Comparison of Dilute Acid and Sulfite Pretreatments on Acacia

Oct 15, 2014 - School of Forestry and Resource Conservation, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan ...
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Comparison of Dilute Acid and Sulfite Pretreatments on Acacia confusa for Biofuel Application and the Influence of Its Extractives Ting-Feng Yeh,* Mao-Ju Chang, and Wan-Jung Chang School of Forestry and Resource Conservation, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan ABSTRACT: Chemical components of lignocellulosic biomass may impede biofuel processing efficiency. To understand whether the heartwood of Acacia confusa is suitable for biofuel application, extractive-free heartwood of A. confusa was subjected to dilute acid (DA) or sulfite pretreatments. Sugar recoveries were used to evaluate the performance of different pretreatments. Cell wall properties, such as 4-O-alkylated lignin structures, S/G ratios, and xylan contents, of the pretreated samples showed significant correlations with the enzymatic saccharification of glucan. The 4% bisulfite-pretreated samples produced higher total sugar recoveries than DA-treated samples. The highest total sugar recoveries from DA and sulfite pretreatment were 52.0% (170 °C for 20 min) and 65.3% (4% NaHSO3 and 1% H2SO4), respectively. The results also demonstrated that the existence of extractives in the heartwood of A. confusa hindered the sugar recoveries from both the pretreatments and enzymatic saccharification. Total sugar recoveries were reduced 11.7−17.7% in heartwood samples with extractives. KEYWORDS: Acacia conf usa, dilute acid pretreatment, enzymatic saccharification, extractives, sugar recovery, sulfite pretreatment

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sulfite pretreatment,12 green liquor pretreatment,13 ammonia explosion,14 and many others.8−10 Dilute acid pretreatment is one of the most thoroughly investigated pretreatment techniques because it is rather inexpensive and effective. It is usually conducted using diluted sulfuric acid or gaseous sulfur dioxide (in steam explosion) at high temperatures (140−200 °C), although some other acids have also been tested.8,10,11 Dilute acid pretreatment mainly hydrolyzes the hemicelluloses in biomass and, hence, creates pores for subsequent enzymatic saccharification. Dilute acid pretreatment can achieve satisfactory cellulose saccharification for a wide range of agricultural residues and some hardwood species but is not effective for softwoods.10 A sulfite pretreatment technique, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), has been reported to have great abilities to tackle softwoods,12 and it is also a promising technique for hardwoods.15 Biomass was treated with bisulfite salts in acidic conditions (pH 2−4) and 160−180 °C for a short time, followed by a disk-milling process for size reduction. The fibrous substrates were then enzymatically hydrolyzed to sugars. An excellent recovery of fermentable sugars with a low amount of fermentation inhibitors was reported for both hardwood and softwood species.16,17 To understand the chemical modifications of the woody biomass during the pretreatment processes and their structural characteristics retained after the pretreatments will provide insightful information into the mechanism of overcoming biomass recalcitrance.18 This can further assist in optimizing the pretreatment processes for the production of cellulosic bioethanol from specific lignocellulosics. The information

INTRODUCTION

Acacia confusa Merr., belonging to the family of Leguminosae, is an indigenous species of Taiwan, and it is distributed over the hills and lowlands. It has great abilities for soil and water conservation as well as remarkable capability for carbon sequestration.1 Its heartwood normally comprises the majority of the cross-section of the stem with aesthetic textures and excellent wood properties, and hence, this tree is one of the major hardwood plantation species in Taiwan. Its plantation area is more than 21 200 ha, and this tree can produce at least 52.9 tons of biomass per hectare.2 It was an important source for feedstocks, charcoals, and construction materials decades ago. However, many of its applications were replaced by modern petrochemical-based materials. Recent studies have found that the crude extractives of heartwood or other tissues from this species were rich in varieties of phenolic compounds, such as benzoic acids, tannins, flavonoids, and flavonoid glycosides. 3,4 Many of these phytochemicals have outstanding bioactivities and can be used for various biomedical purposes, such as antioxidation,5 hepatoprotection,3 antihyperuricemia,6 and anti-inflammatory7 activities. However, after these valuable extractives were removed from A. confusa heartwood, the extractive-free wood residues were underutilized. To achieve the utilization of whole trees and also to resolve the demand for biofuel application, these wood residues can be considered as a potential lignocellulosic biomass for bioethanol production. Current biomass conversion typically relies on combined efforts of chemical and enzymatic treatments to process the lignocelluloses into sugars for fermentation to ethanol.8−10 Hence, certain kinds of pretreatments are required to break down the polymeric structures of the lignin and carbohydrates in biomass to produce materials suitable for enzymatic saccharification. These techniques include dilute acid pretreatment,11 © 2014 American Chemical Society

