Bioresource Technology 136 (2013) 288294

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Bioresource Technology
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Association of wet disk milling and ozonolysis as pretreatment for enzymatic saccharication of sugarcane bagasse and straw
Rodrigo da Rocha Olivieri de Barros a, Raquel de Sousa Paredes a, Takashi Endo b, Elba Pinto da Silva Bon a,, Seung-Hwan Lee b,c,
a b c

Enzyme Technology Laboratory, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil Biomass Renery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagami-Yama, Higashi Hiroshima, Hiroshima 739-0046, Japan Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea

h i g h l i g h t s
 Association of ozonolysis and WDM for the pretreatment of sugarcane baggasse and straw.  Ozonolysis after WDM resulted in higher saccharication yield than WDM after ozonolysis.  Energy consumption was lowest in ozonolysis after WDM.  Saccharication yield was affected by morphology and specic surface area.

a r t i c l e

i n f o

a b s t r a c t
Ozonolysis was studied separately and in combination with wet disk milling (WDM) for the pretreatment of sugarcane bagasse and straw, with the aim of improving their enzymatic saccharication. The glucose yields for ozonolysis followed by WDM were 89.7% for bagasse and 63.1% for straw, whereas the use of WDM followed by ozonolysis resulted in glucose yields of 81.1% for bagasse and 92.4% for straw, with shorter WDM time. This last procedure allowed a substantial decrease in energy consumption in comparison to the use of WDM alone or of ozonolysis followed by WDM. Higher overall saccharication yields with shorter milling times were observed when ozonolysis was carried out before WDM. This effect might be related to the higher specic surface area. Additionally, a ner morphology was observed by the association of the two treatments in comparison to the sole use of ozonolysis or WDM. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 26 December 2012 Received in revised form 26 February 2013 Accepted 1 March 2013 Available online 14 March 2013 Keywords: Sugarcane bagasse Sugarcane straw Ozone treatment Wet disk milling Enzymatic saccharication

1. Introduction Signicant effort has been exerted globally to meet the demand for fuels and chemicals and gradually replace fossil fuels with those obtainable from renewable resources such as biomass. From this perspective, fuels and chemicals can be produced from lignocellulosic materials, which are the most abundant renewable resources on earth (Koukiekolo et al., 2005). However, the availability and selection of biomass feedstock, the development of economically feasible pretreatment technologies, as well as the cost and availability of the necessary enzyme pools for efcient saccharication, are still under development for the commercialization of these new products.
Corresponding author at: Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea. Tel.: +55 82 10 7174 6240. E-mail addresses: elba1996@gmail.com (E.P.S. Bon), lshyhk@kangwon.ac.kr (S.-H. Lee).
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.03.009

Biorenery offers the possibility of different products that can be obtained from the enzymatic hydrolysates of lignocellulosic biomass, including bioethanol, butanol, biohydrogen, oil, and methane. The production chain starts with biomass pretreatment followed by enzymatic saccharication to produce the hydrolysates; the last step usually involves fermentation processes or anaerobic digestion (Cheng et al., 2011; Triolo et al., 2011; Guo et al., 2013; Huang et al., 2012) Sugarcane bagasse and straw are high-potential feedstock for second-generation (2G) bioethanol production in Brazil because of their abundance in the Brazilian sugar mills. Brazil has also acquired signicant ethanol production expertise since the 1970s, after the implementation of the Prolcool program. Nevertheless, the full use of these raw materials in 2G ethanol production is limited, because most of the sugarcane residues are used as energy sources via combustion in Brazilian sugar mill operations (Cardona et al., 2010; Cerqueira Leite et al., 2009; Pandey et al., 2000). The development of a suitable pretreatment coupled to the relevant parameters for the enzymatic saccharication is of foremost

