456.

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ton of maize stover would consume 93 kg of NaOH, which costs about $41.90 ($450/ton NaOH). Therefore, the cost of NaOH consumption is $69.60 to produce 1 ton of monosaccharides from maize stover; this is high in consideration with the fuel ethanol production. Because BPS is a waste generated in aluminum production, the main cost for using BPS in the pretreatment stage is transportation expense. This cost could be very low if the bioethanol production plant is constructed near the aluminum plant. Thus, if NaOH is replaced by BPS, the cost for pretreatment will be reduced significantly. 4. Conclusions In this study, a Bayer process sand (BPS) solution was used to pretreat maize stover. Compared with NaOH pretreatment, BPS pretreatment resulted in a similar enzymatic hydrolysis efficiency, suggesting that BPS should replace NaOH. This will provide savings in pretreatment cost. The effects of the BPS loading, enzyme loading, pretreatment temperature, and pretreatment duration were investigated. With pretreatment at a BPS loading of 0.093 g NaOH equiv/g maize stover, temperature of 35 °C for 24 h, and with enzymatic hydrolysis at the cellulase loading of 15 FPU/g glucan, 95.6% of the pretreated maize stover was hydrolyzed. In combination with the pretreatment and enzymatic hydrolysis stages, 93.2% of glucan in the raw maize stover was converted to glucose and 94.5% of xylan was converted to xylose. Acknowledgment. We appreciate the financial support from the EU Marie Curie Transfer of Knowledge (Contract no. MTKD-CT2004-014432; ABIOS). The authors gratefully acknowledge Dr. David Elliott for technical assistance in carrying out the SEM analysis. EF801032X