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or 8% (w/w) NaHSO3 and 1 or 2% (w/w) H2SO4 at 180 °C for 30 min (4B1A, 4B2A, 8B1A, and 8B2A; Table 1). The initial pH values of the SP liquors were between pH 2.1 and 2.6. After individual pretreatment, the sample was filtered by a sintered glass filter to separate the solid residues [pretreatment residues (PRs)] and pretreatment hydrolysates (PHs). An additional double-distilled water (ddH2O) rinse (50 mL) of the PRs to remove residual pretreatment chemicals and inhibitors was also filtered, collected, and combined into PHs. An aliquot of the pretreatment hydrolysates was used for determination of the neutral sugar contents and other components. Enzymatic Saccharification. The PRs were further vibrationmilled (Wig-L-Bug) for 2.5 min, and the milled solid residues (0.5 g) were subjected to enzymatic saccharification. Enzymatic saccharification was carried out at 1% (w/v) consistency with the enzyme loading of 10.4 filter paper units (FPU)/g of substrate at pH 4.5 (sodium acetate buffer) and 45 °C for 72 h. The enzyme (FiberZyme LBL CONC, Dyadic International, Inc.) was a preparation of carbohydrate hydrolases with cellulase and xylanase activities. After the enzymatic saccharification, the mixtures were centrifuged to collect the supernatant. An aliquot of the supernatant [enzymatic hydrolysates (EHs)] was used for determination of the neutral sugar contents. The overall performance of individual pretreatment was evaluated by the sugar recovery (%).

related to whether A. confusa is suited to produce sugars for bioenergy application is limited, and this hinders the effective utilization of this species. Therefore, the objective of this study was to evaluate the potential of the extractive-free A. confusa heartwood for producing fermentable sugars based on two acidbased pretreatments: dilute acid (DA) and sulfite pretreatment (SP). The changes of the chemical characteristics in the cell wall of this species during these pretreatments were evaluated. Original A. confusa heartwood with high extractive content was also investigated for comparison.

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MATERIALS AND METHODS

Wood Materials. Shavings of a 40-year-old A. confusa heartwood were soaked in ethanol at ambient temperature for 3 weeks to remove valuable extracts. The yield of this ethanolic extract was 8.2%.4 After airdried at ambient temperature, these shavings were ground to 40−60 mesh wood meals. The wood meals were further extracted with toluene/ ethanol (2:1, v/v) and ethanol in a Soxhlet apparatus for 48 h. The extractive yield of this further extraction was 1.1%. A total of 9.3% extractives were obtained from all of the extraction processes. The extractive-free wood meals were further dried over P2O5 under vacuum for later experiments. Pretreatments. All pretreatments were performed in a cylindrical stainless reactor (25 mL) with a dry-heating system. The solid/liquid ratio (w/v) for all pretreatments was 1:10. For dilute acid pretreatments, 1 g of wood meals was reacted with 0.1% (w/w) H2SO4 at 150 or 170 °C for 10 or 20 min (150C10M, 150C20M, 170C10M, and 170C20M; Table 1). For sulfite pretreatments, 1 g of wood meals was reacted with 4