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importance. Pretreatment, which is a necessary step to reduce the natural recalcitrance of lignocellulosic materials, is estimated to be responsible for up to 20% of the total production cost. Moreover, pretreatment has a pervasive impact on all other major operation steps in the overall biomass conversion process, from the choice of feedstock through size reduction, enzymatic hydrolysis, and fermentation, as well as product recovery, residue processing, and coproducts potential (Yang and Wyman, 2008). Ozone, which is a powerful oxidant, has been widely used for pulp bleaching in the paper industry (Roncero et al., 2003; Shatalov and Pereira, 2008). The diversity of ozone applications has substantially increased over the last two decades, nding use, for example, in the treatment of ground and industrial wastewaters (Amat et al., 2005; Coca et al., 2005). Ozonolysis for biomass pretreatment has also shown its utility because of its delignication efciency. It can also cause a slight degradation of the hemicellulose component of the lignocellulosic biomass (Quesada et al., 1999; Sun and Cheng, 2002). Ozone is highly reactive toward compounds with conjugated double bonds and functional groups with high electron densities. Therefore, lignin is the moiety most likely to be oxidized in the ozonolysis of lignocellulosic materials because of its high C@C bond content. Ozone attacks biomass components, releasing soluble low molecular weight compounds, with a preponderance of organic acids such as formic and acetic acids, which can result in a severe pH drop. However, this process is advantageous because the aforementioned degradation products do not interfere with the subsequent enzymatic hydrolysis and fermentation steps. Additionally, the low energy consumption of ozonolysis is attractive, because it takes place at room temperature (Contreras Iglesias, 2002). Indeed, ozonolysis has been known as an effective pretreatment method for various lignocellulosic materials, degrading mainly lignin, for example, in wheat and cotton straw (BenGhedalia and Miron, 1981; Ben-Ghedalia et al., 1983), bagasse, green hay, peanut, pine (Neely, 1984), and poplar sawdust (Vidal and Molinier, 1988). Mechanical pretreatments of biomass aim primarily to increase the surface area by reducing the feedstock particle size, combined with brillation or reduction in the crystallinity degree. This approach facilitates the accessibility of enzymes to the substrate, increasing saccharication rates and yields. Usually, milling processes are very energy intensive, depending on the material characteristics and the target particle size (Laser et al., 2002). WDM is a recently introduced biomass pretreatment process able to produce milled biomass with low energy consumption when compared to conventional ball milling treatment. This technique has been shown to increase the degree of biomass brillation and the space between the microbrils, thus promoting the accessibility of the cellulolytic enzyme pool to cellulose (Hideno et al., 2009). The disk mill is a type of crusher that can be used to grind, cut, shear, berize, pulverize, granulate, or blend. In general, the suspended material is fed between opposing disks or plates that can be grooved, serrated, or spiked. For biomass processing using WDM, a water suspension (15% of solids) of the lignocellulosic material is passed between two ceramic nonporous disks that are separated by a distance of 20100 lm and that have a rotational speed of around 1800 rpm. This process can be repeated according to the required number of WDM cycles; very small particle sizes with high specic surface areas (SSAs) have been observed after a minimum of ve cycles (Endo et al., 2008; da Silva et al., 2010). In this study, ozonolysis was evaluated in association with wet disk milling (WDM) to improve sugarcane bagasse and straw enzymatic saccharication and reduce energy consumption because of sum of benets of each individual treatment. It was reasonable to expect that the loosening of the cell wall structure by delignication via ozonolysis would improve the biomass brillation upon

WDM, reduce the milling time, and also, increase the SSA. Even though mechanical treatment is an environment-friendly process, because it does not use chemicals such as acids or alkalis, it is an energy-intensive process (Hendriks and Zeeman, 2009). Thus, it needs to be used in combination with other treatments to save energy and reduce costs. In previous studies from our laboratory, WDM treatment favored the brillation of some materials such as rice straw and sugarcane bagasse (Hideno et al., 2009; da Silva et al., 2010). Moreover, hot-compressed water (HCW) in the presence or absence of alkali catalyst was studied, aiming to improve mechanical brillation by WDM (Miura et al. 2012). HCW treatment loosens the structure of lignocellulosic materials mostly via hemicellulose removal and structural changes of the lignin, resulting in the improvement of WDM performance. The brillated product so obtained showed high cellulose surface area and improved enzymatic saccharication (Lee et al., 2010; Miura et al., 2012). 2. Methods 2.1. Materials Sugarcane bagasse and straw were kindly supplied by Complexo Bioenergtico Itarum S.A (Estate of Gois, Brazil). The materials were dried at room temperature, coarsely cut using a cutter mill, and sieved to select particles less than 2 mm in size. Enzymatic hydrolysis was performed using Acremonium cellulase (Meiji Seika Co, Japan) and Optimash BG with -xylosidase activity (Genencor International, USA). Other chemicals were purchased from commercial sources. 2.2. Chemical composition of sugarcane bagasse and straw The water content of the milled sugarcane bagasse and straw was determined using a halogen moisture analyzer (HG63, Mettler-Toledo AG Co., Greifensee, Switzerland). Cellulose, hemicellulose, and lignin contents were measured according to Sluiter, 2008, with minor changes. The dried samples (30 mg) were hydrolyzed with 72% sulfuric acid (0.3 mL) at 30 C for 1 h, followed by dilution of the acid to 4% by adding distilled water (8.4 mL) and incubation in an autoclave at 121 C for 1 h. The treated sample was cooled and vacuum ltered through a glass ber lter (Whatman F/A 47 mm, Whatman International Ltd., England, UK). The soluble fraction was neutralized with calcium carbonate and analyzed using a high-performance liquid chromatography (HPLC) system (JASCO, Tokyo, Japan), equipped with an Aminex HPX-87P column at 80 C with a ow rate of 1.0 mL H2O/min, to determine the concentration of the monomeric sugars derived from the cellulose and hemicellulose fractions. Lignin content was determined by gravimetric analysis of the residue from the biomass acid hydrolysis. Ash content was determined by burning 1 g of sample in a tarred crucible for 6 h using a mufe furnace at 600 C until a constant weight was obtained (Sluiter, 2011). Other biomass components were estimated by subtracting the amounts of glucan, xylan, arabinan, lignin, and ash from the initial biomass dry weight. The raw materials normalized compositions are shown in Table 1. Values represent means of independent triplicates and standard deviations are shown parenthetically. 2.3. Ozonolysis treatment An ozone generator, model ED-OG-R5 (EcoDesign Inc., Japan), was fed with an oxygen ow of 0.5 L/min, resulting in an ozone concentration of 204 g/m3 inside the treatment vessel. Samples of bagasse and straw (2040 g), previously humidied to 60%

