Ind. Eng. Chem. Res. 2010, 49, 1473–1479
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Peroxide-Acetic Acid Pretreatment To Remove Bagasse Lignin Prior to Enzymatic Hydrolysis He Tan,‡ Rallming Yang,*,† Weidong Sun,‡ and Shuangfei Wang‡ Institute of Paper Science and Technology, Georgia Institute of Technology, 500 10th Street, Atlanta, Georgia 30332, and College of Light Industries and Food Engineering, GuangXi UniVeristy, 100 Daixue Road, Nanning, Guangxi, People’s Republic of China
This paper presents results on the features of bagasse pretreatment with peroxide and acetic acid (peroxide-HAc) aiming at selective removal of lignin to enhance enzymatic hydrolysis. Surface response methodology was employed to study the effects of major process parameters on delignification and to establish a model for the prediction of lignin removal in the process. Enzymatic hydrolysis following the pretreatment was conducted to evaluate the enhancement of biohydrolysis by the pretreatment. Results revealed that peroxide-HAc pretreatment of bagasse retained most of the carbohydrate constituents, although the delignification rate was low. Peroxide-HAc concentration, temperature, and time demonstrated significant effects on bagasse lignin removal. The relationship between lignin removal and the process parameters could be well described by a mathematical model derived from the experimental data. After treatment with 69.1% peroxide-HAc at 80 °C for 26.5 h, 97.08% of lignin could be removed while keeping 68.24% hemicelluloses intact. Over 93.58% of carbohydrate in treated bagasse could be hydrolyzed with exoglucanase in a dosage of 138 FPU/g carbohydrates at 35 °C within 48 h. It was thus demonstrated that treated bagasse has a much higher response toward enzymatic hydrolysis than its untreated counterpart. Introduction Bagasse is the residue after sugarcane juice is extracted. It is one of the major biomasses available for industrial uses.1 Among the various utilizations proposed for this renewable fibrous resource, generation of simple sugars from bagasse for fuel ethanol production attracts increasing research interest.2,3 Enzymatic hydrolysis can turn the cellulosic materials into simple sugars in an environmentally friendly way.4 However, because of the presence of lignin, raw bagasse is digested directly by enzymes with difficulty.5–7 Pretreatment to remove lignin thus becomes a crucial step for an effective enzymatic hydrolysis.8 Various pretreatment procedures have been investigated for lignin removal to enhance hydrolysis. Among those, pretreatment with peracetic acid has received intensive study.9 This process is not only considered to be environmentally friendly, but it is also very effective.10,11 Peracetic acid can oxidative cleave the aromatic ring in lignin to produce carboxylic acids and their lactones.9 The oxidation begins in the phenolic moieties either with hydroxylation of the available ortho or para positions, or with displacement of the para-substituted group. The orthohydroxylation leads to the formation of catechols, which are first converted to ortho-quinones and then undergo ring cleavage to form muconic acids. The para displacement generates hydroquinones, which are further oxidized to para-quinones following by the formation of maleic and fumaric acids.9,12–14 Nonphenolic lignin units are reportedly stable toward peracetic acid.15 However, if such units contain β-aryl ether linkage, they will be eventually degraded because peracetic acid can induce the cleavage of the linkage resulting in a new phenolic moiety.16 It is also found that nonphenolic lignin units can be degraded through the newly formed phenolic functionality arising from demethoxylation.13,16 Therefore, for etherified guaiacyl lignin * To whom correspondence should be addressed. Tel.: (404) 8945306. Fax: (404) 894-4778. † Georgia Institute of Technology. ‡ GuangXi Univeristy.