sugar recovery (%) = (sugar from PHs + sugar from EHs) × 100/sugar in the original wood Analytical Methods. All analyses were conducted with three replicates, and all calculations were based on the oven-dried weight basis. The lignin content was determined according to the Klason lignin method,19 combining both the Klason lignin and acid-soluble lignin to be the total lignin content. The extinction coefficient for acid-soluble lignin was 110 L g−1 cm−1 at 205 nm.19 Neutral sugar contents of samples were determined from the acidsoluble fraction of lignin determinations by the alditol−acetate method20 and quantified by gas chromatography with a flame ionization detector (GC−FID, Agilent 7890A) and a DB-225 GC column. For the sugar contents released from the pretreatment processes (PHs) or enzymatic saccharification (EHs), aliquots of the corresponding hydrolysates were used for determining the sugar contents by the same alditol−acetate method. Furfural contents in PHs were also determined by GC−FID (Agilent 7890A) with a Supelcowax 10 GC column. An aliquot of the PHs was diluted in acetone and used for determination of the furfural contents according to a GC method.21 Lignin monomeric composition was determined by the nitrobenzene oxidation (NBO) using original wood or pretreated residues according to a previous protocol,22 and 5-iodovanillin was used as the internal standard.23 The amounts of each lignin degradation product were quantified by GC−FID (Agilent 7890A) equipped with a DB-5 GC

Table 1. Lists of Different Pretreatment Conditions Used

a

H2SO4 (0.1%, w/w). bAt 180 °C and 30 min.

Table 2. Chemical Composition of A. confusa Cell Walls before and after Different Pretreatmentsa composition (%)b samplec

pretreatment yield (%)d

total lignin

glucan

xylan

all polysaccharides

79.1 ± 1.7 A 77.9 ± 2.2 A 71.7 ± 0.6 B 70.1 ± 0.4 B 64.1 ± 0.2 C 64.1 ± 0.2 C 63.7 ± 1.1 C 62.5 ± 0.3 C

24.7 ± 0.4 D 25.3 ± 0.3 D 28.2 ± 1.3 B,C 26.4 ± 0.4 C,D 29.0 ± 0.0 A,B 31.3 ± 0.3 A 29.7 ± 0.1 A,B 24.4 ± 0.2 D 28.5 ± 0.6 B,C

36.5 ± 0.8 C 33.0 ± 1.1 C 37.6 ± 0.6 C 36.9 ± 0.4 C 45.2 ± 0.2 B 55.1 ± 1.2 A 53.7 ± 1.7 A 56.9 ± 0.2 A 46.0 ± 0.7 B

16.1 ± 0.3 A 16.8 ± 0.2 A 10.7 ± 0.8 B 4.1 ± 0.3 C,D 5.4 ± 0.3 C 2.4 ± 0.6 D 2.6 ± 0.1 D 2.7 ± 1.1 D 1.7 ± 0.0 D

57.1 ± 1.5 A,B 53.0 ± 1.4 B,C 51.6 ± 1.2 B,C 41.1 ± 0.1 D 51.8 ± 0.9 C 58.9 ± 0.7 A 59.3 ± 0.8 A 59.8 ± 0.6 A 48.0 ± 0.7 C

e

wood 150C10M 150C20M 170C10M 170C20M 4B1A 4B2A 8B1A 8B2A

Values in the same column with different letters are statistically different under Tukey’s HSD test (α = 0.05) [mean ± standard error (SE); n = 3; numbers are rounded up to the first decimal place]. bPercentage based on original or pretreated samples subjected for chemical analyses. cPlease refer to Table 1 for the sample name. dPercentage of starting materials recovered as insoluble residues. eExtractive-free. a