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Table 1 Chemical compositions of untreated sugarcane bagasse and straw. Chemical composition (%) Glucan Xylan Arabinan Galactan Lignin Ash Others Sugarcane bagasse 43.95 (1.85) 21.80 (0.68) 2.48 (0.16) 0.81 (0.07) 27.82 (0.71) 0.99 (0.05) 2.14 Sugarcane straw 40.80 (0.37) 21.68 (0.04) 3.60 (0.20) 0.72 (0.10) 24.79 (0.11) 4.92 (0.27) 3.49

were determined from the BrunauerEmmettTeller (BET) plot of a nitrogen-adsorption isotherm using a BELSORP-max (BEL Japan, Osaka, Japan) (Brunauer et al., 1938). Prior to both analyses, the samples were thoroughly washed with t-butyl alcohol and freeze-dried to preserve their morphological properties.

3. Results and discussion 3.1. Ozonolysis Table 2 shows normalized ozone consumption data for the ozonolysis of sugarcane bagasse and straw for treatment times from 30 to 120 min. It also shows the corresponding WSF solids content and the residual lignin content in the treated materials. Table 2 also summarizes the yields for the enzymatic saccharication of the cellulose and hemicelluloses of the pretreated sugarcane bagasse and straw in comparison to the yields of untreated materials. The stepwise increase in the ozone treatment time resulted in higher ozone consumption as well as biomass degradation, as shown by the increase in the amount of water-soluble solids. Sugarcane bagasse and straw responded differently with regard to ozone consumption, such that a lower consumption was observed for bagasse (0.187 g ozone/g biomass) in comparison to straw (0.213 g ozone/g biomass) for 60 min treatment. Nevertheless, the amount of watersoluble solids was higher for bagasse (27.60%) in comparison to that of straw (19.50%). Accordingly, the residual lignin was lower for bagasse (12.40%) in comparison to straw (15.39%). After 60 min exposure to ozone, glucose and xylose saccharication yields increased to 59.24% and 32.38% for bagasse and 46.95% and 27.84% for straw, respectively, in comparison to that for untreated materials. Because bagasse lignin was revealed to be more prone to ozonolysis than straw, bagasse saccharication yields were higher. Considering the saccharication time course, glucose yields did not signicantly increase after 60 min, while xylose yields decreased after 60 min for bagasse and after 90 min for straw. The harmful effect on xylose yield as a result of longer treatment times could be because of hemicellulose degradation by ozone. Nevertheless, ozone is mainly known to degrade lignin; hemicellulose and cellulose can be slightly affected (Quesada et al., 1999; Roncero et al., 2003; Shatalov and Pereira, 2008; Sun and Cheng, 2002). Binder et al. (1980) reported that, although delignication was benecial to the saccharication process, it was deleterious for lignin removal beyond 60%. Another study related to ozone treatment for lignocellulosic materials showed that enzymatic hydrolysis yields of up to 88.6% and 57% were obtained compared to 29% and 16% in nonozonated wheat and rye straws, respectively (Garca-Cubero et al., 2009). By analyzing the morphological structure of the ozone-treated samples, it was possible to identify a disturbed structure in treated bagasse, in addition to the presence of a nanoscopic brous morphology, which was less apparent for the ozone-treated straw (Complementary Fig. 1). However, these nanoscopic bers appeared only in some areas (maybe from primary cell wall) of the treated material, in other regions, the treated samples showed a type of morphology similar to the raw materials. Lignin content in primary cell wall is lower than in secondary wall, so that delignication by ozonolysis could easily release cellulose microbrills from primary cell wall. However, the amount of primary cell wall is smaller than the secondary cell wall, thus it is limited to affect to increase the specic surface area. This probably can directly compromise the SSA values and saccharication yields of glucose and xylose. Lignin removal data indicated that ozone was signicantly more effective toward bagasse delignication (15.42%) than for straw (9.40%). The substantial lignin removal also allowed the visualization of the biomass microbers, although some areas of the

