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Association of wet disk milling and ozonolysis as pretreatment for enzymatic saccharification of sugarcane bagasse and straw
Accepted Manuscript Association of wet disk milling and ozonolysis as pretreatment for enzymatic saccharification of sugarcane bagasse and straw Rodrigo da Rocha Olivieri de Barros, Raquel de Sousa Paredes, Takashi Endo, Elba Pinto da Silva Bon, Seung-Hwan Lee PII: DOI: Reference:
S0960-8524(13)00364-7 http://dx.doi.org/10.1016/j.biortech.2013.03.009 BITE 11468
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
26 December 2012 26 February 2013 1 March 2013
Please cite this article as: de Barros, R.d.R., Paredes, R.d.S., Endo, T., da Silva Bon, E.P., Lee, S-H., Association of wet disk milling and ozonolysis as pretreatment for enzymatic saccharification of sugarcane bagasse and straw, Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.03.009
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Association of wet disk milling and ozonolysis as pretreatment for
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enzymatic saccharification of sugarcane bagasse and straw
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Rodrigo da Rocha Olivieri de Barros1), Raquel de Sousa Paredes1), Takashi Endo2), Elba
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Pinto da Silva Bon 1)*, Seung-Hwan Lee2, 3)*
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*Corresponding author: e-mail: [email protected] and [email protected]
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1) Enzyme Technology Laboratory, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil 2) Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagami-Yama, Higashi Hiroshima, Hiroshima 739-0046, Japan 3) Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
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Abstract
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Ozonolysis was studied separately and in combination with wet disk milling (WDM) for
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the pretreatment of sugarcane bagasse and straw, with the aim of improving their
20
enzymatic saccharification. The glucose yields for ozonolysis followed by WDM were
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89.7% for bagasse and 63.1% for straw, whereas the use of WDM followed by
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ozonolysis resulted in glucose yields of 81.1% for bagasse and 92.4% for straw, with
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shorter WDM time. This last procedure allowed a substantial decrease in energy
24
consumption in comparison to the use of WDM alone or of ozonolysis followed by
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WDM. Higher overall saccharification yields with shorter milling times were observed
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when ozonolysis was carried out before WDM. This effect might be related to the
1
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higher specific surface area. Additionally, a finer morphology was observed by the
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association of the two treatments in comparison to the sole use of ozonolysis or WDM.
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Keywords: sugarcane bagasse, sugarcane straw, ozone treatment, wet disk milling,
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enzymatic saccharification.
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1. Introduction
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Significant effort has been exerted globally to meet the demand for fuels and chemicals
35
and gradually replace fossil fuels with those obtainable from renewable resources such
36
as biomass. From this perspective, fuels and chemicals can be produced from
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lignocellulosic materials, which are the most abundant renewable resources on earth
38
(Koukiekolo et al., 2005). However, the availability and selection of biomass feedstock,
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the development of economically feasible pretreatment technologies, as well as the cost
40
and availability of the necessary enzyme pools for efficient saccharification, are still
41
under development for the commercialization of these new products.
42
Biorefinery offers the possibility of different products that can be obtained from the
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enzymatic hydrolysates of lignocellulosic biomass, including bioethanol, butanol,
44
biohydrogen, oil, and methane. The production chain starts with biomass pretreatment
45
followed by enzymatic saccharification to produce the hydrolysates; the last step usually
46
involves fermentation processes or anaerobic digestion (Cheng et al., 2011; Triolo et al.,
47
2011; Guo et al., 2012; Huang et al., 2012) Sugarcane bagasse and straw are high-
48
potential feedstock for second-generation (2G) bioethanol production in Brazil because
49
of their abundance in the Brazilian sugar mills. Brazil has also acquired significant
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ethanol production expertise since the 1970s, after the implementation of the Pro-Álcool
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program. Nevertheless, the full use of these raw materials in 2G ethanol production is 2
52
limited, because most of the sugarcane residues are used as energy sources via
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combustion in Brazilian sugar mill operations (Cardona et al., 2010; Leite et al., 2009;
54
Pandey et al., 2000).
55
The development of a suitable pretreatment coupled to the relevant parameters for the
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enzymatic saccharification is of foremost importance. Pretreatment, which is a
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necessary step to reduce the natural recalcitrance of lignocellulosic materials, is
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estimated to be responsible for up to 20% of the total production cost. Moreover,
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pretreatment has a pervasive impact on all other major operation steps in the overall
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biomass conversion process, from the choice of feedstock through size reduction,
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enzymatic hydrolysis, and fermentation, as well as product recovery, residue processing,
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and coproducts potential (Yang and Wyman, 2008).
63
Ozone, which is a powerful oxidant, has been widely used for pulp bleaching in the
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paper industry (Roncero et al., 2003; Shatalov and Pereira, 2008). The diversity of
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ozone applications has substantially increased over the last two decades, finding use, for
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example, in the treatment of ground and industrial wastewaters (Amat et al., 2005; Coca
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et al., 2005). Ozonolysis for biomass pretreatment has also shown its utility because of
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its delignification efficiency. It can also cause a slight degradation of the hemicellulose
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component of the lignocellulosic biomass (Quesada et al., 1999; Sun and Cheng, 2002).
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Ozone is highly reactive toward compounds with conjugated double bonds and
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functional groups with high electron densities. Therefore, lignin is the moiety most
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likely to be oxidized in the ozonolysis of lignocellulosic materials because of its high
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C=C bond content. Ozone attacks biomass components, releasing soluble low molecular
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weight compounds, with a preponderance of organic acids such as formic and acetic
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acids, which can result in a severe pH drop. However, this process is advantageous
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because the aforementioned degradation products do not interfere with the subsequent
3
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enzymatic hydrolysis and fermentation steps. Additionally, the low energy consumption
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of ozonolysis is attractive, because it takes place at room temperature (Contreras, 2002).
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Indeed, ozonolysis has been known as an effective pretreatment method for various
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lignocellulosic materials, degrading mainly lignin, for example, in wheat and cotton
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straw (Ben-Ghedalia and Miron, 1981; 1983), bagasse, green hay, peanut, pine (Neely,
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1984), and poplar sawdust (Vidal and Molinier, 1988).
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Mechanical pretreatments of biomass aim primarily to increase the surface area by
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reducing the feedstock particle size, combined with fibrillation or reduction in the
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crystallinity degree. This approach facilitates the accessibility of enzymes to the
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substrate, increasing saccharification rates and yields. Usually, milling processes are
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very energy intensive, depending on the material characteristics and the target particle
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size (Laser et al., 2002). WDM is a recently introduced biomass pretreatment process
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able to produce milled biomass with low energy consumption when compared to
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conventional ball milling treatment. This technique has been shown to increase the
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degree of biomass fibrillation and the space between the microfibrils, thus promoting
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the accessibility of the cellulolytic enzyme pool to cellulose (Hideno et al., 2009). The
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disk mill is a type of crusher that can be used to grind, cut, shear, fiberize, pulverize,
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granulate, or blend. In general, the suspended material is fed between opposing disks or
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plates that can be grooved, serrated, or spiked. For biomass processing using WDM, a
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water suspension (1–5% of solids) of the lignocellulosic material is passed between two
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ceramic nonporous disks that are separated by a distance of 20–100 µm and that have a
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rotational speed of around 1800 rpm. This process can be repeated according to the
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required number of WDM cycles; very small particle sizes with high specific surface
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areas (SSAs) have been observed after a minimum of five cycles (Endo et al., 2008; Da
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Silva et al., 2010).