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column. H-type lignin was calculated from p-hydroxybenzaldehyde; Gtype lignin was calculated from the sum of vanillin and vanillic acid; and S-type lignin was from the sum of syringaldehyde and syringic acid. No p-hydroxybenzoic acid was found in the nitrobenzene oxidation products. The cellulose crystallinity indexes (CrIs) of different samples were calculated from the X-ray diffractogram (XRD) of the wood meal with the mesh size ranging from 40 to 60 with or without pretreatments. The XRDs were recorded on a Bruker D2 PHASER diffractometer. All scans were collected at 30 kV and 10 mA in the range of 5° < 2θ < 40° using Cu Kα radiation (λ = 0.1544 nm). A peak fitting software (PeakFit, Systat Software, Inc.) was used to deconvolute the individual peaks, assuming Gaussian function for each peak and a broad peak at around 20.04° assigned to the amorphous component. Peak fittings were continued until the maximum F values were achieved. In all cases, the F values were greater than 2700, which corresponding to an r2 > 0.98. The CrI was calculated by dividing the diffractogram area because of the crystalline components (sum of peak areas from 002, 021, 101, 101̅, and 040 peaks) by the total area of the original diffractogram.24 Properties of different treatment groups were compared all together by multiple mean comparisons [Tukey’s honest significant difference (HSD) test; α = 0.05] to test the significant differences among groups (SAS 9.3, SAS Institute, Inc., Cary, NC). Correlation analysis of the specific property pair was also performed with the same software using the multivariate function.

Figure 1. Lignin composition of samples before and after different pretreatments. Percentage based on moles of lignin from each sample assuming the molecular weight of one lignin C9 unit to be 210 g/mol. H, p-hydroxybenzaldehyde; G, sum of vanillin and vanillic acid; and S, sum of syringaldehyde and syringic acid. Mean ± SE; n = 3. Please refer to Table 1 for the sample name.

RESULTS AND DISCUSSION Cell Wall Chemical Composition before and after Pretreatments. Table 2 shows changes in the chemical composition before and after pretreatments. A typical A. confusa wood contains about 24.7% lignin and 57.1% polysaccharides. The pretreatment yields of the DA pretreatments were between 70.1 and 79.1%. As the reaction temperatures increased, the yields were reduced. The total lignin contents of the DApretreated residues slightly increased as the reaction times became longer. The glucan contents of the DA-pretreated residues did not change, except for the 170C20M sample. However, if the glucan contents of the DA-pretreated residues were calculated back to the glucan contents of the original wood, about 13−28% original glucan was actually lost during the DA pretreatment processes. The xylan contents reduced dramatically as the reaction temperatures increased from 150 to 170 °C. The increased glucan and lignin and reduced xylan contents in pretreated residues were also reported from the dilute-acidpretreated Populus trichocarpa18,25 and mixed hardwood species.17,26 The pretreatment yields of the sulfite pretreatments (62.5− 64.1%) were lower than those in the DA pretreatments, as displayed in Table 2. The pretreatment yields from different sulfite pretreatments were similar to each other. The lignin contents of the SP-pretreated residues were increased when compared to that of the original wood, except for the 8B1A sample. The glucan contents of the SP-pretreated residues (46.0−56.9%) were much higher when compared to that of the original wood, and these contents were also higher than those of the DA-pretreated residues. If the glucan contents of the SPpretreated residues were calculated back to the glucan contents of the original wood, about 1−6% original glucan was lost during the sulfite pretreatment processes except for the 8B2A sample. These glucan losses were lower than those in the DA-pretreated processes. The xylan contents of the SP-pretreated residues (1.7−2.7%) were lower than those of the DA-pretreated residues (4.1−16.8%). These results are consistent with the observation from other researchers, who applied SPORL on various hardwood species.15,17

