Enzymatic Hydrolysis of Steam-Treated Sugarcane Bagasse: Effect of

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Enzymatic hydrolysis of steam-treated sugarcane bagasse: effect of enzyme loading and substrate total solids on its fractal kinetic modeling and rheological properties Douglas Henrique Fockink, Mateus Barbian Urio, Jorge Hernan Sanchez Toro, and Luiz Pereira Ramos Energy Fuels, Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 22, 2017

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Energy & Fuels

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Enzymatic hydrolysis of steam-treated sugarcane bagasse: effect of enzyme

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loading and substrate total solids on its fractal kinetic modeling and rheological

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properties

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Douglas H. Fockink1; Mateus B. Urio1; Jorge H. Sánchez2; Luiz P. Ramos1,3*

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1

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University of Paraná - UFPR, P. O. Box 1908 – Curitiba, PR – 81531-980 – Brazil

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2

Research Center in Applied Chemistry (CEPESQ), Department of Chemistry, Federal

Pulp and Paper Research Group, Department of Chemical Engineering, Universidad

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Pontificia Bolivariana – UPB, P.O. Box 56006 – Medellín – Colombia

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3

INCT Energy & Environment (INCT E&A), Federal University of Paraná, Curitiba, Brazil

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ABSTRACT

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Rheological studies and fractal kinetic modeling were applied to investigate the enzymatic

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hydrolysis of steam-exploded sugarcane bagasse (195°C, 7.5 min) using Cellic CTec3

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cellulases (Novozymes). Initially, a central composite rotatable design (CCRD) was

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performed to evaluate the effect of different enzyme loadings and substrate total solids (TS)

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on both substrate apparent viscosity and kinetic parameters of enzymatic hydrolysis.

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Hydrolysis at 20% TS for 12 and 96 h using 38.6 FPU g-1 glucan released 52 and 110 g L-1

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glucose equivalents from the steam-exploded material, respectively, with cellobiose being

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always below 1.7% of these readings. Fractal kinetic modeling provided a good fit of both

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glucan and xylan conversions and the fractal kinetic parameters k and h had a strong

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correlation with changes in both substrate TS and enzyme loading. At the center point of the

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CCRD, Cellic CTec3 caused a decrease of one order of magnitude in the substrate apparent

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viscosity at every 6 h of hydrolysis. Cellic HTec3 had a boosting effect on the enzymatic

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hydrolysis of cane bagasse glucans regardless of the low hemicellulose content of the steam-

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treated material. Glucan hydrolysis was improved by 8% when 10% Cellic HTec3 was

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added to a hydrolysis mixture containing Cellic CTec3 at 38.6 FPU g-1 glucan. With this, a

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total production of 120 g L-1 glucose was achieved at 72 h using 20% TS.

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Key-words: Sugarcane bagasse, enzymatic hydrolysis, apparent viscosity, fractal kinetics,

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glucose yield.

33 34 35

1. INTRODUCTION

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Renewable liquid fuels such as ethanol are recognized as sustainable alternatives to

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overcome the negative environmental impact of fossil fuels in the transportation sector.

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However, most of the fuel ethanol produced to date comes from sucrose or starch and these

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primary feedstocks may not meet the sustainable criteria of modern biorefineries as well as

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the rising demand for this biofuel worldwide. Ethanol can also be produced from

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lignocellulosic materials but their highly ordered and tightly packed microfibrillar structure

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is very hard to access. Therefore, a pretreatment method is needed to improve the

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accessibility of both glucans (mostly cellulose) and hemicelluloses to enzymatic hydrolysis,

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this without releasing substantial amounts of inhibitory compounds for the subsequent step

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of fermentation. Besides, the lignin type, content and distribution also represent additional

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barriers affecting the enzymatic hydrolysis of lignocellulosic materials.1 For these and other

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reasons, pretreatment is still considered one of the most challenging steps for the optimal

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performance of cellulosic ethanol production.2, 3 2 ACS Paragon Plus Environment

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Different pretreatment strategies have been developed to date for a wide variety of

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lignocellulosic materials.4 Among these, steam explosion is recognized as one of the most