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In this study, ozonolysis was evaluated in association with wet disk milling (WDM)
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to improve sugarcane bagasse and straw enzymatic saccharification and reduce energy
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consumption because of sum of benefits of each individual treatment. It was reasonable
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to expect that the loosening of the cell wall structure by delignification via ozonolysis
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would improve the biomass fibrillation upon WDM, reduce the milling time, and also,
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increase the SSA. Even though mechanical treatment is an environment-friendly process,
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because it does not use chemicals such as acids or alkalis, it is an energy-intensive
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process (Hendriks and Zeeman, 2009). Thus, it needs to be used in combination with
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other treatments to save energy and reduce costs. In previous studies from our
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laboratory, WDM treatment favored the fibrillation of some materials such as rice straw
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and sugarcane bagasse (Hideno et al., 2009; Silva et al., 2010). Moreover, hot-
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compressed water (HCW) in the presence or absence of alkali catalyst was studied,
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aiming to improve mechanical fibrillation by WDM (Miura et al. 2012). HCW treatment
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loosens the structure of lignocellulosic materials mostly via hemicellulose removal and
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structural changes of the lignin, resulting in the improvement of WDM performance.
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The fibrillated product so obtained showed high cellulose surface area and improved
118
enzymatic saccharification (Lee et al., 2010; Miura et al., 2012).
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2. Methods
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2.1. Materials
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Sugarcane bagasse and straw were kindly supplied by Complexo Bioenergético Itarumã
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S.A (Estate of Goiás, Brazil). The materials were dried at room temperature, coarsely
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cut using a cutter mill, and sieved to select particles less than 2 mm in size. Enzymatic
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hydrolysis was performed using Acremonium cellulase (Meiji Seika Co, Japan) and
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Optimash™ BG with β-xylosidase activity (Genencor® International, USA). Other 5
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chemicals were purchased from commercial sources.
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2.2. Chemical composition of sugarcane bagasse and straw
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The water content of the milled sugarcane bagasse and straw was determined using a
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halogen moisture analyzer (HG63, Mettler-Toledo AG Co., Greifensee, Switzerland).
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Cellulose, hemicellulose, and lignin contents were measured according to Sluiter et al.,
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2008, with minor changes. The dried samples (30 mg) were hydrolyzed with 72%
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sulfuric acid (0.3 mL) at 30 °C for 1 h, followed by dilution of the acid to 4% by adding
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distilled water (8.4 mL) and incubation in an autoclave at 121 °C for 1 h. The treated
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sample was cooled and vacuum filtered through a glass fiber filter (Whatman F/A 47
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mm, Whatman International Ltd., England, UK). The soluble fraction was neutralized
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with calcium carbonate and analyzed using a high-performance liquid chromatography
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(HPLC) system (JASCO, Tokyo, Japan), equipped with an Aminex HPX-87P column at
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80 °C with a flow rate of 1.0 mL H2O/min, to determine the concentration of the
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monomeric sugars derived from the cellulose and hemicellulose fractions. Lignin
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content was determined by gravimetric analysis of the residue from the biomass acid
143
hydrolysis. Ash content was determined by burning 1 g of sample in a tarred crucible
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for 6 h using a muffle furnace at 600 °C until a constant weight was obtained (Sluiter et
145
al., 2005). Other biomass components were estimated by subtracting the amounts of
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glucan, xylan, arabinan, lignin, and ash from the initial biomass dry weight. The raw
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materials’ normalized compositions are shown in Table 1. Values represent means of
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independent triplicates and standard deviations are shown parenthetically.
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2.3. Ozonolysis treatment
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An ozone generator, model ED-OG-R5 (EcoDesign Inc., Japan), was fed with an
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oxygen flow of 0.5 L/min, resulting in an ozone concentration of 204 g/m3 inside the
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treatment vessel. Samples of bagasse and straw (20–40 g), previously humidified to
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60% water content, were submitted to ozone treatment at 40 °C for 30, 60, 90 and 120
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min under agitation. The amount of ozone consumed by the biomass sample, for each
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experiment, was monitored through the measurement of the electrical current (voltage)
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using a UV ozone monitor (OZM 5000G OKITROTEC, Japan). A control experiment
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was carried out with the amount of water corresponding to 60% biomass moisture
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content, allowing the measurement of ozone consumption by water. Ozone consumption
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was determined by subtracting the residual ozone of the control experiment from that of
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each experiment and was expressed as the consumption rate per gram of dry biomass.
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After the treatment, the samples were washed with water (400–800 mL). The solids
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content of the water soluble fraction (WSF), representing organic acids and soluble
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solids, was quantified by evaporation.
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2.4. Wet disk milling treatment
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WDM was performed using a Supermasscolloider (MKZA6-2, Masuko Sangyo Co.,
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Ltd., Saitama, Japan) equipped with two ceramic nonporous disks. The clearance of the
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two disks was adjusted to 20–40 μm and the rotation speed was 1800 rpm. A water
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suspension of ozone-treated or ozone-untreated sugarcane bagasse or straw (60 g in 3 L)
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was fed to the equipment. Milling operation cycles in the range 1–5 for the ozone-
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treated or 1–7 for the ozone-untreated material (control experiments) were carried out.
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WDM time for each milling cycle was calculated taking into account the weight of
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materials. The milling energy consumption for each milling cycle was calculated taking
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into account the data for voltage, current, and operation time.
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2.5. Enzymatic hydrolysis and HPLC analysis
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Enzymatic saccharification was carried out using a treated or untreated biomass
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concentration of 2.5% (w/w) and an enzyme load of 15 FPU Acremonium cellulase per
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gram of biomass supplemented with 0.2% (v/v) OptimashTM BG as a source of
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hemicellulase, in acetate buffer (pH 5.0). Reactions were incubated under agitation at
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50 °C for 72 h. The sugar quantification was performed using a HPLC system (JASCO,
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Tokyo, Japan) following the same conditions as described in Section 2.2.
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Enzymatic hydrolysis yield calculations were based on glucan and xylan contents
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in the raw materials, taking into account the recovered mass of glucose and xylose and
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also the mass losses in the water soluble fractions after each biomass pretreatment.
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2.6. Morphology and surface area
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The morphologies of the treated and untreated samples were observed by scanning
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electron microscopy (SEM) using an S-4800 SEM (Hitachi High Technologies Co.,
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Tokyo, Japan). Tiny pieces of each sample were coated with a thin layer of osmium. The
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SSAs were determined from the Brunauer–Emmett–Teller (BET) plot of a nitrogen-
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adsorption isotherm using a BELSORP-max (BEL Japan, Osaka, Japan) (Brunauer et al.,
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1938). Prior to both analyses, the samples were thoroughly washed with t-butyl alcohol
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and freeze-dried to preserve their morphological properties.
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3. Results and Discussion
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3.1. Ozonolysis
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Table 2 shows normalized ozone consumption data for the ozonolysis of sugarcane
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bagasse and straw for treatment times from 30 to 120 min. It also shows the
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corresponding WSF solids content and the residual lignin content in the treated 8
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materials. Table 2 also summarizes the yields for the enzymatic saccharification of the
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cellulose and hemicelluloses of the pretreated sugarcane bagasse and straw in
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comparison to the yields of untreated materials.
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The stepwise increase in the ozone treatment time resulted in higher ozone
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consumption as well as biomass degradation, as shown by the increase in the amount of
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water-soluble solids. Sugarcane bagasse and straw responded differently with regard to
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ozone consumption, such that a lower consumption was observed for bagasse (0.187 g
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ozone/g biomass) in comparison to straw (0.213 g ozone/g biomass) for 60 minutes
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treatment. Nevertheless, the amount of water-soluble solids was higher for bagasse
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(27.60%) in comparison to that of straw (19.50%). Accordingly, the residual lignin was
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lower for bagasse (12.40%) in comparison to straw (15.39%). After 60 min exposure to
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ozone, glucose and xylose saccharification yields increased to 59.24% and 32.38% for
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bagasse and 46.95% and 27.84% for straw, respectively, in comparison to that for
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untreated materials. Because bagasse lignin was revealed to be more prone to
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ozonolysis than straw, bagasse saccharification yields were higher. Considering the
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saccharification time course, glucose yields did not significantly increase after 60 min,
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while xylose yields decreased after 60 min for bagasse and after 90 min for straw. The
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harmful effect on xylose yield as a result of longer treatment times could be because of
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hemicellulose degradation by ozone. Nevertheless, ozone is mainly known to degrade
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lignin; hemicellulose and cellulose can be slightly affected (Quesada et al., 1999;
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Roncero et al., 2003; Shatalov and Pereira, 2008; Sun and Cheng, 2002). Binder et al.