A. confusa lignin were S- and G-type lignin, and they were about 38.6 and 14.7% based on moles of lignin, respectively. H-type lignin was the minor component and was only about 0.9% (Figure 1). The S/G ratio of A. confusa lignin was about 2.6, and the total yield of the nitrobenzene oxidation products was about 54.2%. The total yields of the nitrobenzene oxidation products decreased as the reaction temperatures or times increased during the DA pretreatments, indicating that the noncondensed lignin structures, such as 4-O-alkylated, α-O-4, and β-O-4 lignin structures,22 were reduced. This suggests that the lignin structures retained in the DA-pretreated residues were more condensed-type than that of the original wood. Recent evidence also revealed that the condensed lignin structures were increased after dilute acid pretreatments in spruce.16 The DA pretreatments removed more S-type lignin than G-type lignin from the original wood lignin structures as the reaction temperatures or times increased (Figure 1). This caused the S/G ratios of lignin to decrease gradually from 2.7 (150C10M) to 2.4 (170C20M) as the reaction conditions became severe. The H-type lignin was rather stable (∼1%) when compared to S- or G-type lignin under these pretreatments. Current results are consistent with the previous reports that β-O-4 linkages and S/G ratios were reduced during the DA pretreatment on hardwood species and the relative content of H-type lignin was rather unchanged during this process.18,26 During the sulfite pretreatments, A. confusa lignin was partially sulfonated, and the sulfonated lignin structures were removed by dissolving in the pretreatment liquors. The removals of noncondensed S- and G-type lignin structures in the sulfite pretreatments were more significant when compared to those in the DA pretreatments (Figure 1). The removal of the S-type lignin in the sulfite pretreatments seemed higher than that of the G-type lignin, causing the S/G ratios to reduce to 2.2−2.4 compared to that of the original wood (2.6; Figure 1). The total yields of the nitrobenzene oxidation products from the SPpretreated samples were reduced in the range from 24% (8B2A) to 34% (8B1A), indicating that the residual lignin in acid bisulfite-pretreated samples was a more condensed type of lignin than that in DA-pretreated samples or modified lignin structures,

Nitrobenzene oxidation was used to profile the lignin composition of A. confusa wood before and after pretreatments, as illustrated in Figure 1. The major monomeric components of

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cannot rule out the possibility that the aggregation of cellulose fibrils might occur because of thermally induced crystallization and aggregate growth under hydrothermal conditions.28 The increments in CrIs of the SP-pretreated residues were reduced as the charges of bisulfite or acid increased. The reduced increments in CrIs of the SP-pretreated residues were likely attributed to the relatively increased amorphous components resulting from the condensed lignin aggregation. Composition of Hydrolysates Released from Pretreatments. The monosugar components of the pretreatment hydrolysates were further analyzed and expressed as polysaccharide forms, as shown in Figure 3. Both DA and sulfite pretreatments hydrolyzed lots of xylan into the hydrolysates (Figure 3). In the DA pretreatments, 3.4% xylan was recovered from the 150C10M PHs and about 9−10% xylan was recovered from other PHs (Figure 3A). In the sulfite pretreatments, about 12−13% xylan was recovered from the 4% bisulfite-charged PHs and only about 6% xylan was recovered from the 8% bisulfitecharged PHs. The furfural contents in the SP hydrolysates were about 3.9−5.4% (Figure 3B), which were higher than those in the DA hydrolysates (∼2.0%; Figure 3A). Studies have shown that xylan, in comparison to other polysaccharides, can easily be hydrolyzed under acidic conditions and release xylose into the pretreatment hydrolysates,8 and these xyloses might continue to be broken down into furfural and other degradation products.30 Hence, by considering the mass balance of xylan, the lower xylan contents of 8% SP hydrolysates compared to those of 4% SP hydrolysates indicated that further degradation might occur during the pretreatment processes. The DA pretreatments broke down about 1.7−3.3% lignin into the pretreatment hydrolysates (Figure 3A). The same extinction coefficient for acid-soluble lignin was assumed and used to calculate the sulfonated acid-soluble lignin in the SP hydrolysates. The acid-soluble lignin contents in the SP hydrolysates were about 4.6−6.9% (Figure 3B), which were higher than those in the DA hydrolysates. This indicated that more lignins were actually removed from the original wood during the sulfite pretreatments when compared to those during the dilute acid pretreatments. The disrupted and sulfonated

which could not be analyzed by the nitrobenzene oxidation. The occurrence of the lignin condensation reaction was reported in acidic sulfite pulping27 and the SPORL-pretreated spruce,16 suggesting that a similar condensation reaction might occur in the SP-pretreated A. confusa and, hence, increase the condensed lignin structures. The CrI of original wood without any pretreatment was estimated to be 59.1% (Figure 2). DA pretreatments increased