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efficient for hardwoods and agricultural residues and its selective fractionation is the core

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technology for several cellulosic ethanol production facilities available worldwide.5-7 In this

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process, lignocellulosic materials, with or without pre-impregnation with an exogenous

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catalyst, are exposed to pressurized steam followed by a rapid decompression that results in

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the explosive breakdown of the plant cell wall structure,8 promoting hemicellulose

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depolymerization (mainly converted to water soluble oligomers) and structural changes in

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lignin due to the use of relatively high pretreatment temperatures.9

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Enzymatic hydrolysis of pretreated materials is also a key step towards the efficient

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conversion of biomass into fuels and chemicals.2 However, technological improvements are

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still needed to make the process economically viable.10 A potential route to reduce both

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capital and production costs is to perform hydrolysis at high total solids (TS > 15%) with the

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aims of lowering the use of water and increasing the sugar concentration in the liquid

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stream. Economic analyses suggested that the concentration of fermentable sugars in

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biomass hydrolysates must be high enough to produce ethanol concentrations above 4 wt.%.

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This way, the energy consumption in the distillation step is reduced considerably and the

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economics of the overall production process is greatly improved.11

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Processing at high total solids (TS) is not an easy task since pretreated biomass

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slurries are highly viscous and exhibit strong non-Newtonian flow properties.12-14 The low

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availability of free water in these reaction systems introduces mechanical challenges that are

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related to mixing and pumping,15 creating heat and mass transfer limitations that hinder the

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optimal performance and uniform distribution of the enzymes, respectively.16 In addition,

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soluble sugars rapidly accumulate in the reaction medium at high substrate TS, decreasing


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the water mobility and causing enzyme inhibition at the early stages of hydrolysis.14, 17, 18 As

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a results, kinetic models for homogeneous reaction systems that are based on the Fick's law

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of diffusion do not apply to the enzymatic hydrolysis of cellulose.19 By contrast, fractal

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kinetics has been successfully used to fit such experimental data 20-22 but its application to

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enzymatic hydrolysis with advanced cellulase systems such as Cellic® CTec3 (Novozymes)

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at various substrate TS and enzyme loadings has not been reported as yet.

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In the present study, sugarcane bagasse was pretreated by steam explosion at reaction

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conditions that were pre-optimized by Pitarelo et al. 23 for optimal glucan recovery and

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enzymatic hydrolysis. Then, a central composite rotatable design (CCRD) was employed to

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evaluate the effect of different enzyme loadings and substrate TS on changes in both

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substrate apparent viscosity and glucan conversion. The apparent viscosity was monitored

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during the first 24 h of hydrolysis using different substrate TS and Cellic CTec3 enzyme

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loadings. In addition, the fractal kinetic model was applied to fit the hydrolysis data and to

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establish a correlation between the fractal kinetic parameters h and k and the susceptibility of

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cane bagasse polysaccharides to enzymatic hydrolysis. The influence of Cellic HTec3

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hemicellulases was also evaluated in order to assess their synergism with Cellic CTec3 in

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the hydrolysis of steam-treated substrates.

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2. MATERIAL AND METHODS

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2.1. Material Sugarcane bagasse was obtained from the São Martinho Mill (São Paulo, SP, Brazil)

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with the logistical support of the Cane Technology Center - CTC (Piracicaba, SP, Brazil).

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This sample was considered representative because it was collected directly from an

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industrial site. Besides, little variance is observed in the compositional analysis of industrial 4 ACS Paragon Plus Environment

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samples of cane bagasse when suitable analytical methods are used for characterization.24, 25

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The commercial enzymes used for hydrolysis (Cellic CTec3 and Cellic HTec3) were kindly

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donated by Novozymes Latin America (Araucária, PR, Brazil).