223
(1980) reported that, although delignification was beneficial to the saccharification
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process, it was deleterious for lignin removal beyond 60%. Another study related to
225
ozone treatment for lignocellulosic materials showed that enzymatic hydrolysis yields
226
of up to 88.6% and 57% were obtained compared to 29% and 16% in nonozonated
9
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wheat and rye straws, respectively (García-Cubero et al., 2009).
228
By analyzing the morphological structure of the ozone-treated samples, it was
229
possible to identify a disturbed structure in treated bagasse, in addition to the presence
230
of a nanoscopic fibrous morphology, which was less apparent for the ozone-treated
231
straw (Complementary Fig. 1). However, these nanoscopic fibers appeared only in some
232
areas (maybe from primary cell wall) of the treated material, in other regions, the treated
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samples showed a type of morphology similar to the raw materials. Lignin content in
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primary cell wall is lower than in secondary wall, so that delignification by ozonolysis
235
could easily release cellulose microfibrills from primary cell wall. However, the amount
236
of primary cell wall is smaller than the secondary cell wall, thus it is limited to affect to
237
increase the specific surface area. This probably can directly compromise the SSA
238
values and saccharification yields of glucose and xylose. Lignin removal data indicated
239
that ozone was significantly more effective toward bagasse delignification (15.42%)
240
than for straw (9.40%). The substantial lignin removal also allowed the visualization of
241
the biomass microfibers, although some areas of the treated materials retained
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characteristics similar to the untreated materials, presenting a smooth and aggregated
243
surface. In this context, it is worth mentioning that the low xylose concentrations might
244
be linked to xylan degradation, as mentioned. The use of 60 min ozonolysis alone
245
resulted in a prominent SSA increase (Fig. 1) resulting from the generation of a porous
246
structure by the removal of lignin. However, the SSA increase was different for
247
sugarcane bagasse and straw, because a sevenfold increase (from 2.414 m2/g to
248
18.016 m2/g) was measured for bagasse, and a 23-fold increase (from 1.571 m2/g to
249
36.285 m2/g) was measured for straw. These differences could be related to the nature of
250
the different plant tissues of the sugarcane plant, as well as the prior exposure of the
251
bagasse to the high processing pressures of sugarcane juice extraction.
10
252
Because the consumption of large amounts of ozone makes the process expensive, in
253
this work, ozone treatments for 30 and 60 min were selected to be further associated
254
with WDM, with the aim to improve saccharification yields.
255 256
3.2. Wet disk milling treatment
257
Figure 2 shows the effect of WDM time (or cycle number) on glucose and xylose yields
258
for WDM sugarcane bagasse and straw. Considering cellulose hydrolysis, seven cycles
259
of milling increased the digestibility of bagasse by 50% (1.181 min/g) and of straw by
260
46% (1.241 min/g). However, because untreated straw was more prone to
261
saccharification than bagasse, its final conversion of 73.85% was higher than that of
262
bagasse (62.25%). Considering the hydrolysis of hemicelluloses, the same number of
263
cycles increased the digestibility of bagasse by 33% (1.181 min/g) and of straw by 39%
264
(1.241 min/g). However, because untreated straw was more prone to saccharification
265
than bagasse, its final conversions of 73.85% and 53.42% were higher than those of
266
bagasse, 62.25% and 42.63%, to glucose and xylose, respectively. Because milling
267
cycles were performed via batch processing, although digestibility increased in response
268
to the number of cycles, it was not possible to identify a steady increase from 1 to 7
269
cycles. Nevertheless, significant improvements were observed after 3, 4, and 7 cycles.
270
Hideno et al. (2009) also reported very similar results, obtaining a 78.5% yield of
271
glucose and 41.5% yield of xylose after 10 WDM cycles using rice straw biomass.
272
The morphology of WDM-treated bagasse and straw samples indicated similar
273
defibrillating effects on both materials; nevertheless, most of the fibers remained
274
unfibrillated on micron scale, with some disrupted microfibers branching out.
275
(Complementary Fig. 1) Moreover, the fibers presented themselves attached to each
276
other by the presence of lignin that was not removed by WDM. The comparison of
11
277
ozonolysis to WDM indicated that WDM was more efficient for the degradation of
278
glucan and xylan from bagasse and straw, and that WDM was more indicated for straw
279
in comparison to bagasse.
280
Concerning the surface area (Fig. 1) of WDM-treated samples, the values increased
281
proportionally to the WDM cycle number, showing a 14-fold increase (from 2.414 m2/g
282
to 34.344 m2/g) for bagasse and a 25-fold increase (from 1.571 m2/g to 39.451 m2/g) for
283
sugarcane straw, after 7 WDM cycles. This improvement in terms of yields when
284
compared to ozone treatment might have resulted from the particle size reduction, the
285
easy homogenization of samples, and the increase in surface area, which are strongly
286
dependent on WDM cycle number.
287 288
3.3. Association of wet disk milling and ozonolysis
289
Figure 3 shows the effect of normalized WDM time (min/g) on the normalized
290
concentration of glucose and xylose (mg sugar/g of biomass) after 72 h saccharification
291
for pretreated bagasse and straw with four different combinations of WDM and
292
ozonolysis. Figure 3 also shows the data for the sole use of WDM (control experiments).
293
The higher saccharification yields of glucose and xylose resulting from the combined
294
pretreatments, in comparison to the sole use of WDM, showed that they were, in general,
295
more effective. Because high sugar concentrations were achieved for a lower number of
296
WDM cycles and, by extension, less milling time, there was a reduction in the energy
297
consumption in the milling processes.
298
Overall, bagasse saccharification yields of glucose, 89.67% (437.88 mg glucose/g
299
bagasse) and xylose, 48.75% (120.76 mg xylose/g bagasse), after 60 min ozonolysis
300
followed by WDM (four cycles and 1.2 min/g) were higher than those obtained by
301
WDM (four cycles and 0.2 min/g) followed by 60 min ozonolysis (81.14% (396.24 mg
12
302
glucose/g bagasse) and 50.50% (125.11 mg xylose/g bagasse)). However, the milling
303
time and consequent energy consumption for the ozonolysis/WDM combination were
304
sixfold higher than that for the WDM/ozonolysis combination. Therefore, pending on
305
the process priorities, the best option for bagasse pretreatment would involve the
306
WDM/ozonolysis combination, despite the lower 10% glucose yield.
307
The overall straw glucose and xylose saccharification yields of 63.07% (285.94 mg
308
glucose/g straw) and 36.60% (90.16 mg xylose/g straw), respectively, after 60 min
309
ozonolysis followed by WDM (four cycles and 0.8 min/g) were lower than those
310
obtained by WDM (four cycles and 0.2 min/g) followed by 60 min ozonolysis, 92.40%
311
(418.88 mg glucose/g straw) and xylose, 52.33%(128.93 mg xylose/ g straw), indicating
312
that bagasse and straw responded differently to the same process conditions.
313
The morphology of sugarcane bagasse and straw treated by the combination of
314
WDM followed by ozonolysis showed a very disorganized (no hierarchically ordered)
315
nanoscopic fibrous structure (Complementary Fig. 1). In addition, there was no
316
evidence of lignin accumulation. It was possible to observe fine microfibrils, 50–100
317
nm in diameter, separated from the micron-scale microfibril aggregates. As discussed
318
above, as ozonolysis degrades lignin, it weakness the adhesive strength between
319
cellulose microfibrils. When WDM is applied before ozonolysis, it enhances the SSAs,
320
which results in a higher delignification potential during ozonolysis and the production
321
of a highly microfibrillated structure. This fine fibrous morphology results in a
322
significant increase in the surface area, favoring enzyme adsorption and, by extension,
323
increasing the polymer degradation rate.