units, the methoxyl group in the C3 position of the aromatic ring can split off to form a new phenolic functionality, which guides the hydroxylation at the C6 position. Further reactions of the hydroxylated product will give the para-quinone products and ultimately lead to ring rupture. Etherified syringyl lignin units are found to be degraded in a similar way but with a higher reactivity.17 Bagasse lignin consists of guaiacyl, syringyl, and coumaric basic units, and it is expected to degrade in peracetic acid medium smoothly. Teixeira et al.2 pretreated bagasse with peracetic acid in order to facilitate the following enzymatic hydrolysis. After mixing with peracetic acid solution in a liquor to a materials ratio of 8:1 (by weight), bagasse was stored at room temperature for 7 days. The treated bagasse was found to be hydrolyzed by enzymes by over 90%. Zhao et al.18 further investigated the parameters that affected the bagasse lignin degradation in the pretreatment with peracetic acid under the catalysis of sulfuric acid. It was found that the peracetic acid charge, ratio of liquor to bagasse mass, temperature, and time had significant effects on lignin removal and on the following enzymatic hydrolysis efficiency. According to the analysis of variances (ANOVA), the authors claimed that there were strong interactions between the peracetic acid charge and the liquor volume, treatment time, and treatment temperature. As new bio-based technologies emerge, fermentation to produce ethanol no longer limits the uses of C6-glucose but also includes C5-xylose.19 Since lignin and its analogues in bagasse are the constituents that affect enzymatic hydrolysis, pretreatment should be conducted in a selective way to remove these problematic substances only. Although peracetic acid treatment with strong mineral acid as catalyst can remove lignin efficiently, the mineral acid also catalyzes the hydrolysis of carbohydrates, especially the hemicellulose constituents.18 Since bagasse is rich in hemicelluloses, the removal of this fraction of materials leads to substantial lost of C5-sugars. An equal volume mixture of hydrogen peroxide and acetic acid (peroxide-HAc) has long been recognized as an effective reagent for lignin removal to isolate plant fibers20 and for use
10.1021/ie901529q 2010 American Chemical Society Published on Web 12/30/2009
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in the new pulping process.21 Because of the absence of strong mineral acid, the procedure is more selective in terms of lignin removal. Moreover, the procedure is simple. Acetic acid and peroxide can be directly mixed to form the reagent without any preparation. This paper describes the use of peroxide-HAc as an active reagent to remove bagasse lignin following an enzymatic hydrolysis. Box-Behnken surface response methodology is used to obtain a quadratic mathematic model to describe the relationship between lignin removal and the major process variables, namely peroxide-HAc concentration, pretreatment temperature, and time. Experimental Section Materials and Chemicals. Sugarcane bagasse was provided by a local sugarcane mill in Guangxi, China. The material was ground to pass a 40 mesh screen and extracted with refluxing acetone for 4 h. The extractives-free bagasse was stored at 5 °C until use. Hydrogen peroxide solution (30%) and glacial acetic acid were purchased from Sigma-Aldrich. Sugar standards and authentic compounds for degraded lignin products evaluations were purchased from Sigma-Aldrich and VWR International. MSTFA (N-methyl-N- trimethylsilyltrifluoroacetamide) was purchased from Pierce Chemical Co. The enzyme used for the hydrolysis was a crude extract containing mainly exoglucanase from Trichoderma longibrachiatum. It is commercially available from Nalco Co. in the United States under the brand name Pergalase A40. The received enzyme was tested and was found to have an activity of 15 FPU/mL. It was stored at 5 °C until use. Pretreatment. A liquor to material ratio of 8:1 (by weight) was used for all the experiments. Equal volumes (50/50) of peroxide solution (30%) and glacial acetic acid were mixed to form peroxide-HAc solution. For the treatment with sulfuric acid as catalyst, 0.5% (w/w) sulfuric acid (98% concentration) was added to this peroxide-HAc solution. Peroxide-HAc concentration in the treatment liquor was determined by the weight percentage of this solution in the treatment liquor. Therefore, 5 g of extracted bagasse was weighed into a 50 mL digestion tube. After 40 mL of pretreatment solution containing the desired concentration of peroxide-HAc was added, the tube was shaken to ensure