Data are shown as mean values of three independent experiments and the standard deviations are shown parenthetically.

water content, were submitted to ozone treatment at 40 C for 30, 60, 90 and 120 min under agitation. The amount of ozone consumed by the biomass sample, for each experiment, was monitored through the measurement of the electrical current (voltage) using a UV ozone monitor (OZM 5000G OKITROTEC, Japan). A control experiment was carried out with the amount of water corresponding to 60% biomass moisture content, allowing the measurement of ozone consumption by water. Ozone consumption was determined by subtracting the residual ozone of the control experiment from that of each experiment and was expressed as the consumption rate per gram of dry biomass. After the treatment, the samples were washed with water (400800 mL). The solids content of the water soluble fraction (WSF), representing organic acids and soluble solids, was quantied by evaporation. 2.4. Wet disk milling treatment WDM was performed using a Supermasscolloider (MKZA62, Masuko Sangyo Co., Ltd., Saitama, Japan) equipped with two ceramic nonporous disks. The clearance of the two disks was adjusted to 2040 lm and the rotation speed was 1800 rpm. A water suspension of ozone-treated or ozone-untreated sugarcane bagasse or straw (60 g in 3 L) was fed to the equipment. Milling operation cycles in the range 15 for the ozone-treated or 17 for the ozoneuntreated material (control experiments) were carried out. WDM time for each milling cycle was calculated taking into account the weight of materials. The milling energy consumption for each milling cycle was calculated taking into account the data for voltage, current, and operation time. 2.5. Enzymatic hydrolysis and HPLC analysis Enzymatic saccharication was carried out using a treated or untreated biomass concentration of 2.5% (w/w) and an enzyme load of 15 FPU Acremonium cellulase per gram of biomass supplemented with 0.2% (v/v) Optimash BG as a source of hemicellulase, in acetate buffer (pH 5.0). Reactions were incubated under agitation at 50 C for 72 h. The sugar quantication was performed using a HPLC system (JASCO, Tokyo, Japan) following the same conditions as described in Section 2.2. Enzymatic hydrolysis yield calculations were based on glucan and xylan contents in the raw materials, taking into account the recovered mass of glucose and xylose and also the mass losses in the water soluble fractions after each biomass pretreatment. 2.6. Morphology and surface area The morphologies of the treated and untreated samples were observed by scanning electron microscopy (SEM) using an S-4800 SEM (Hitachi High Technologies Co., Tokyo, Japan). Tiny pieces of each sample were coated with a thin layer of osmium. The SSAs

R.R.O. Barros et al. / Bioresource Technology 136 (2013) 288294 Table 2 Ozone consumption, water-soluble fraction, lignin content, and saccharication yields after the ozone treatment of sugarcane bagasse and straw. Material Ozone treatment time (min.) Consumed ozone amount (g/g-biomass) Water soluble fraction amount (%) Liginin content in ozonetreated product (%) Saccharication yields (%) for 72 h. Glucose mg/g of biomass Bagasse 0 30 60 90 120 0 30 60 90 120 0.078 0.187 0.296 0.377 0.080 0.213 0.317 0.490 20.60 27.60 31.00 33.35 8.66 19.50 19.90 23.65 27.82 16.23 12.40 11.49 10.00 24.79 17.86 15.39 14.24 13.01 60.80 246.14 289.31 306.64 308.72 124.40 180.49 212.84 215.63 204.92 % (based on glucan) 12.45 50.40 59.24 62.79 63.22 27.44 39.81 46.95 47.57 45.20 Xylose mg/g of biomass 24.40 33.46 32.38 29.41 26.80 35.60 66.13 68.59 68.25 62.91

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% (based on xylan) 9.85 33.46 32.38 29.41 26.80 14.45 26.84 27.84 27.70 25.54

Straw

Complementary Fig. 1. Scanning electron microscopy micrograms of raw bagasse (a) and straw (e), ozone-treated bagasse (b) and straw (f) for 60 min, WDM (seven cycles)treated bagasse (c) and straw (g), and WDM followed by ozone-treated bagasse (d) and straw (h).