324
For the combination treatment, the value of SSA (Fig. 3) was comparable with those
325
obtained by only ozonolysis and WDM, even though the WDM time was shorter. In
326
particular, the SSAs of the materials treated by ozonolysis (after 60 min) followed by
13
327
WDM (four cycles) were found to be 66.474 m2/g and 39.380 m2/g for bagasse and
328
straw, respectively, which was 27-fold higher than that of raw bagasse and 25-fold
329
higher than that obtained from raw straw. This higher SSA can improve enzymatic
330
accessibility, resulting in higher saccharification yields.
331
The energy efficiency of the WDM/ozonolysis combination could be related to the
332
higher ozonolysis effectiveness of the milled material owing to its higher surface area.
333
For the combined WDM and ozonolysis treatments, the estimated energy consumption
334
was 87 kWh/Kg and 93 kWh/Kg for bagasse and straw, respectively. The average
335
ozonolysis energy consumption was 39 kWh/Kg for both materials. Considering the
336
lower ozonolysis energy consumption, it is reasonable to conclude that the
337
WDM/ozonolysis combination would be advantageous for reducing the high WDM
338
energy consumption through shorter milling times.
339
Interestingly, for the three types of pretreatments, cellulose hydrolysis was prevalent
340
compared to that for hemicellulose, despite the full amorphous nature of the
341
hemicellulose. This morphology, which hinders enzyme accessibility to the cellulose
342
surface, might be one reason why enzymatic saccharification did not surpass 75% by the
343
use of ozonolysis or WDM alone.
344
Besides WDM, ball milling treatments have been applied in other studies. In 2010,
345
an investigation using sugarcane biomass showed that after 60 min ball milling, glucose
346
and xylose yields of 78.7% and 72.1% were obtained for bagasse, and after 90 min, ball
347
milling afforded glucose and xylose yields of 77.6% and 56.8% for straw, respectively.
348
In both cases, the enhancement in cellulose digestibility was related to the reduction of
349
cellulose crystallinity to a nearly amorphous level (Da Silva et al., 2010).
350
In another similar study, after 120 min pretreatment of Eucalyptus using a planetary
351
ball milling process, the digestibility of both glucan and xylan increased to 76.7% and
14
352
63.9%, respectively, even at a substrate concentration of 20% and an enzyme dosage of
353
4 FPU/g of substrate. These results indicated that, in addition to its high energy
354
consumption, ball milling was extremely efficient in enhancing biomass reactivity to
355
enzymes (Inoue et al., 2008).
356 357
4. Conclusions
358
The responses of bagasse and straw toward ozonolysis and WDM were different
359
because ozonolysis was better suited to bagasse and WDM to straw. The ozonolytic
360
optimum treatment time for both materials, considering glucose yields, was 60 min.
361
WDM effectiveness increased in response to the cycle number for both materials. When
362
an initial WDM fibrillation step was used, ozonolytic lignin removal was improved,
363
resulting in increases in the surface area and the effectiveness of enzymatic
364
saccharification for a shorter WDM time. It was also observed that this approach
365
resulted in lower energy consumption, as compared to the ozonolysis/WDM sequence.
366 367 368
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24. Neely, W.C., 1984. Factors affecting the pretreatment of biomass with gaseous
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25. Pandey, A., Soccol, C.R., Nigam, P., Soccol, V.T., 2000. Biotechnological potential
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29. Sluiter, A., 2008. Determination of Ash in Biomass: Laboratory Analytical
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30. Sluiter, A., 2011. Determination of Structural Carbohydrates and Lignin in Biomass:
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33. Vidal, P.F., Molinier, J., 1988. Ozonolysis of lignin — Improvement of in vitro
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4565(88)90012-1.
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of
poplar
sawdust.
Biomass
16
(1),
1–17.
doi:10.1016/0144-
482
21
483 484 485 486 487 488 489 490 491 492
Table captions Table 1. Chemical compositions of untreated sugarcane bagasse and straw. Table 2. Ozone consumption, water-soluble fraction, lignin content, and saccharification yields after the ozone treatment of sugarcane bagasse and straw.
22
493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518
Figures captions Figure 1. Specific surface areas of raw and pretreated samples of sugarcane bagasse and straw. Figure 2. Data for enzymatic saccharification 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 saccharification yields based on the initial amounts of glucan and xylan in the raw materials. Figure 3. Normalized glucose ((a) and (c)) and xylose ((b) and (d)) concentrations as a function of wet disk milling time upon enzymatic saccharification of sugarcane bagasse and straw, after treatment by WDM (control experiment) and four combinations of WDM and ozonolysis. WDM (S); ozone treatment for 30 min () and 60 min (z) followed by WDM treatment; WDM treatment followed by ozone treatment for 30 min ( ) and 60 min ({). Complementary Figure 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).
23
519 520
Highlights
521
• Association of ozonolysis and WDM for the pretreatment of sugarcane baggasse and straw
522 523 524 525 526 527
• Ozonolysis after WDM resulted in higher saccharification yield than WDM after ozonolysis. • Energy consumption was lowest in ozonolysis after WDM.
528 529
• Saccharification yield was affected by morphology and specific surface area
530
24
531 532 533
Table 1. Chemical compositions of untreated sugarcane bagasse and straw. Chemical composition (%)
534 535
Sugarcane Bagasse
Sugarcane Straw
Glucan
43.95 (± 1.85)
40.80 (± 0.37)
Xylan
21.80 (± 0.68)
21.68 (± 0.04)
Arabinan
2.48 (± 0.16)
3.60 (± 0.20)
Galactan
0.81 (± 0.07)
0.72 (± 0.10)
Lignin
27.82 (± 0.71)
24.79 (± 0.11)
Ash
0.99 (± 0.05)
4.92 (± 0.27)
Others
2.14
3.49
Data are shown as mean values of three independent experiments and the standard deviations are shown parenthetically.
25
536 537 538 539 540
Table 2. Ozone consumption, water-soluble fraction, lignin content, and saccharification yields after the ozone treatment of sugarcane bagasse and straw. Saccharification yields (%) for 72h. Material
Bagasse
Straw
541
Ozone treatment time (min.)
Consumed ozone amount (g/g-biomass)
Water soluble fraction amount (%)
Liginin content in ozone-treated product (%)
0
-
-
30
0.078
60 90
Glucose
Xylose
mg/g of biomass
% (based on glucan)
mg/g of biomass
% (based on xylan)
27.82
60.80
12.45
24.40
9.85
20.60
16.23
246.14
50.40
33.46
33.46
0.187
27.60
12.40
289.31
59.24
32.38
32.38
0.296
31.00
11.49
306.64
62.79
29.41
29.41
120
0.377
33.35
10.00
308.72
63.22
26.80
26.80
0
-
-
24.79
124.40
27.44
35.60
14.45
30
0.080
8.66
17.86
180.49
39.81
66.13
26.84
60
0.213
19.50
15.39
212.84
46.95
68.59
27.84
90
0.317
19.90
14.24
215.63
47.57
68.25
27.70
120
0.490
23.65
13.01
204.92
45.20
62.91
25.54
26
542 543
544 545 546 547
Figure 1. Specific surface areas of raw and pretreated samples of sugarcane bagasse and straw.
27
548 549 550
551 552 553 554 555 556
Figure 2. Data for enzymatic saccharification 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 saccharification yields based on the initial amounts of glucan and xylan in the raw materials.
28
557
558 559 560 561 562 563 564
Figure 3. Normalized glucose ((a) and (c)) and xylose ((b) and (d)) concentrations as a function of wet disk milling time upon enzymatic saccharification of sugarcane bagasse and straw, after treatment by WDM (control experiment) and four combinations of WDM and ozonolysis. WDM (S); ozone treatment for 30 min () and 60 min (z) followed by WDM treatment; WDM treatment followed by ozone treatment for 30 min ( ) and 60 min ({).
29
565 566 567
568 569 570 571 572 573
Complementary Figure 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).