a homogeneous mixing of the contents. The tube was then placed in the preset water bath to begin the pretreatment. The tube was shaken occasionally during the process. After the treatment, the content was filtered through No. 4 Whatman filter paper, and the residues were washed until neutral. The washed bagasse was analyzed for lignin content and sugar compositions (if applied). Lignin Measurement. The lignin content of the materials was measured as acid-insoluble lignin in a modified Tappi method T222 (acid-insoluble lignin measurement method)22 to accommodate a small scale of sample size. In brief, 0.18 g (oven-dry, o.d.) of treated bagasse was dissolved in 1.5 mL of 72% sulfuric acid (specific gravity at 25 °C is 1.6338). The content was mixed carefully by using a glass bar until it became thick past matter. The tube was then transferred to a multiplehold heater that was set at 30 °C for 1 h, and this was followed by another 1 h of hydrolysis at 121 °C after the desired amount of water was added to adjust the acid content to 3%. Acidinsoluble lignin was determined from the solid residues and the hydrolysate was used to determine the sugars composition (if applied). Acid-insoluble lignin data determined from this procedure is comparable with that from Tappi method T222. Due to the interference of acid hydrolysis byproduct in the
hydrolysate, the acid-soluble lignin content of the cellulosic materials was not determined. Enzymatic Hydrolysis. A 0.55 g sample of treated bagasse (o.d. weight) was weighed into a flask followed by the addition of 100 mL of distilled water. After the bagasse was soaked for 2 h at room temperature, 5 mL of Pergalase A40 was added into the suspension. The pH of the initial mixture was 6.7, and it was not controlled and monitored in the process. The flask was set in a shaking water bath at 35 °C. In a desired interval, a 2 mL aliquot was taken and forced to pass a 0.25 µm polyethersulfone membrane cartridge from Whatman Inc. The aqueous samples were analyzed for sugar content as outlined below. Simple Sugar Analysis. The contents of individual simple sugars, namely arabinose, galactose, glucose, xylose, and mannose, were determined by using a Dionex LC20 system equipped with an ED40 electrochemical detector. The hemicelluloses content was determined as the sum of arabinose, galactose, xylose, and mannose after multiplying the pentoses with 0.88 and the hexoses with 0.90 to compensate for the water addition during the hydrolysis of carbohydrates. The column used for sugar separation was a Dionex CarboPac PA10. During the analysis, 20 mM NaOH solution was first applied for 10 min to condition the column, and the system was equilibrated for 10 min by applying 2 mM NaOH solution prior to the sample injection. All the simple sugars were separated and eluted out of the column by using 2 mM NaOH solution in 36 min. Lignin Degradation Product Analysis. A 5 mL volume of spent pretreatment liquor was extracted using three separate 10 mL aliquots of dichloromethane. The solvent extract was dried over anhydride sodium sulfate overnight. The solid was then filtered off and washed with 5 mL of fresh dichloromethane three times. The combined solvent extract was evaporated by using a rotary vacuum evaporator. The residues were dissolved in dichloromethane and quantitatively transferred to a 5 mL volumetric flask. A 200 µL volume of this sample solution and 5 µL of heptadecanoic acid internal standard (4.0 mg/mL) were measured into a 1 mL vial. The solvent was removed under a stream of nitrogen at 70 °C. The residues were derivatized with 50 µL of MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide) at 30 °C for 30 min. A 1 µL volume of the derivatized mixture was injected into a GC/MS for analysis. The column was a DB5 60 m capillary column. The GC was programmed with the following conditions: initial temperature 150 °C; initial time 5 min; rate 20 °C/min; final temperature 280 °C; inject port temperature 250 °C. Identifications of lignin degradation products were based on an MS spectrum library (Palisade Complete Mass Spectra Library, 600K Edition). Quantification of an individual compound was done using the peak areas ratio of the compound to the internal standard, and the response factor derived from the injection of a known amount of authentic compound. Experimental Design. Initial experiments have found that lignin removal with peroxide-HAc pretreatment was affected by the peroxide-HAc concentration, temperature, and time. The effects of the variables on lignin removal were not linear. In order to establish the relationship between lignin removal and independent variables with a minimum number of experiments, Box-Behnken surface response design23 was employed to set up teh experiment plan. Data were used to construct the secondorder polynomial mathematic model with the following form:
Ind. Eng. Chem. Res., Vol. 49, No. 4, 2010 n
lignin removal ) A0 +
∑BX
i i
i)1
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n
+
∑CX
2
i i
+
i)1
n
∑
DijXiXj (i < j)
i)1,j)1
where lignin removal denotes a dependent variable and Xi represent coded or natural variables. The second term in the equation represents the linear influence of the variables, while the third and fourth terms reflect the quadratic and interaction effects of the variables, respectively. A0, Bi, Ci, and Dij are unknown constants, and they will be determined from the experimental data. The independent variables were coded by using the equation Xni )
xni - xn0 (xmax - xmin)/2
where Xni is a coded value for number n variable at i level (i ) -1, 0, 1); xni is the natural value of the n variable at i level. xn0 is the natural value of the n variable at the middle level (0 level). xmax is the natural value of the n variable at high level (1 level), whereas xmin is the natural value of the n variable at low level (-1 level). Design Expert software trial version 6.0.10 (Stat-Ease, Minneapolis, MN) was used for the regression analysis and for the determination of optimal pretreatment conditions. Both the significances of the regression coefficients and the model were checked using an F test, and the quality of fit of the model was expressed by the coefficient of determination, R2. Results and Discussion Features of Peroxide-HAc Pretreatment. A. Delignification. Figure 1 demonstrates the increase in lignin removal of treated bagasse with increasing concentration of peroxideHAc. Lignin removal steadily increases to over 95% after 24 h reaction as the concentration of the reagents increases up to 65%. Further increase in reagent concentration does not improve lignin removal significantly. Table 1 displays the treatment of bagasse in peroxide-HAc medium with and without sulfuric acid. The yield and lignin content of treated bagasse decreased with an increasing treatment time, indicating the removal of lignin and carbohydrates. The delignification rate in the treatment without sulfuric acid is substantially low compared to that in the pretreatment with sulfuric acid as catalyst. As indicated, after 3 h treatment without sulfuric acid, bagasse lignin was only removed by around 20%. However, when sulfuric acid was added as catalyst, lignin removal reached almost 97%. Sun et al.24 reported a similar result in the pretreatment of maize stems with peracetic acid without the addition of sulfuric acid. After treatment at 50 °C for 6 h, lignin removal was 33.3%. However, with the addition of sulfuric acid as catalyst, Zhao et al.18 recently reported over 90% bagasse lignin removal in 3 h. The high delignification rate in the presence of a strong mineral acid is attributed to the high concentration of H+, as indicated by the kinetic equation13 rate ) K2[H2O2][nucleophile-] + K3[H2O2][nucleophile][H+] K3 in the equation is believed to be about 2 orders of magnitude larger than K2. Therefore, a high H+ concentration can substantially increase the delignification rate. Although the conversion rate is low, a simple mixing of acetic acid and peroxide was found to generate peracetic acid.10
Figure 1. Increase in lignin removal as a function of peroxide-HAc content in treatment liquid. Pretreatment temperature 60 °C; time 24 h. Lignin removal, %, was calculated by the formula 100[(lignin in raw bagasse lignin in treated bagasse)/lignin in raw bagasse].
Consequently, it is expected that lignin degradation in a peroxide-HAc medium follows a similar pathway in peracetic acid delignification. Table 2 lists a series of lignin degradation products extracted from the pretreatment spent liquid. The identifications of p-quinone, 3-methoxyl-p-quinone, and 3,5dimethoxyl-p-quinone clearly demonstrate the occurrence of side chain displacement and hydroxylation. According to Gierer,25 the heterolytic cleavage of peracetic acid in acidic medium can generate hydroxonium ion, HO +, which is a strong electrophilic species. A series of reactions can occur between the hydroxonium ion and lignin, including the displacement of side chains. Working with lignin model compounds, Nimz et al.26 demonstrated that guaiacyl and syringal lignin units were mostly transformed into quinones by reactions with peracetic acid. The identification of quinone products in peroxide-HAc pretreatment spent liquid indicates the involvement of peracetic acid in