Figure 2. CrIs of samples before and after different pretreatments. Mean ± SE; n = 3. Please refer to Table 1 for the sample name.

the CrIs of the pretreated residues when compared to that of the original wood. The increases of these CrIs were positively correlated to the reaction temperatures and times, and the highest CrI was 69.7% (170C20M). The sulfite pretreatments also increased the CrIs of the pretreated residues (61.5−66.8%) compared to that of the original wood. The increasing values of CrIs in pretreated residues were consistent with previous reports that dilute acid or acid bisulfite pretreatments usually elevated the crystallinity of lignocellulosics.28,29 The increase of the biomass CrI was usually attributed to that of the amorphous hemicelluloses and partial lignin, and amorphous regions of native cellulose were removed and disrupted during the pretreatment process; hence, these would relatively increase the crystalline portion of the pretreated residue. However, current results

Figure 3. Chemical components in different pretreatment hydrolysates: (A) dilute acid pretreatments and (B) sulfite pretreatments. Mean ± SE; n = 3. Please refer to Table 1 for the sample name. 10771

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lignin could facilitate cellulose susceptibility to the following enzymatic hydrolysis.16 The glucan contents recovered from the pretreatment hydrolysates were not high. It was 0.1−0.4% from the DA hydrolysates (Figure 3A) and 0.8−1.2% from the SP hydrolysates (Figure 3B). Thus, about 12−28% original glucan was degraded to other products during the pretreatments, especially in the DA pretreatments (cf. Table 2 and Figure 3). These degradation products were not measured in the current study. However, previous studies have suggested that fermentation inhibitor compounds, such as 5-hydroxymethylfurfural, 5methylfurfual, levulinic acid, and formic acid, might come from glucose degradation.16,30 In spruce, DA pretreatments produced more than twice these inhibitor compounds when compared to those from SPORL pretreatments.16 These could attribute to the low mass balances of glucan in the DA-pretreated samples (cf. Table 2 and Figure 3). Enzymatic Saccharification and Cell Wall Properties. Without any pretreatment, A. confusa wood released about 15% xylose or glucose based on the xylose or glucose contents of the original wood during the enzymatic saccharification (Figure 4).

Figure 5. Correlation coefficient between enzymatic saccharification of glucan and the cell wall properties. NBO, nitrobenzene oxidation; S/G, S/G ratio; and CrI, crystallinity index. (∗) p < 0.05.

pretreated residues would increase the enzymatic saccharification of glucan (r = −0.7122; Figure 5). Similar results were reported that decreasing the xylan content in the DA-pretreated corn stover31 and SPORL-pretreated Douglas fir32 would improve the extent of cellulose digestibility. Xylan is the major hemicellulose of A. confusa, and hemicelluloses are spatially associated with cellulose. Removal of hemicelluloses is important to enhance the cellulose digestibility. The total yield of nitrobenzene oxidation reflects the portion of the noncondensed lignin in the overall lignin structures.22 The correlation analysis indicated that, if the pretreated residues had less noncondensed lignin (more condensed lignin), the pretreated residues were more enzymatically hydrolyzable (r = −0.6952; Figure 5). Similarly, if the lignin S/G ratios of these pretreated residues were lower, the pretreated residues were more enzymatically hydrolyzable (r = −0.6908; Figure 5). The influence of lignin composition on cell wall digestibility still remains uncertain, and the results can be very different using various biomass and pretreatment techniques.33−37 In the current study, the results can be explained by considering the cellulose accessible surface after the pretreatments. Our results indicated that both DA and sulfite pretreatments could break down the lignin barriers to loosen the ultrastructures of the cell walls and the sulfite pretreatments removed more lignin than the DA processes (Figure 3). The removal of lignin was more on the noncondensed lignin (high S/G ratio moieties), and this action left more condensed lignin (low S/G ratio moieties) retained in the SP-pretreated residues (Figure 1). The S/G ratios of the SPpretreated residues were lower than those of the DA-pretreated residues (Figure 1), indicating that more severe lignin removal and loosened cell walls might exist in the SP-pretreated residues compared to those in the DA-pretreated residues. A previous study in comparing the cell wall structure changes after DA and SPORL pretreatments has demonstrated that the cellulose accessible surfaces from the SPORL-pretreated pine cell wall were about 3 times more than those from the DA-pretreated samples.38 The cellulose accessible surface was actually created because of the lignin removal and loosened structures during the pretreatments. More cellulose accessible surfaces in the SPpretreated residues might be created in comparison to those in the DA-pretreated residues, and hence, the enzymatic saccharification of the SP-pretreated residues (low S/G ratio and more condensed lignin) was increased in comparison to those in the DA-pretreated residues. The sulfonated lignin structures of the SP-pretreated residues might also play a significant role in the enzymatic efficiency and enhance the glucose yields.12