sugarcane bagasse and straw, because a sevenfold increase (from 2.414 to 18.016 m2/g) was measured for bagasse, and a 23-fold increase (from 1.571 to 36.285 m2/g) was measured for straw. These differences could be related to the nature of the different plant tissues of the sugarcane plant, as well as the prior exposure of the bagasse to the high processing pressures of sugarcane juice extraction. Because the consumption of large amounts of ozone makes the process expensive, in this work, ozone treatments for 30 and 60 min were selected to be further associated with WDM, with the aim to improve saccharication yields. 3.2. Wet disk milling treatment Fig. 2 shows the effect of WDM time (or cycle number) on glucose and xylose yields for WDM sugarcane bagasse and straw. Considering cellulose hydrolysis, seven cycles of milling increased the digestibility of bagasse by 50% (1.181 min/g) and of straw by 46% (1.241 min/g). However, because untreated straw was more prone to saccharication than bagasse, its nal conversion of 73.85% was higher than that of bagasse (62.25%). Considering the hydrolysis of hemicelluloses, the same number of cycles increased the digestibility of bagasse by 33% (1.181 min/g) and of straw by 39% (1.241 min/g). However, because untreated straw was more prone to saccharication than bagasse, its nal conversions of 73.85% and 53.42% were higher than those of bagasse, 62.25% and 42.63%, to glucose and xylose, respectively. Because milling cycles were

Fig. 1. Specic surface areas of raw and pretreated samples of sugarcane bagasse and straw.

treated materials retained characteristics similar to the untreated materials, presenting a smooth and aggregated surface. In this context, it is worth mentioning that the low xylose concentrations might be linked to xylan degradation, as mentioned. The use of 60 min ozonolysis alone resulted in a prominent SSA increase (Fig. 1) resulting from the generation of a porous structure by the removal of lignin. However, the SSA increase was different for

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Fig. 2. Data for enzymatic saccharication of sugarcane bagasse (a) and sugarcane straw (b) upon WDM treatment as a function of WDM time (min/g biomass) and number of cycles. Figure shows data for normalized sugar concentrations and saccharication yields based on the initial amounts of glucan and xylan in the raw materials.

Fig. 3. Normalized glucose ((a) and (c)) and xylose ((b) and (d)) concentrations as a function of wet disk milling time upon enzymatic saccharication of sugarcane bagasse and straw, after treatment by WDM (control experiment) and four combinations of WDM and ozonolysis. WDM (N); ozone treatment for 30 min (j) and 60 min (d) followed by WDM treatment; WDM treatment followed by ozone treatment for 30 min (h) and 60 min (s).

performed via batch processing, although digestibility increased in response to the number of cycles, it was not possible to identify a steady increase from 1 to 7 cycles. Nevertheless, signicant improvements were observed after 3, 4, and 7 cycles. Hideno et al. (2009) also reported very similar results, obtaining a 78.5% yield of glucose and 41.5% yield of xylose after 10 WDM cycles using rice straw biomass. The morphology of WDM-treated bagasse and straw samples indicated similar debrillating effects on both materials; nevertheless, most of the bers remained unbrillated on micron scale, with some disrupted microbers branching out. (Complementary Fig. 1) Moreover, the bers presented themselves attached to each other by the presence of lignin that was not removed by WDM. The com-

parison of ozonolysis to WDM indicated that WDM was more efcient for the degradation of glucan and xylan from bagasse and straw, and that WDM was more indicated for straw in comparison to bagasse. Concerning the surface area (Fig. 1) of WDM-treated samples, the values increased proportionally to the WDM cycle number, showing a 14-fold increase (from 2.414 to 34.344 m2/g) for bagasse and a 25-fold increase (from 1.571 to 39.451 m2/g) for sugarcane straw, after 7 WDM cycles. This improvement in terms of yields when compared to ozone treatment might have resulted from the particle size reduction, the easy homogenization of samples, and the increase in surface area, which are strongly dependent on WDM cycle number.