the process. Among others, aromatic aldehydes including vanillin are common lignin degradation products. However, no aldehydes but aromatic carboxylic acids are found in the spent liquor. This is probably due to the Baeyer-Villiger oxidation that converts the aldehyde intermediates into the corresponding carboxylic acid products.27 This phenomenon has been confirmed in the studies using model compounds.26,28 Table 2 also indicates that there are still free p-hydroxycinnamic acid and ferulic acid in the spent liquid. Due to the presence of unsaturated side chains, the two compounds are expected to be degraded quickly. The detection of the two compounds suggests that lignin degradation under the experimental conditions is very mild. This observation is in line with the reported results from Sun et al.24 After the treatment of fibrous materials with peracetic acid, the authors were able to recover the dissolved lignin by pH adjustment of the spent liquor. Nitrobenzene oxidation of the reclaimed lignin yielded over 29% products, indicating that little modification occurred to the dissolved lignin during this type of pretreatment. B. Carbohydrate Degradation. In spite of the low delignification rate, pretreatment without mineral acid as catalyst has a high selectivity. As indicated in Table 1, over 71% hemicelluloses are lost in sulfuric acid catalyzed pretreatment when 97% lignin is removed. As a comparison, this loss is only 34% in the treatment without sulfuric acid while a similar percentage of lignin is removed. It has been reported that bagasse hemicelluloses dominate with L-arabino-(4-O-methyl-D-glu curono)-D-xylan.29,30 The molar ratio of the arabinose unit to the xylose unit can range from 0.019 to 0.247.31 Data shown in Table 2 are generally in line with the reported values. Hemicelluloses are believed to bind chemically with both cellulose
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Table 1. Change in Bagasse Composition after Pretreatment for Various Times sugar composition,a % time, h
yield, %
lignin, %
arabinose
galactose
glucose
xylose
mannose, %
hemicellulose,b %
49.72
25.96
0.26
25.84
26.00 26.13 26.03 26.67 27.43 27.35 26.86 26.73
0.23 0.26 0.19 0.27 0.24 0.22 0.28 0.26
25.37 25.26 25.02 25.73 25.86 25.42 25.17 24.65
0.25 0.22 0.21 0.25 0.26 0.39 0.39
24.94 23.62 23.53 23.69 22.88 17.33 14.03
Raw Bagasse 0.0
23.80
2.21
0.61
Peroxide-HAc Treatmentc 0.5 1.0 3.0 7.0 10.0 15.0 22.0 24.0
97.83 96.45 94.61 89.25 85.36 81.02 76.35 68.47
22.39 21.07 20.15 18.35 16.26 13.87 4.32 0.81
2.02 1.90 1.62 1.72 1.11 0.65 0.98 0.34
0.57 0.40 0.58 0.56 0.58 0.64 0.47 0.66
49.55 49.03 50.11 52.24 54.13 56.92 67.07 72.56
Peroxide-HAc Treatment with Sulfuric Acidd 0.2 0.5 1.2 1.5 2.2 2.7 3.0
97.83 88.67 78.98 75.46 68.75 58.96 52.25
21.99 19.12 16.76 15.35 10.87 2.79 1.27
2.05 1.83 1.67 1.03 0.76 0.22 0.11
0.60 0.60 0.54 0.30 0.23 0.28 0.00
49.49 53.65 55.08 57.09 62.12 76.85 82.29
25.41 24.17 24.30 25.32 24.74 18.80 15.43
a Sugar compositions are percentage of hydrolyzed fibrous material. b Hemicellulose was determined from arabinose, galactose, xylose, and mannose by multiplying pentose with 0.88 and hexose with 0.9 to compensate for the water addition during hydrolysis of polymer to simple sugars. c Experiments were conducted at 80 °C using 70% peroxide-HAc in various time frames from 0.5 to 24 h. d Peroxide-HAc contained 0.5% (w/w) sulfuric acid as catalyst in various time frames from 0.2 to 3 h. Other conditions were the same as in footnotec.
Table 2. GC/MS Identified Lignin Degradation Products degradation product
yield,a %
p-quinone 4-hydroxybenzoic acid 3-methoxyl-p-quinoneb 4-hydroxyl-3-methoxyl benzoic acid 3,5-dimethoxyl-p-quinoneb catechol 4-hydroxy-3,5-dimethoxyl benzoic acid p-hydroxycinnamic acid ferulic acid
0.17 0.11 0.07 0.15 0.02 0.03 0.07 0.14 0.05
a Based on lignin. b Use the same GC response factor of p-quinone for the content calculation.
Figure 2. Xylose reduction as a function of treatment time. Pretreatment temperature 80 °C; peroxide-HAc concentration 70%. Treatment liquor contained 0.5% sulfuric acid.
and lignin in the cell wall.29 Xylan, the major hemicelluloses component, can be categorized into two fractions: an easy hydrolysis fraction and the difficult hydrolysis fraction.31 Figure 2 indicates that the xylose content of the bagasse during sulfuric acid containing pretreatment undergoes three stage reductions. In the first 0.5 h when only 29% lignin is removed, there is about 17% reduction in xylose content. After this stage, xylose reduction slows down but continues until around 2 h pretreatment. In this second stage, there is another 17% xylose is lost.