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2.2. Sugarcane bagasse pretreatment and chemical characterization Pretreatment was performed in a 10-L high pressure batch steam reactor at 195 °C

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for 7.5 min using sugarcane bagasse with a final moisture content of 50 wt.%. Water-

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washing was applied on the resulting pretreated material to remove water-soluble

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hemicellulose and lignin components.23

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The composition of both untreated and treated materials was characterized following

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the National Renewable Energy Laboratory (NREL) standard procedures. The total

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moisture, ash and total extractive contents were determined according to NREL/TP-510-

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42621, 26 NREL/TP-510-42622 27 and NREL/TP-510-42619 28, respectively. Carbohydrate

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and total lignin contents (acid-soluble and acid-insoluble lignin) were determined as

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recommended by NREL/TP-510-42618.29 Substrate acid hydrolysates were analyzed by

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high performance liquid chromatography (Shimadzu HPLC, LC-20AD series) using an

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Agilent Hi-Plex H column (300 x 7.7 mm) with its corresponding guard column (50 x 7.7

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mm) at 65 °C. Column elution was performed with 5 mmol L-1 H2SO4 at a flow rate of 0.6

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mL min-1. Sample injection (20 µL) was carried out automatically using a Shimadzu SIL-

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10AF autosampler. Quantitative analysis was carried out by external calibration using

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differential refractometry (Shimadzu RID-10A) for detecting cellobiose, glucose, xylose,

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arabinose and acetic acid, and UV spectrophotometry (Shimadzu SPD-M10AVP) at 280 nm

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for detecting furfural and hydroxymethylfurfural. Each of these analytes was subsequently

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converted to their original polysaccharide component by taking into account their


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corresponding anhydrous mass correction factors.

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2.3 Enzymatic hydrolysis Enzymatic hydrolysis of steam-exploded cane bagasse was initially investigated

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through a central composite rotatable design (CCRD) that involved four experiments at the

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vertices and four experiments at the axial points of a 22 factorial design plus three replicates

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at its center point for a total of 11 experiments. Enzyme loadings (Cellic CTec3) of 7.7 and

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38.6 FPU g-1 glucan and substrate TS of 10 and 20% (w v-1) were adopted in the vertices of

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this factorial design as shown in Table 1 (Design A). Statistical analysis was carried out with

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the software Statistica 8.0 and the proposed CCRD was used to generate models that were

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able to describe changes in the fractal kinetic parameters k and h for both glucan and xylan

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conversions (see below for details).

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Table 1

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Total cellulase activity measured as filter paper units (FPU) was determined by the

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I.U.P.A.C. method 30 with the adaptations proposed by Schwald et al. 31. All hydrolysis

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experiments were carried out at 50 °C and 150 rpm in 125 mL Erlenmeyer flasks using 50

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mmol L-1 sodium acetate buffer (pH 5.2). Aliquots were collected in reaction times of 0, 3,

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6, 9, 12, 24, 48, 72 and 96 h, centrifuged at 10,000 g and analyzed in the same HPLC system

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mentioned above. External calibration was used for the quantitative analysis of cellobiose,

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glucose and xylose; however, cellobiose and glucose concentrations were accounted

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together as glucose equivalents ([GlcEq] = [glucose] + 1.0526 [cellobiose]). Glucan

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conversion was calculated by expressing GlcEq release in relation to the amount of glucans


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(mostly cellulose) that was present in the original steam-exploded material. However, small

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volume variations in the substrate hydrolysates were not taken into account and, for this

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reason, the resulting glucan conversions might have been a little overestimated.

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Another simple 22 experimental design (two variables in two levels plus three

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replicates at the center point) was carried out to evaluate the influence of Cellic HTec3

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hemicelullases on hydrolysis efficiency (Table 1, Design B). These experiments were also

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carried out in a rotary shaker incubator at 20% (w v-1) TS using 50 mmol L-1 sodium acetate

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buffer (pH 5.2) at 50 °C and 150 rpm. Aliquots were collected in 12, 24, 48, 72 and 96 h,

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centrifuged at 10,000 g and analyzed by HPLC. Quantitative analysis of cellobiose, glucose

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and xylose was carried out by external calibration.

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2.4 Fractal kinetics modeling The release of sugars (GlcEq and xylose) were fitted by fractal kinetics 20 using the pseudo first-order reaction equation originally proposed by Kopelman 32 (Equation 1),

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P(t) = S0[1-exp(-k · t1-h)]

(1)

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were P(t) is the product concentration (g L-1), S0 is the initial concentration of glucan or

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xylan (represented as glucose or xylose in g L-1), k is the time dependent rate coefficient, h is

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the fractal exponent, and t is the reaction time (h).