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3.3. Association of wet disk milling and ozonolysis Fig. 3 shows the effect of normalized WDM time (min/g) on the normalized concentration of glucose and xylose (mg sugar/g of biomass) after 72 h saccharication for pretreated bagasse and straw with four different combinations of WDM and ozonolysis. Fig. 3 also shows the data for the sole use of WDM (control experiments). The higher saccharication yields of glucose and xylose resulting from the combined pretreatments, in comparison to the sole use of WDM, showed that they were, in general, more effective. Because high sugar concentrations were achieved for a lower number of WDM cycles and, by extension, less milling time, there was a reduction in the energy consumption in the milling processes. Overall, bagasse saccharication yields of glucose, 89.67% (437.88 mg glucose/g bagasse) and xylose, 48.75% (120.76 mg xylose/g bagasse), after 60 min ozonolysis followed by WDM (four cycles and 1.2 min/g) were higher than those obtained by WDM (four cycles and 0.2 min/g) followed by 60 min ozonolysis (81.14% (396.24 mg glucose/g bagasse) and 50.50% (125.11 mg xylose/g bagasse)). However, the milling time and consequent energy consumption for the ozonolysis/WDM combination were sixfold higher than that for the WDM/ozonolysis combination. Therefore, pending on the process priorities, the best option for bagasse pretreatment would involve the WDM/ozonolysis combination, despite the lower 10% glucose yield. The overall straw glucose and xylose saccharication yields of 63.07% (285.94 mg glucose/g straw) and 36.60% (90.16 mg xylose/g straw), respectively, after 60 min ozonolysis followed by WDM (four cycles and 0.8 min/g) were lower than those obtained by WDM (four cycles and 0.2 min/g) followed by 60 min ozonolysis, 92.40% (418.88 mg glucose/g straw) and xylose, 52.33%(128.93 mg xylose/g straw), indicating that bagasse and straw responded differently to the same process conditions. The morphology of sugarcane bagasse and straw treated by the combination of WDM followed by ozonolysis showed a very disorganized (no hierarchically ordered) nanoscopic brous structure (Complementary Fig. 1). In addition, there was no evidence of lignin accumulation. It was possible to observe ne microbrils, 50 100 nm in diameter, separated from the micron-scale microbril aggregates. As discussed above, as ozonolysis degrades lignin, it weakness the adhesive strength between cellulose microbrils. When WDM is applied before ozonolysis, it enhances the SSAs, which results in a higher delignication potential during ozonolysis and the production of a highly microbrillated structure. This ne brous morphology results in a signicant increase in the surface area, favoring enzyme adsorption and, by extension, increasing the polymer degradation rate. For the combination treatment, the value of SSA (Fig. 3) was comparable with those obtained by only ozonolysis and WDM, even though the WDM time was shorter. In particular, the SSAs of the materials treated by ozonolysis (after 60 min) followed by WDM (four cycles) were found to be 66.474 and 39.380 m2/g for bagasse and straw, respectively, which was 27-fold higher than that of raw bagasse and 25-fold higher than that obtained from raw straw. This higher SSA can improve enzymatic accessibility, resulting in higher saccharication yields. The energy efciency of the WDM/ozonolysis combination could be related to the higher ozonolysis effectiveness of the milled material owing to its higher surface area. For the combined WDM and ozonolysis treatments, the estimated energy consumption was 87 and 93 kWh/kg for bagasse and straw, respectively. The average ozonolysis energy consumption was 39 kWh/kg for both materials. Considering the lower ozonolysis energy consumption, it is reasonable to conclude that the WDM/ozonolysis combination would be advantageous for reducing the high WDM energy consumption through shorter milling times.

Interestingly, for the three types of pretreatments, cellulose hydrolysis was prevalent compared to that for hemicellulose, despite the full amorphous nature of the hemicellulose. This morphology, which hinders enzyme accessibility to the cellulose surface, might be one reason why enzymatic saccharication did not surpass 75% by the use of ozonolysis or WDM alone. Besides WDM, ball milling treatments have been applied in other studies. In 2010, an investigation using sugarcane biomass showed that after 60 min ball milling, glucose and xylose yields of 78.7% and 72.1% were obtained for bagasse, and after 90 min, ball milling afforded glucose and xylose yields of 77.6% and 56.8% for straw, respectively. In both cases, the enhancement in cellulose digestibility was related to the reduction of cellulose crystallinity to a nearly amorphous level (da Silva et al., 2010). In another similar study, after 120 min pretreatment of Eucalyptus using a planetary ball milling process, the digestibility of both glucan and xylan increased to 76.7% and 63.9%, respectively, even at a substrate concentration of 20% and an enzyme dosage of 4 FPU/g of substrate. These results indicated that, in addition to its high energy consumption, ball milling was extremely efcient in enhancing biomass reactivity to enzymes (Inoue et al., 2008).