Figure 4. Enzymatic released sugar of different samples after 72 h of enzymatic saccharification. Percentage based on xylose or glucose contents in the substrates subjected to enzymatic hydrolysis. Mean ± SE; n = 3. Please refer to Table 1 for the sample name.

Both DA and sulfite pretreatments increased the enzymatic saccharification of pretreated residues when compared to that of the original wood without any pretreatment. Because the xylan contents in many of the pretreated residues were rather low (Table 2), the enzymatic saccharification of glucan was more significant than that of xylan in the pretreated residues. A generally higher glucose yield (44.0−58.0%) was obtained from the SP-pretreated residues when compared to that from the DApretreated residues (40.9−44.6%; Figure 4). These enzymatic saccharification yields are rather comparable to those obtained in the previous studies, in which various pretreatments, including dilute acid, SPORL, green liquor, autohydrolysis, and ozone pretreatments, were applied on various poplar woods.17,25 To further understand the cell wall properties that might have influences on the enzymatic saccharification of glucan, correlation analysis was further performed to profile the relationships (Figure 5). Among the cell wall properties measured in this study, only the xylan content, total yield of nitrobenzene oxidation, and S/G ratio were found to have significant correlations (p < 0.05) to the enzymatic saccharification of glucan. Other properties, such as total lignin, glucan content, and CrIs, were tested without significant correlations (p > 0.13). Our results indicated that lower xylan contents in the 10772

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Total Sugar Recovery. To further evaluate the overall performance of different pretreatments, sugar recovery, combining the sugar contents from both PHs and EHs, was calculated. Without any pretreatment, A. confusa wood yielded only about 15% sugar recovery based on xylose, glucose, or total sugar of the original wood (Figure 6). All pretreatments could enhance the

Figure 7. Sugar recovery (%) based on xylose, glucose, and total sugars of the original wood with and without extractives after specific pretreatments and enzymatic saccharification. Please refer to Table 1 for the sample name.

enzymatic saccharification. Without any pretreatment, the original wood samples performed similarly either with or without extractives, and the total sugar recoveries were 13.0% for wood with extractives and 14.2% for wood without extractives, respectively (Figure 7). For the 170C20M-pretreated sample, the existence of extractives reduced the sugar recovery majorly from the PHs, especially in the recovery of xylose (Figure 7). For the 4B1A-pretreated sample, the existence of extractives reduced the sugar recoveries from both the pretreatment hydrolysates and enzymatic hydrolysates (Figure 7). The combined reductions of total sugar recoveries were 11.7% in the 170C20M-pretreated sample and 17.7% in the 4B1A-pretreated sample, indicating that the existence of extractives would reduce the sugar recoveries from both the DA and sulfite pretreatments. The major constituents in the extractives of A. confusa heartwood are phenolic compounds, flavonoids, and condensed tannins.4,41 These compounds are chemically reactive species able to react with other molecules or through condensation reactions under acid treatments or acidic sulfite conditions.27,42 These side reactions rerouted the pretreatment reactions, which were originally arranged to decrease the recalcitrance of cell walls to facilitate followed enzymatic saccharification. The nonproductive adsorption of enzymes onto the condensation adducts from extractives can also be disadvantageous to enzymatic saccharification and, hence, reduce overall sugar recovery efficiency. Therefore, it is worth performing an extraction process in advance to remove the valuable extractives from A. confusa heartwood for biomedical purposes, and the resulting extractive-free wood residues will then go for deconstructing the cell walls for bioethanol utilization to maximize the benefits. In summary, extractive-free A. confusa heartwood was subjected to different dilute acid and sulfite pretreatments. Reduced 4-O-alkylated lignin structures, lower S/G ratios, decreased xylan contents, and increased cellulose CrIs were found in all pretreated residues. All pretreated samples showed better sugar recoveries than the untreated wood. Cell wall properties, such as 4-O-alkylated lignin structures, S/G ratios, and xylan contents, of the pretreated residues showed significant correlations with the enzymatic saccharification of glucan. Increased cellulose accessible surfaces in the SP-pretreated samples compared to those in the DA-pretreated samples might enhance the enzymatic saccharification of the SP-pretreated samples. As a result, 4% bisulfite-pretreated samples yielded