574 575
30
S0960-8524(13)00364-7 http://dx.doi.org/10.1016/j.biortech.2013.03.009 BITE 11468
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
26 December 2012 26 February 2013 1 March 2013
Please cite this article as: de Barros, R.d.R., Paredes, R.d.S., Endo, T., da Silva Bon, E.P., Lee, S-H., Association of wet disk milling and ozonolysis as pretreatment for enzymatic saccharification of sugarcane bagasse and straw, Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.03.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2
Association of wet disk milling and ozonolysis as pretreatment for
3
enzymatic saccharification of sugarcane bagasse and straw
4 5
Rodrigo da Rocha Olivieri de Barros1), Raquel de Sousa Paredes1), Takashi Endo2), Elba
6
Pinto da Silva Bon 1)*, Seung-Hwan Lee2, 3)*
7 8
*Corresponding author: e-mail: [email protected] and [email protected]
9 10 11 12 13 14 15
1) Enzyme Technology Laboratory, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil 2) Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagami-Yama, Higashi Hiroshima, Hiroshima 739-0046, Japan 3) Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
16 17
Abstract
18
Ozonolysis was studied separately and in combination with wet disk milling (WDM) for
19
the pretreatment of sugarcane bagasse and straw, with the aim of improving their
20
enzymatic saccharification. The glucose yields for ozonolysis followed by WDM were
21
89.7% for bagasse and 63.1% for straw, whereas the use of WDM followed by
22
ozonolysis resulted in glucose yields of 81.1% for bagasse and 92.4% for straw, with
23
shorter WDM time. This last procedure allowed a substantial decrease in energy
24
consumption in comparison to the use of WDM alone or of ozonolysis followed by
25
WDM. Higher overall saccharification yields with shorter milling times were observed
26
when ozonolysis was carried out before WDM. This effect might be related to the
1
27
higher specific surface area. Additionally, a finer morphology was observed by the
28
association of the two treatments in comparison to the sole use of ozonolysis or WDM.
29 30
Keywords: sugarcane bagasse, sugarcane straw, ozone treatment, wet disk milling,
31
enzymatic saccharification.
32 33
1. Introduction
34
Significant effort has been exerted globally to meet the demand for fuels and chemicals
35
and gradually replace fossil fuels with those obtainable from renewable resources such
36
as biomass. From this perspective, fuels and chemicals can be produced from
37
lignocellulosic materials, which are the most abundant renewable resources on earth
38
(Koukiekolo et al., 2005). However, the availability and selection of biomass feedstock,
39
the development of economically feasible pretreatment technologies, as well as the cost
40
and availability of the necessary enzyme pools for efficient saccharification, are still
41
under development for the commercialization of these new products.
42
Biorefinery offers the possibility of different products that can be obtained from the
43
enzymatic hydrolysates of lignocellulosic biomass, including bioethanol, butanol,
44
biohydrogen, oil, and methane. The production chain starts with biomass pretreatment
45
followed by enzymatic saccharification to produce the hydrolysates; the last step usually
46
involves fermentation processes or anaerobic digestion (Cheng et al., 2011; Triolo et al.,
47
2011; Guo et al., 2012; Huang et al., 2012) Sugarcane bagasse and straw are high-
48
potential feedstock for second-generation (2G) bioethanol production in Brazil because
49
of their abundance in the Brazilian sugar mills. Brazil has also acquired significant
50
ethanol production expertise since the 1970s, after the implementation of the Pro-Álcool
51
program. Nevertheless, the full use of these raw materials in 2G ethanol production is 2
52
limited, because most of the sugarcane residues are used as energy sources via
53
combustion in Brazilian sugar mill operations (Cardona et al., 2010; Leite et al., 2009;
54
Pandey et al., 2000).
55
The development of a suitable pretreatment coupled to the relevant parameters for the
56
enzymatic saccharification is of foremost importance. Pretreatment, which is a
57
necessary step to reduce the natural recalcitrance of lignocellulosic materials, is
58
estimated to be responsible for up to 20% of the total production cost. Moreover,
59
pretreatment has a pervasive impact on all other major operation steps in the overall
60
biomass conversion process, from the choice of feedstock through size reduction,
61
enzymatic hydrolysis, and fermentation, as well as product recovery, residue processing,
62
and coproducts potential (Yang and Wyman, 2008).
63
Ozone, which is a powerful oxidant, has been widely used for pulp bleaching in the
64
paper industry (Roncero et al., 2003; Shatalov and Pereira, 2008). The diversity of
65
ozone applications has substantially increased over the last two decades, finding use, for
66
example, in the treatment of ground and industrial wastewaters (Amat et al., 2005; Coca
67
et al., 2005). Ozonolysis for biomass pretreatment has also shown its utility because of
68
its delignification efficiency. It can also cause a slight degradation of the hemicellulose
69
component of the lignocellulosic biomass (Quesada et al., 1999; Sun and Cheng, 2002).
70
Ozone is highly reactive toward compounds with conjugated double bonds and
71
functional groups with high electron densities. Therefore, lignin is the moiety most
72
likely to be oxidized in the ozonolysis of lignocellulosic materials because of its high
73
C=C bond content. Ozone attacks biomass components, releasing soluble low molecular
74
weight compounds, with a preponderance of organic acids such as formic and acetic
75
acids, which can result in a severe pH drop. However, this process is advantageous
76
because the aforementioned degradation products do not interfere with the subsequent
3
77
enzymatic hydrolysis and fermentation steps. Additionally, the low energy consumption
78
of ozonolysis is attractive, because it takes place at room temperature (Contreras, 2002).
79
Indeed, ozonolysis has been known as an effective pretreatment method for various
80
lignocellulosic materials, degrading mainly lignin, for example, in wheat and cotton
81
straw (Ben-Ghedalia and Miron, 1981; 1983), bagasse, green hay, peanut, pine (Neely,
82
1984), and poplar sawdust (Vidal and Molinier, 1988).
83
Mechanical pretreatments of biomass aim primarily to increase the surface area by
84
reducing the feedstock particle size, combined with fibrillation or reduction in the
85
crystallinity degree. This approach facilitates the accessibility of enzymes to the
86
substrate, increasing saccharification rates and yields. Usually, milling processes are
87
very energy intensive, depending on the material characteristics and the target particle
88
size (Laser et al., 2002). WDM is a recently introduced biomass pretreatment process
89
able to produce milled biomass with low energy consumption when compared to
90
conventional ball milling treatment. This technique has been shown to increase the
91
degree of biomass fibrillation and the space between the microfibrils, thus promoting
92
the accessibility of the cellulolytic enzyme pool to cellulose (Hideno et al., 2009). The
93
disk mill is a type of crusher that can be used to grind, cut, shear, fiberize, pulverize,
94
granulate, or blend. In general, the suspended material is fed between opposing disks or
95
plates that can be grooved, serrated, or spiked. For biomass processing using WDM, a
96
water suspension (1–5% of solids) of the lignocellulosic material is passed between two
97
ceramic nonporous disks that are separated by a distance of 20–100 µm and that have a
98
rotational speed of around 1800 rpm. This process can be repeated according to the
99
required number of WDM cycles; very small particle sizes with high specific surface
100
areas (SSAs) have been observed after a minimum of five cycles (Endo et al., 2008; Da
101
Silva et al., 2010).
4
102
In this study, ozonolysis was evaluated in association with wet disk milling (WDM)
103
to improve sugarcane bagasse and straw enzymatic saccharification and reduce energy
104
consumption because of sum of benefits of each individual treatment. It was reasonable
105
to expect that the loosening of the cell wall structure by delignification via ozonolysis
106
would improve the biomass fibrillation upon WDM, reduce the milling time, and also,
107
increase the SSA. Even though mechanical treatment is an environment-friendly process,
108
because it does not use chemicals such as acids or alkalis, it is an energy-intensive
109
process (Hendriks and Zeeman, 2009). Thus, it needs to be used in combination with
110
other treatments to save energy and reduce costs. In previous studies from our
111
laboratory, WDM treatment favored the fibrillation of some materials such as rice straw
112
and sugarcane bagasse (Hideno et al., 2009; Silva et al., 2010). Moreover, hot-
113
compressed water (HCW) in the presence or absence of alkali catalyst was studied,
114
aiming to improve mechanical fibrillation by WDM (Miura et al. 2012). HCW treatment
115
loosens the structure of lignocellulosic materials mostly via hemicellulose removal and
116
structural changes of the lignin, resulting in the improvement of WDM performance.