4. Conclusions The responses of bagasse and straw toward ozonolysis and WDM were different because ozonolysis was better suited to bagasse and WDM to straw. The ozonolytic optimum treatment time for both materials, considering glucose yields, was 60 min. WDM effectiveness increased in response to the cycle number for both materials. When an initial WDM brillation step was used, ozonolytic lignin removal was improved, resulting in increases in the surface area and the effectiveness of enzymatic saccharication for a shorter WDM time. It was also observed that this approach resulted in lower energy consumption, as compared to the ozonolysis/WDM sequence. References
Amat, A.M., Arques, A., Miranda, M.A., Lpez, F., 2005. Use of ozone and/or UV in the treatment of efuents from board paper industry. Chemosphere 60 (8), 1111 1117. http://dx.doi.org/10.1016/j.chemosphere.2004.12.062. Ben-Ghedalia, D., Shefet, G., Dror, Y., 1983. Chemical treatments for increasing the digestibility of cotton straw: 1. Effect of ozone and sodium hydroxide treatments on rumen metabolism and on the digestibility of cell walls and organic matter. J. Agric. Sci. 100 (02), 393. http://dx.doi.org/10.1017/ S0021859600033542. Ben-Ghedalia, D., Miron, J., 1981. The effect of combined chemical and enzyme treatments on the saccharication and in vitro digestion rate of wheat straw. Biotechnol. Bioeng. 23 (4), 823831. http://dx.doi.org/10.1002/bit.260230412. Binder, A., Pelloni, L., Fiechter, A., 1980. Delignication of straw with ozone to enhance biodegradability. Eur. J. Appl. Microbiol. Biotechnol. 11 (1), 15. http:// dx.doi.org/10.1007/BF00514070. Brunauer, S., Emmett, P.H., Teller, E., 1938. J. Am. Chem. Soc. 60 (2), 309319. http:// dx.doi.org/10.1021/ja01269a023. Cardona, C.A., Quintero, J.A., Paz, I.C., 2010. Production of bioethanol from sugarcane bagasse: status and perspectives: special issue on lignocellulosic bioethanol: current status and perspectives. Bioresour. Technol. 101 (13), 47544766. http://dx.doi.org/10.1016/j.biortech.2009.10.097. Cerqueira Leite, R.C.d., Verde Leal, M.R.L., Barbosa Cortez, L.A., Grifn, W.M., Gaya Scandifo, M.I., 2009. Can Brazil replace 5% of the 2025 gasoline world demand with ethanol?: 4th Dubrovnik conference 4th Dubrovnik conference on sustainable development of energy. Water Environ. Energy 34 (5), 655661. http://dx.doi.org/10.1016/j.energy.2008.11.001. Cheng, C.-L., Lo, Y.-C., Lee, K.-S., Lee, D.-J., Lin, C.-Y., Chang, J.-S., 2011. Biohydrogen production from lignocellulosic feedstock. Special Issue: Biofuels-III: Biohydrogen 102 (18), 85148523. http://dx.doi.org/10.1016/j.biortech.2011. 04.059. Coca, M., Pea, M., Gonzlez, G., 2005. Variables affecting efciency of molasses fermentation wastewater ozonation. Chemosphere 60 (10), 14081415. http:// dx.doi.org/10.1016/j.chemosphere.2005.01.090. Contreras Iglesias, S. 2002. Degradation and biodegradability enhancement of nitrobenzene and 2,4-dichlorophenol by means of advanced oxidation processes based on ozone. PhD Thesis, Spain.

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R.R.O. Barros et al. / Bioresource Technology 136 (2013) 288294 Miura, T., Lee, S.-H., Inoue, S., Endo, T., 2012. Improvement of enzymatic saccharication of sugarcane bagasse by dilute-alkali-catalyzed hydrothermal treatment and subsequent disk milling. Bioresour. Technol. 105, 9599. http:// dx.doi.org/10.1016/j.biortech.2011.11.118. Neely, W.C., 1984. Factors affecting the pretreatment of biomass with gaseous ozone. Biotechnol. Bioeng. 26 (1), 5965. http://dx.doi.org/10.1002/ bit.260260112. Pandey, A., Soccol, C.R., Nigam, P., Soccol, V.T., 2000. Biotechnological potential of agro-industrial residues. I: Sugarcane bagasse. Bioresour. Technol. 74 (1), 69 80. http://dx.doi.org/10.1016/S0960-8524(99)00142-X. Quesada, J., Rubio, M., Gmez, D., 1999. Ozonation of lignin rich solid fractions from corn stalks. J. Wood Chem. Technol. 19 (12), 115137. http://dx.doi.org/ 10.1080/02773819909349603. Roncero, M.B., Torres, A.L., Colom, J.F., Vidal, T., 2003. TCF bleaching of wheat straw pulp using ozone and xylanase. Part A: Paper quality assessment. Bioresour. Technol. 87 (3), 305314. http://dx.doi.org/10.1016/S0960-8524(02)00224-9. Shatalov, A.A., Pereira, H., 2008. Arundo donax L. reed: new perspectives for pulping and bleaching. 5. Ozone-based TCF bleaching of organosolv pulps. Bioresour. Technol. 99 (3), 472478. http://dx.doi.org/10.1016/j.biortech.2007.01.014. Sluiter, A. 2008. Determination of Ash in Biomass: Laboratory Analytical Procedure (LAP); Issue Date: 7/17/2005. <http://www.nrel.gov/biomass/pdfs/42622.pdf>. Accessed 14 December 2012. Sluiter, A. 2011. Determination of Structural Carbohydrates and Lignin in Biomass: Laboratory Analytical Procedure (LAP); Issue Date: April 2008; Revision Date: July 2011 (Version 07-08-2011). <http://www.nrel.gov/biomass/pdfs/ 42618.pdf>. Accessed 14 December 2012. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review: reviews issue. Bioresour. Technol. 83 (1), 111. http:// dx.doi.org/10.1016/S0960-8524(01)00212-7. Triolo, J.M., Sommer, S.G., Mller, H.B., Weisbjerg, M.R., Jiang, X.Y., 2011. A new algorithm to characterize biodegradability of biomass during anaerobic digestion: inuence of lignin concentration on methane production potential. Bioresour. Technol. 102 (20), 93959402. http://dx.doi.org/10.1016/ j.biortech.2011.07.026. Vidal, P.F., Molinier, J., 1988. Ozonolysis of lignin improvement of in vitro digestibility of poplar sawdust. Biomass 16 (1), 117. http://dx.doi.org/10.1016/ 0144-4565(88)90012-1. Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels, Bioprod. Bioren. 2 (1), 2640. http://dx.doi.org/10.1002/ bbb.49.