Figure 6. Sugar recovery (%) based on xylose, glucose, and total sugar of the original wood after individual pretreatment and enzymatic saccharification. Please refer to Table 1 for the sample name.

sugar recovery (>30%) based on xylose, glucose, or total sugar of the original wood. The recoveries of xylose exceeded 65% for many of the pretreatments in either the DA or sulfite pretreatment. These high xylose recoveries (Figure 6) mainly resulted from the xylose contents in the PHs (cf. Table 2 and Figure 3). All glucose recoveries from different pretreatments were more than 30%, and the highest glucose recovery was from the 4% NaHSO3 and 1% H2SO4 pretreatment (51.2%; Figure 6). Current glucose recoveries seem lower than the high glucose recovery suggested by the previous report, which showed a nearcomplete cellulose conversion to glucose in aspen using SPORL pretreatment.15 About 60−80% original lignin was still remained in the pretreated residues (Table 2), and this residual condensedtype lignin and also the ultrastructures of the pretreated cell walls were still barriers limiting the full accessibility of enzymes during enzymatic saccharification. This indicates that further efforts either chemically or mechanically to increase the accessibility of the hydrolytic enzymes will help to enhance the glucose recoveries of current results. Studies even suggested that posttreatments after the pretreatment process, such as oxygen delignification39 and mechanical refining,12,40 would open up the surface areas and pore volumes, which would ultimately improve the enzymatic hydrolysis and finally increase the sugar recovery. In the DA pretreatments, total sugar recoveries increased when the reaction temperatures and times increased. In the sulfite pretreatments, 4% bisulfite-pretreated samples yielded better total sugar recoveries than 8% bisulfite-pretreated samples. Overall, these total sugar recoveries were comparable to the results of the DA and SPORL pretreatments on various poplar species.17 In the current study, the best total sugar recoveries from the DA and sulfite pretreatments are 52.0% (170C20M) and 65.3% (4B1A). Comparison of Samples with and without Extractives. A high extractive content (9.3%) was obtained from the A. confusa heartwood. The best two pretreatments in the current study, 170C20M and 4B1A, were further chosen to evaluate the influences of extractives on the pretreatment and enzymatic saccharification. Figure 7 shows the sugar recoveries of wood, with and without extractives, after each pretreatment and 10773

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higher sugar recovery than the DA-pretreated samples. The best sugar recovery was from the 4% NaHSO3 and 1% H2SO4 pretreated sample. Current results also revealed that the existence of extractives in A. confusa reduced the sugars released from both the pretreatments and enzymatic saccharification.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +886-2-33664655. Fax: +886-2-23654520. E-mail: [email protected]. Funding

This study was sponsored part by a grant (Grant 100AS-8.4.1FB-e1(5)) from the Forestry Bureau, Council of Agriculture, Taiwan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors sincerely thank Dyadic International, Inc. (Jupiter, FL) for providing the enzymes. The authors gratefully acknowledge Prof. Shang-Tzen Chang for instrument access and suggestions to the manuscript. The authors also thank Prof. Jinn P. Chu for access to the X-ray diffractometer.

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REFERENCES

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