117
The fibrillated product so obtained showed high cellulose surface area and improved
118
enzymatic saccharification (Lee et al., 2010; Miura et al., 2012).
119 120
2. Methods
121
2.1. Materials
122
Sugarcane bagasse and straw were kindly supplied by Complexo Bioenergético Itarumã
123
S.A (Estate of Goiás, Brazil). The materials were dried at room temperature, coarsely
124
cut using a cutter mill, and sieved to select particles less than 2 mm in size. Enzymatic
125
hydrolysis was performed using Acremonium cellulase (Meiji Seika Co, Japan) and
126
Optimash™ BG with β-xylosidase activity (Genencor® International, USA). Other 5
127
chemicals were purchased from commercial sources.
128 129
2.2. Chemical composition of sugarcane bagasse and straw
130
The water content of the milled sugarcane bagasse and straw was determined using a
131
halogen moisture analyzer (HG63, Mettler-Toledo AG Co., Greifensee, Switzerland).
132
Cellulose, hemicellulose, and lignin contents were measured according to Sluiter et al.,
133
2008, with minor changes. The dried samples (30 mg) were hydrolyzed with 72%
134
sulfuric acid (0.3 mL) at 30 °C for 1 h, followed by dilution of the acid to 4% by adding
135
distilled water (8.4 mL) and incubation in an autoclave at 121 °C for 1 h. The treated
136
sample was cooled and vacuum filtered through a glass fiber filter (Whatman F/A 47
137
mm, Whatman International Ltd., England, UK). The soluble fraction was neutralized
138
with calcium carbonate and analyzed using a high-performance liquid chromatography
139
(HPLC) system (JASCO, Tokyo, Japan), equipped with an Aminex HPX-87P column at
140
80 °C with a flow rate of 1.0 mL H2O/min, to determine the concentration of the
141
monomeric sugars derived from the cellulose and hemicellulose fractions. Lignin
142
content was determined by gravimetric analysis of the residue from the biomass acid
143
hydrolysis. Ash content was determined by burning 1 g of sample in a tarred crucible
144
for 6 h using a muffle furnace at 600 °C until a constant weight was obtained (Sluiter et
145
al., 2005). Other biomass components were estimated by subtracting the amounts of
146
glucan, xylan, arabinan, lignin, and ash from the initial biomass dry weight. The raw
147
materials’ normalized compositions are shown in Table 1. Values represent means of
148
independent triplicates and standard deviations are shown parenthetically.
149 150
2.3. Ozonolysis treatment
151
An ozone generator, model ED-OG-R5 (EcoDesign Inc., Japan), was fed with an
6
152
oxygen flow of 0.5 L/min, resulting in an ozone concentration of 204 g/m3 inside the
153
treatment vessel. Samples of bagasse and straw (20–40 g), previously humidified to
154
60% water content, were submitted to ozone treatment at 40 °C for 30, 60, 90 and 120
155
min under agitation. The amount of ozone consumed by the biomass sample, for each
156
experiment, was monitored through the measurement of the electrical current (voltage)
157
using a UV ozone monitor (OZM 5000G OKITROTEC, Japan). A control experiment
158
was carried out with the amount of water corresponding to 60% biomass moisture
159
content, allowing the measurement of ozone consumption by water. Ozone consumption
160
was determined by subtracting the residual ozone of the control experiment from that of
161
each experiment and was expressed as the consumption rate per gram of dry biomass.
162
After the treatment, the samples were washed with water (400–800 mL). The solids
163
content of the water soluble fraction (WSF), representing organic acids and soluble
164
solids, was quantified by evaporation.
165 166
2.4. Wet disk milling treatment
167
WDM was performed using a Supermasscolloider (MKZA6-2, Masuko Sangyo Co.,
168
Ltd., Saitama, Japan) equipped with two ceramic nonporous disks. The clearance of the
169
two disks was adjusted to 20–40 μm and the rotation speed was 1800 rpm. A water
170
suspension of ozone-treated or ozone-untreated sugarcane bagasse or straw (60 g in 3 L)
171
was fed to the equipment. Milling operation cycles in the range 1–5 for the ozone-
172
treated or 1–7 for the ozone-untreated material (control experiments) were carried out.
173
WDM time for each milling cycle was calculated taking into account the weight of
174
materials. The milling energy consumption for each milling cycle was calculated taking
175
into account the data for voltage, current, and operation time.
176
7
177
2.5. Enzymatic hydrolysis and HPLC analysis
178
Enzymatic saccharification was carried out using a treated or untreated biomass
179
concentration of 2.5% (w/w) and an enzyme load of 15 FPU Acremonium cellulase per
180
gram of biomass supplemented with 0.2% (v/v) OptimashTM BG as a source of
181
hemicellulase, in acetate buffer (pH 5.0). Reactions were incubated under agitation at
182
50 °C for 72 h. The sugar quantification was performed using a HPLC system (JASCO,
183
Tokyo, Japan) following the same conditions as described in Section 2.2.
184
Enzymatic hydrolysis yield calculations were based on glucan and xylan contents
185
in the raw materials, taking into account the recovered mass of glucose and xylose and
186
also the mass losses in the water soluble fractions after each biomass pretreatment.
187 188
2.6. Morphology and surface area
189
The morphologies of the treated and untreated samples were observed by scanning
190
electron microscopy (SEM) using an S-4800 SEM (Hitachi High Technologies Co.,
191
Tokyo, Japan). Tiny pieces of each sample were coated with a thin layer of osmium. The
192
SSAs were determined from the Brunauer–Emmett–Teller (BET) plot of a nitrogen-
193
adsorption isotherm using a BELSORP-max (BEL Japan, Osaka, Japan) (Brunauer et al.,
194
1938). Prior to both analyses, the samples were thoroughly washed with t-butyl alcohol
195
and freeze-dried to preserve their morphological properties.
196 197
3. Results and Discussion
198
3.1. Ozonolysis
199
Table 2 shows normalized ozone consumption data for the ozonolysis of sugarcane
200
bagasse and straw for treatment times from 30 to 120 min. It also shows the
201
corresponding WSF solids content and the residual lignin content in the treated 8
202
materials. Table 2 also summarizes the yields for the enzymatic saccharification of the
203
cellulose and hemicelluloses of the pretreated sugarcane bagasse and straw in
204
comparison to the yields of untreated materials.
205
The stepwise increase in the ozone treatment time resulted in higher ozone
206
consumption as well as biomass degradation, as shown by the increase in the amount of
207
water-soluble solids. Sugarcane bagasse and straw responded differently with regard to
208
ozone consumption, such that a lower consumption was observed for bagasse (0.187 g
209
ozone/g biomass) in comparison to straw (0.213 g ozone/g biomass) for 60 minutes
210
treatment. Nevertheless, the amount of water-soluble solids was higher for bagasse
211
(27.60%) in comparison to that of straw (19.50%). Accordingly, the residual lignin was
212
lower for bagasse (12.40%) in comparison to straw (15.39%). After 60 min exposure to
213
ozone, glucose and xylose saccharification yields increased to 59.24% and 32.38% for
214
bagasse and 46.95% and 27.84% for straw, respectively, in comparison to that for
215
untreated materials. Because bagasse lignin was revealed to be more prone to
216
ozonolysis than straw, bagasse saccharification yields were higher. Considering the
217
saccharification time course, glucose yields did not significantly increase after 60 min,
218
while xylose yields decreased after 60 min for bagasse and after 90 min for straw. The
219
harmful effect on xylose yield as a result of longer treatment times could be because of
220
hemicellulose degradation by ozone. Nevertheless, ozone is mainly known to degrade
221
lignin; hemicellulose and cellulose can be slightly affected (Quesada et al., 1999;
222
Roncero et al., 2003; Shatalov and Pereira, 2008; Sun and Cheng, 2002). Binder et al.