da Silva, A.S., Inoue, H., Endo, T., Yano, S., Bon, E.P.S., 2010. Milling pretreatment of sugarcane bagasse and straw for enzymatic hydrolysis and ethanol fermentation. Bioresour. Technol. 101 (19), 74027409. http://dx.doi.org/ 10.1016/j.biortech.2010.05.008. Endo, T., Tanaka, N., Yamasaki, R., Teramoto, Y., Lee, S.H. 2008. Wet mechanochemical treatment for enzymatic saccharication of wood. In: 15th Annual Meeting of the Cellulose Society of Japan, vol. 1011, Kyoto, p117118. Garca-Cubero, M.T., Gonzlez-Benito, G., Indacoechea, I., Coca, M., Bolado, S., 2009. Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw. Bioresour. Technol. 100 (4), 16081613. http://dx.doi.org/10.1016/ j.biortech.2008.09.012. Guo, T., He, A.-y., Du, T.-f., Zhu, D.-w., Liang, D.-f., Jiang, M., Wei, P., Ouyang, P.-k., 2013. Butanol production from hemicellulosic hydrolysate of corn ber by a Clostridium beijerinckii mutant with high inhibitor-tolerance. Bioresour. Technol. 135, 379385. Hendriks, A., Zeeman, G., 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100 (1), 1018. http://dx.doi.org/ 10.1016/j.biortech.2008.05.027. Hideno, A., Inoue, H., Tsukahara, K., Fujimoto, S., Minowa, T., Inoue, S., Endo, T., Sawayama, S., 2009. Wet disk milling pretreatment without sulfuric acid for enzymatic hydrolysis of rice straw. Bioresour. Technol. 100 (10), 27062711. http://dx.doi.org/10.1016/j.biortech.2008.12.057. Huang, C., Chen, X.-f., Xiong, L., Chen, X.-d., Ma, L.-l., 2012. Oil production by the yeast Trichosporon dermatis cultured in enzymatic hydrolysates of corncobs. Bioresour Technol 110 (0), 711714. http://dx.doi.org/10.1016/j.biortech.2012.01.077 Inoue, H., Yano, S., Endo, T., Sakaki, T., Sawayama, S., 2008. Combining hotcompressed water and ball milling pretreatments to improve the efciency of the enzymatic hydrolysis of eucalyptus. Biotechnol. Biofuels 1 (1), 2. http:// dx.doi.org/10.1186/1754-6834-1-2. Koukiekolo, R., Cho, H.-Y., Kosugi, A., Inui, M., Yukawa, H., Doi, R.H., 2005. Degradation of corn ber by clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA. Appl. Environ. Microbiol. 71 (7), 3504 3511. http://dx.doi.org/10.1128/AEM.71.7.3504-3511.2005. Laser, M., Schulman, D., Allen, S.G., Lichwa, J., Antal, M.J., Lynd, L.R., 2002. A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol. Bioresour. Technol. 81 (1), 3344. http:// dx.doi.org/10.1016/S0960-8524(01)00103-1. Lee, S.H., Chang, F., Inoue, S., Endo, T., 2010. Increase in enzyme accessibility by generation of nanospace in cell wall supramolecular structure. Bioresour. Technol. 101 (19), 72187223. http://dx.doi.org/10.1016/j.biortech.2010.04.069.