Table 3. Independent Variables of the Process and Their Corresponding Levels symbols independent variables a
peroxide-HAc, % temperature, °C time, h
levels
coded
uncoded
-1
0
+1
X1 X2 X3
x1 x2 x3
50 60 20
60 70 24
70 80 28
a Percentage (by weight) of mixed solution containing equal volume (50/50) of glacial acetic acid and 30% hydrogen peroxide in the pretreatment solution.
In the third stage xylose reduction accelerates again when 68% lignin is removed. In this stage, 35% xylan is lost. The first rapid reduction phase in xylose may be attributed to the loss of the easy hydrolysis portion of xylan. Following this, the difficult hydrolysis fraction of xylan begins to dissolve. After a significant portion of lignin is removed, the binding of xylan to lignin and cellulose may have been cleaved. This leads to another stage of rapid decomposition of xylan. When no sulfuric acid is used, although the loss in xylan is much slower, the hydrolysis of carbohydrates speeds up after a significant amount of lignin is removed. For the traditional fermentation process generating ethanol from glucose, it may be desirable to remove hemicelluloses as much as possible through the pretreatments. However, as the novel fermentation process is developed, both C5- and C6-sugars can be used to generate fuel ethanol. The use of peroxide-HAc for bagasse lignin removal thus results in a substantial increase in fuel ethanol production. Effects of Pretreatment Variables. With the help of experimental design software, a Box-Behnken surface response experiment plan with three factor three levels is set up and presented in Table 3. Lignin removal of the treated bagasse is chosen as the response. Table 4 lists experimental results, from which ANOVA data were generated after the analysis using the Design Expert software, and the data are listed in Table 5. As indicated, all three process parameters are significant on the first-order level. Temperature and time are also significant on the second-order level. As to the interactions between
Ind. Eng. Chem. Res., Vol. 49, No. 4, 2010 Table 4. Experimental Design Showing Randomized Run Order, Combinations of Variable Levels, and the Response run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 validation 1 validation 2 validation 3
peroxide-HAc X1
temperature X2
time X3
response lignin removal, %
-1 0 -1 1 0 0 0 0 1 0 1 -1 0 -1 1
0 1 0 0 0 1 0 -1 -1 -1 0 1 0 -1 1
-1 -1 1 1 0 1 0 1 0 -1 -1 0 0 0 0
87.86 93.32 93.82 95.17 93.91 97.56 94.75 91.51 92.4 86.51 93.32 94.50 94.03 88.07 97.02 96.51 97.68 97.05
parameters, only the one between peroxide-HAc concentration and treatment time shows significant. In order to express as much as possible of the variations by the model, all the terms are included in the model regardless of their significance. As such, the model expressing the lignin removal as a function of pretreatment variables can be established in the form of coded variables as lignin removal (%) ) 94.23 + 1.71X1 + 2.99X2 + 2.13X3 0.45X1X2 - 1.03X1X3 - 0.19X2X3 - 0.46X12 - 0.77X22 1.23X32 where X1, X2, and X3 are coded peroxide-HAc concentration, temperature, and time, respectively. After the variables are converted to their natural forms, the model becomes lignin removal (%) ) -112.424 + 1.653x1 + 1.769x2 + 6.100x3 + 0.005x1x2 - 0.026x1x3 - 0.005x2x3 - 0.005x12 0.008x22 - 0.008x32 where x1, x2, and x3 are the natural forms of peroxide-HAc concentration, temperature, and time, and their units are %, °C, and h, respectively. The Fisher variance ratio (F value) of the model indicated in Table 5 is much higher than the tabular value (74.19 vs 3.44) at the 5% level. Since the F value is the ratio of the mean square of the regression to the mean square of the residue, it reflects how well the model expresses the observed variations. A high ratio is an indicator of a good prediction model. R2 and adjusted R2 of the regression are found to be 0.98 and 0.96, respectively. It confirms that the established model is a good fit to the experiment data. Examination of the internally studentized residuals indicates that none of them is above 2. As this residue should be approximately normal with mean zero and unit variance, a value in the range -3 to +3 is acceptable.32 It follows that the experiment data have no outliers, and the model established from the data set is a good predictor in the range of study. Table 5 also indicates that the lack of fit of the model is insignificant. It suggests that the established model is adequate for the prediction. Figures 3- 5 show the various three-dimensional plots of the response surface model. As indicated in Figure 3, at a constant pretreatment time, lignin removal of treated bagasse increases steadily with the increases in both peroxide-HAc