223
(1980) reported that, although delignification was beneficial to the saccharification
224
process, it was deleterious for lignin removal beyond 60%. Another study related to
225
ozone treatment for lignocellulosic materials showed that enzymatic hydrolysis yields
226
of up to 88.6% and 57% were obtained compared to 29% and 16% in nonozonated
9
227
wheat and rye straws, respectively (García-Cubero et al., 2009).
228
By analyzing the morphological structure of the ozone-treated samples, it was
229
possible to identify a disturbed structure in treated bagasse, in addition to the presence
230
of a nanoscopic fibrous morphology, which was less apparent for the ozone-treated
231
straw (Complementary Fig. 1). However, these nanoscopic fibers appeared only in some
232
areas (maybe from primary cell wall) of the treated material, in other regions, the treated
233
samples showed a type of morphology similar to the raw materials. Lignin content in
234
primary cell wall is lower than in secondary wall, so that delignification by ozonolysis
235
could easily release cellulose microfibrills from primary cell wall. However, the amount
236
of primary cell wall is smaller than the secondary cell wall, thus it is limited to affect to
237
increase the specific surface area. This probably can directly compromise the SSA
238
values and saccharification yields of glucose and xylose. Lignin removal data indicated
239
that ozone was significantly more effective toward bagasse delignification (15.42%)
240
than for straw (9.40%). The substantial lignin removal also allowed the visualization of
241
the biomass microfibers, although some areas of the treated materials retained
242
characteristics similar to the untreated materials, presenting a smooth and aggregated
243
surface. In this context, it is worth mentioning that the low xylose concentrations might
244
be linked to xylan degradation, as mentioned. The use of 60 min ozonolysis alone
245
resulted in a prominent SSA increase (Fig. 1) resulting from the generation of a porous
246
structure by the removal of lignin. However, the SSA increase was different for
247
sugarcane bagasse and straw, because a sevenfold increase (from 2.414 m2/g to
248
18.016 m2/g) was measured for bagasse, and a 23-fold increase (from 1.571 m2/g to
249
36.285 m2/g) was measured for straw. These differences could be related to the nature of
250
the different plant tissues of the sugarcane plant, as well as the prior exposure of the
251
bagasse to the high processing pressures of sugarcane juice extraction.
10
252
Because the consumption of large amounts of ozone makes the process expensive, in
253
this work, ozone treatments for 30 and 60 min were selected to be further associated
254
with WDM, with the aim to improve saccharification yields.
255 256
3.2. Wet disk milling treatment
257
Figure 2 shows the effect of WDM time (or cycle number) on glucose and xylose yields
258
for WDM sugarcane bagasse and straw. Considering cellulose hydrolysis, seven cycles
259
of milling increased the digestibility of bagasse by 50% (1.181 min/g) and of straw by
260
46% (1.241 min/g). However, because untreated straw was more prone to
261
saccharification than bagasse, its final conversion of 73.85% was higher than that of
262
bagasse (62.25%). Considering the hydrolysis of hemicelluloses, the same number of
263
cycles increased the digestibility of bagasse by 33% (1.181 min/g) and of straw by 39%
264
(1.241 min/g). However, because untreated straw was more prone to saccharification
265
than bagasse, its final conversions of 73.85% and 53.42% were higher than those of
266
bagasse, 62.25% and 42.63%, to glucose and xylose, respectively. Because milling
267
cycles were performed via batch processing, although digestibility increased in response
268
to the number of cycles, it was not possible to identify a steady increase from 1 to 7
269
cycles. Nevertheless, significant improvements were observed after 3, 4, and 7 cycles.
270
Hideno et al. (2009) also reported very similar results, obtaining a 78.5% yield of
271
glucose and 41.5% yield of xylose after 10 WDM cycles using rice straw biomass.
272
The morphology of WDM-treated bagasse and straw samples indicated similar
273
defibrillating effects on both materials; nevertheless, most of the fibers remained
274
unfibrillated on micron scale, with some disrupted microfibers branching out.
275
(Complementary Fig. 1) Moreover, the fibers presented themselves attached to each
276
other by the presence of lignin that was not removed by WDM. The comparison of
11
277
ozonolysis to WDM indicated that WDM was more efficient for the degradation of
278
glucan and xylan from bagasse and straw, and that WDM was more indicated for straw
279
in comparison to bagasse.
280
Concerning the surface area (Fig. 1) of WDM-treated samples, the values increased
281
proportionally to the WDM cycle number, showing a 14-fold increase (from 2.414 m2/g
282
to 34.344 m2/g) for bagasse and a 25-fold increase (from 1.571 m2/g to 39.451 m2/g) for
283
sugarcane straw, after 7 WDM cycles. This improvement in terms of yields when
284
compared to ozone treatment might have resulted from the particle size reduction, the
285
easy homogenization of samples, and the increase in surface area, which are strongly
286
dependent on WDM cycle number.
287 288
3.3. Association of wet disk milling and ozonolysis
289
Figure 3 shows the effect of normalized WDM time (min/g) on the normalized
290
concentration of glucose and xylose (mg sugar/g of biomass) after 72 h saccharification
291
for pretreated bagasse and straw with four different combinations of WDM and
292
ozonolysis. Figure 3 also shows the data for the sole use of WDM (control experiments).
293
The higher saccharification yields of glucose and xylose resulting from the combined
294
pretreatments, in comparison to the sole use of WDM, showed that they were, in general,
295
more effective. Because high sugar concentrations were achieved for a lower number of
296
WDM cycles and, by extension, less milling time, there was a reduction in the energy
297
consumption in the milling processes.
298
Overall, bagasse saccharification yields of glucose, 89.67% (437.88 mg glucose/g
299
bagasse) and xylose, 48.75% (120.76 mg xylose/g bagasse), after 60 min ozonolysis
300
followed by WDM (four cycles and 1.2 min/g) were higher than those obtained by
301
WDM (four cycles and 0.2 min/g) followed by 60 min ozonolysis (81.14% (396.24 mg
12
302
glucose/g bagasse) and 50.50% (125.11 mg xylose/g bagasse)). However, the milling
303
time and consequent energy consumption for the ozonolysis/WDM combination were
304
sixfold higher than that for the WDM/ozonolysis combination. Therefore, pending on
305
the process priorities, the best option for bagasse pretreatment would involve the
306
WDM/ozonolysis combination, despite the lower 10% glucose yield.
307
The overall straw glucose and xylose saccharification yields of 63.07% (285.94 mg
308
glucose/g straw) and 36.60% (90.16 mg xylose/g straw), respectively, after 60 min
309
ozonolysis followed by WDM (four cycles and 0.8 min/g) were lower than those
310
obtained by WDM (four cycles and 0.2 min/g) followed by 60 min ozonolysis, 92.40%
311
(418.88 mg glucose/g straw) and xylose, 52.33%(128.93 mg xylose/ g straw), indicating
312
that bagasse and straw responded differently to the same process conditions.
313
The morphology of sugarcane bagasse and straw treated by the combination of
314
WDM followed by ozonolysis showed a very disorganized (no hierarchically ordered)
315
nanoscopic fibrous structure (Complementary Fig. 1). In addition, there was no
316
evidence of lignin accumulation. It was possible to observe fine microfibrils, 50–100
317
nm in diameter, separated from the micron-scale microfibril aggregates. As discussed
318
above, as ozonolysis degrades lignin, it weakness the adhesive strength between
319
cellulose microfibrils. When WDM is applied before ozonolysis, it enhances the SSAs,
320
which results in a higher delignification potential during ozonolysis and the production
321
of a highly microfibrillated structure. This fine fibrous morphology results in a
322
significant increase in the surface area, favoring enzyme adsorption and, by extension,
323
increasing the polymer degradation rate.