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concentration and treatment temperature. However, lignin removal is more profound by elevating temperature than by increasing in concentration of peroxide-HAc. Working on bagasse pulping with organic acids and peroxide, Kham et al.10 also observed the strong effect of temperature on deligninfication, and the authors suggested that the formation of radicals under elevated temperature had made this factor to be a dominant variable. As discussed in the previous section, displacement of lignin side chain involving radical reaction was probably occurring in peroxide-HAc pretreatment. The impact of the temperature variable on lignin removal further supports this hypothesis. The dominant effect of temperature on delignification is also shown in Figure 5, where peroxide-HAc is kept constant at the 60% level with changing temperature and treatment time. The interactions between variables are not very strong. Among these, only the interaction between peroxide-HAc and time is significant. As indicated in Figure 4, at 70 °C pretreatment temperature, lignin removal reaches a maximum as the treatment time is increased at high peroxide-HAc concentration. This observation may be attributed to the rate increase in carbohydrate decomposition after a certain level of lignin is removed. It is well-known that lignin concentrates in middle lamella of plant fiber cells. As lignin is dissolved, more carbohydrate constituents will be directly exposed to the peroxide-HAc medium. Since the carbohydrate constituents are prone to attack by acid, the removal of lignin opening up the path for the acid will eventually increase the hydrolysis rate of carbohydrates. Additionally, the removal of lignin probably leads to the cleavage of the bonding between lignin and carbohydrates. This will further increase the loss in hemicelluloses. When more carbohydrates than lignin are dissolved, the content of residual lignin in the treated bagasse will become high. This is actually in agreement with the results shown in Table 1, where hemicellulose removal speeds up when lignin is removed beyond a certain level. In order to allow a better enzymatic hydrolysis of bagasse, a low lignin content in bagasse is desirable. To obtain lignin removal around 95%, the optimum conditions suggested by the model are as follows: 69.1% peroxide-HAc concentration, 80 °C pretreatment temperature, and 26.5 h pretreatment time. Triplicate runs of validation with the optimum conditions were carried out, and results are shown in Table 4. It indicates that an average of 97.08% lignin removal in the treated bagasse is accomplished with a standard deviation of 0.6. This validates the model in the prediction of lignin removal during peroxide-HAc pretreatment of bagasse. Enzymatic Hydrolysis of Pretreated Bagasse. Treated bagasse from the triplicate runs of pretreatment was mixed together. The lignin content of the resulted bagasse was 0.92%, while 68.24% of the original hemicelluloses was retained. The treated bagasse was hydrolyzed by using 138 FPU of Pergalase A40 for each gram of carbohydrates, as specified in the Experimental Section. Raw bagasse was also hydrolyzed in the same way, except that the enzyme dosage was increased to 179 FPU/g carbohydrates. The released sugars (sum of arabinose, galactose, xylose, glucose, and mannose) in the first 12 h period are shown in Figure 6. It is evident that bagasse after the pretreatment hydrolyzes much faster than its untreated counterpart even though a low dosage of enzyme was used. In the first hour, the sugar concentration in the hydrolysate reached 1.011 g/L, corresponding to 18.38% hydrolysis of starting material. In contrast to the treated bagasse, the raw bagasse was only hydrolyzed by 2.82% to yield a mixture with 0.149 g/L
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Table 5. ANOVA of the Quadratic Model for Degradation of Bagasse Lignin in Peroxide-HAc Medium
a
source
sum of squares
df
model X1 (peroxide-HAc) X2 (temperature) X3 (time) X1X2 X1X3 X2X3 X12 X22 X32 residual lack of fit pure error corrected total intercept
143.98 23.32 71.46 36.34 0.82 4.22 0.14 0.77 2.22 5.59 1.08 0.67 0.41 145.06
9 1 1 1 1 1 1 1 1 1 5 3 2 14
coefficient
mean square
F value
p-value prob > F
16.00 23.32 71.46 36.34 0.82 4.22 0.14 0.77 2.22 5.59 0.22 0.22 0.21
74.19 108.17 331.41 168.52 3.80 19.59 0.67 3.58 10.28 25.91