324
For the combination treatment, the value of SSA (Fig. 3) was comparable with those
325
obtained by only ozonolysis and WDM, even though the WDM time was shorter. In
326
particular, the SSAs of the materials treated by ozonolysis (after 60 min) followed by
13
327
WDM (four cycles) were found to be 66.474 m2/g and 39.380 m2/g for bagasse and
328
straw, respectively, which was 27-fold higher than that of raw bagasse and 25-fold
329
higher than that obtained from raw straw. This higher SSA can improve enzymatic
330
accessibility, resulting in higher saccharification yields.
331
The energy efficiency of the WDM/ozonolysis combination could be related to the
332
higher ozonolysis effectiveness of the milled material owing to its higher surface area.
333
For the combined WDM and ozonolysis treatments, the estimated energy consumption
334
was 87 kWh/Kg and 93 kWh/Kg for bagasse and straw, respectively. The average
335
ozonolysis energy consumption was 39 kWh/Kg for both materials. Considering the
336
lower ozonolysis energy consumption, it is reasonable to conclude that the
337
WDM/ozonolysis combination would be advantageous for reducing the high WDM
338
energy consumption through shorter milling times.
339
Interestingly, for the three types of pretreatments, cellulose hydrolysis was prevalent
340
compared to that for hemicellulose, despite the full amorphous nature of the
341
hemicellulose. This morphology, which hinders enzyme accessibility to the cellulose
342
surface, might be one reason why enzymatic saccharification did not surpass 75% by the
343
use of ozonolysis or WDM alone.
344
Besides WDM, ball milling treatments have been applied in other studies. In 2010,
345
an investigation using sugarcane biomass showed that after 60 min ball milling, glucose
346
and xylose yields of 78.7% and 72.1% were obtained for bagasse, and after 90 min, ball
347
milling afforded glucose and xylose yields of 77.6% and 56.8% for straw, respectively.
348
In both cases, the enhancement in cellulose digestibility was related to the reduction of
349
cellulose crystallinity to a nearly amorphous level (Da Silva et al., 2010).
350
In another similar study, after 120 min pretreatment of Eucalyptus using a planetary
351
ball milling process, the digestibility of both glucan and xylan increased to 76.7% and
14
352
63.9%, respectively, even at a substrate concentration of 20% and an enzyme dosage of
353
4 FPU/g of substrate. These results indicated that, in addition to its high energy
354
consumption, ball milling was extremely efficient in enhancing biomass reactivity to
355
enzymes (Inoue et al., 2008).
356 357
4. Conclusions
358
The responses of bagasse and straw toward ozonolysis and WDM were different
359
because ozonolysis was better suited to bagasse and WDM to straw. The ozonolytic
360
optimum treatment time for both materials, considering glucose yields, was 60 min.
361
WDM effectiveness increased in response to the cycle number for both materials. When
362
an initial WDM fibrillation step was used, ozonolytic lignin removal was improved,
363
resulting in increases in the surface area and the effectiveness of enzymatic
364
saccharification for a shorter WDM time. It was also observed that this approach
365
resulted in lower energy consumption, as compared to the ozonolysis/WDM sequence.
366 367 368
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31. Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol
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production: a review: Reviews Issue. Bioresource Technology 83 (1), 1–11.
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doi:10.1016/S0960-8524(01)00212-7.
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32. Triolo, J.M., Sommer, S.G., Møller, H.B., Weisbjerg, M.R., Jiang, X.Y., 2011. A
474
new algorithm to characterize biodegradability of biomass during anaerobic digestion:
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Influence of lignin concentration on methane production potential. Bioresource
476
Technology 102 (20), 9395–9402. doi:10.1016/j.biortech.2011.07.026.
20
477
33. Vidal, P.F., Molinier, J., 1988. Ozonolysis of lignin — Improvement of in vitro
478
digestibility
479
4565(88)90012-1.
480
34. Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost
481
cellulosic ethanol. Biofuels, Bioprod. Bioref. 2 (1), 26–40. doi:10.1002/bbb.49.
of
poplar
sawdust.
Biomass
16
(1),
1–17.
doi:10.1016/0144-
482
21
483 484 485 486 487 488 489 490 491 492
Table captions Table 1. Chemical compositions of untreated sugarcane bagasse and straw. Table 2. Ozone consumption, water-soluble fraction, lignin content, and saccharification yields after the ozone treatment of sugarcane bagasse and straw.
22
493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518
Figures captions Figure 1. Specific surface areas of raw and pretreated samples of sugarcane bagasse and straw. Figure 2. Data for enzymatic saccharification 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 saccharification yields based on the initial amounts of glucan and xylan in the raw materials. Figure 3. Normalized glucose ((a) and (c)) and xylose ((b) and (d)) concentrations as a function of wet disk milling time upon enzymatic saccharification of sugarcane bagasse and straw, after treatment by WDM (control experiment) and four combinations of WDM and ozonolysis. WDM (S); ozone treatment for 30 min () and 60 min (z) followed by WDM treatment; WDM treatment followed by ozone treatment for 30 min ( ) and 60 min ({). Complementary Figure 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).
23
519 520
Highlights
521
• Association of ozonolysis and WDM for the pretreatment of sugarcane baggasse and straw
522 523 524 525 526 527
• Ozonolysis after WDM resulted in higher saccharification yield than WDM after ozonolysis. • Energy consumption was lowest in ozonolysis after WDM.
528 529
• Saccharification yield was affected by morphology and specific surface area
530
24
531 532 533
Table 1. Chemical compositions of untreated sugarcane bagasse and straw. Chemical composition (%)
534 535
Sugarcane Bagasse
Sugarcane Straw
Glucan
43.95 (± 1.85)
40.80 (± 0.37)
Xylan
21.80 (± 0.68)
21.68 (± 0.04)
Arabinan
2.48 (± 0.16)
3.60 (± 0.20)
Galactan
0.81 (± 0.07)
0.72 (± 0.10)
Lignin
27.82 (± 0.71)
24.79 (± 0.11)
Ash
0.99 (± 0.05)
4.92 (± 0.27)
Others
2.14
3.49
Data are shown as mean values of three independent experiments and the standard deviations are shown parenthetically.
25
536 537 538 539 540
Table 2. Ozone consumption, water-soluble fraction, lignin content, and saccharification yields after the ozone treatment of sugarcane bagasse and straw. Saccharification yields (%) for 72h. Material
Bagasse
Straw
541
Ozone treatment time (min.)
Consumed ozone amount (g/g-biomass)
Water soluble fraction amount (%)
Liginin content in ozone-treated product (%)
0
-
-
30
0.078
60 90
Glucose
Xylose
mg/g of biomass
% (based on glucan)
mg/g of biomass
% (based on xylan)
27.82
60.80
12.45
24.40
9.85
20.60
16.23
246.14
50.40
33.46
33.46
0.187
27.60
12.40
289.31
59.24
32.38
32.38
0.296
31.00
11.49
306.64
62.79
29.41
29.41
120
0.377
33.35
10.00
308.72
63.22
26.80
26.80
0
-
-
24.79
124.40
27.44
35.60
14.45
30
0.080
8.66
17.86
180.49
39.81
66.13
26.84
60
0.213
19.50
15.39
212.84
46.95
68.59
27.84
90
0.317
19.90
14.24
215.63
47.57
68.25
27.70
120
0.490
23.65
13.01
204.92
45.20
62.91
25.54
26
542 543
544 545 546 547
Figure 1. Specific surface areas of raw and pretreated samples of sugarcane bagasse and straw.
27
548 549 550
551 552 553 554 555 556
Figure 2. Data for enzymatic saccharification 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 saccharification yields based on the initial amounts of glucan and xylan in the raw materials.
28
557
558 559 560 561 562 563 564
Figure 3. Normalized glucose ((a) and (c)) and xylose ((b) and (d)) concentrations as a function of wet disk milling time upon enzymatic saccharification of sugarcane bagasse and straw, after treatment by WDM (control experiment) and four combinations of WDM and ozonolysis. WDM (S); ozone treatment for 30 min () and 60 min (z) followed by WDM treatment; WDM treatment followed by ozone treatment for 30 min ( ) and 60 min ({).
29
565 566 567
568 569 570 571 572 573
Complementary Figure 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).
574 575
30