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2.5 Apparent viscosity measurements Rheological measurements were performed in a stress/shear-rate controlled DHR-2 rheometer (TA instruments) using a Peltier concentric cylinder system. The geometry


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consisted of a four-bladed vane rotor, 42 mm in height and 28 mm in diameter, placed in a

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30 mm diameter cup containing the sample, which resulted in a narrow gap. The vane

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shaped rotor was used to reduce wall slip. The temperature was controlled and all

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rheological experiments were carried out at the same temperature used for enzymatic

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hydrolysis, i.e., 50 °C. In order to minimize evaporation, the cup was covered with a solvent

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trap.

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Enzymatic hydrolyses were carried out in shake-flasks under the selected conditions

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of the CCRD (Table 1, Design A). The reaction was stopped after 3, 6, 9, 12 and 24 h of

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hydrolysis and the suspension was transferred immediately to the rheometer for measuring

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changes in its rheological properties. To obtain consistent rheological data and to eliminate

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thixotropic effects after loading the sample into the measuring device, all suspensions were

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pre-sheared to rupture any fiber bundles, followed by a relaxation time for the partial

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recovery their fiber structure. Steady-state flow curves were obtained by a logarithmic shear

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rate sweep of 1-1000 s-1, from which the apparent viscosity of the fiber suspension was

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obtained.

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3. RESULTS AND DISCUSSION

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3.1. Chemical characterization of sugarcane bagasse Both untreated and pretreated sugarcane bagasse were characterized for their

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chemical composition (Table 2) and these values were used to calculate both pretreatment

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and hydrolysis yields. Both glucan (measured as anhydroglucose) and lignin contents were

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higher in the steam-treated materials as a result of the high susceptibility of hemicelluloses

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to acid hydrolysis. The xylan content decreased from 16.7 wt.% in the native material to 3.4

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wt.% after pretreatment. Also, acetyl groups and arabinose were not detectable in the 8 ACS Paragon Plus Environment

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pretreated cane bagasse acid hydrolysates.

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Table 2

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Similar carbohydrate and lignin contents were observed in other studies involving

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steam explosion of sugarcane bagasse, even when more drastic pretreatment conditions were

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applied. Ewanick and Bura 33 carried out pretreatment at 205 °C for 10 min to produce

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substrates with glucan, hemicellulose and total lignin contents of 51.3, 3.6 and 33.9 wt.%,

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respectively. Rocha et al. 34 pretreated sugarcane bagasse for 15 min at 190 °C and obtained

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substrates with 58.0, 4.5 and 33.0 wt.% of glucan, hemicellulose and total lignin contents,

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respectively. Amores et al. 35 found similar glucan (58.1 wt.%) and lignin (28.5 wt.%)

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contents in pretreated samples that were steam-exploded at 200 °C for 5 min but their

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hemicellulose content (14.0 wt.%) was much higher; however, the raw material used for

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pretreatment had a high hemicellulose content as well (25.0 wt.%). Sugar losses after acid

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hydrolysis have been attributed to pentose and hexose dehydration under acidic conditions to

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form furfural and hydroxymethylfurfural (HMF), respectively. In cane bagasse acid

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hydrolysates, HMF derives from hexoses such as glucose and galactose whereas furfural

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comes from pentoses such as xylose and arabinose. On the basis of this clear uncertainty in

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their origin, the quantification of furfural and HMF in acid hydrolysates was indicated in

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Table 2 as unidentified pentoses and hexoses, respectively.36

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The mass recovery yield after steam explosion was 86.2 wt.%, being 65.5 wt.% in

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the water-insoluble fraction and 20.7 wt.% in the water-soluble fraction. The mass losses

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observed during pretreatment (13.8 wt.%) were attributed to the volatilization of extractives

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and byproducts (furfural, HMF and acetic acid) that are hard to recover from the high


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pressure steam reactor.37, 38 Also, furan compounds derived from sugar dehydration and

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phenolic compounds derived from lignin may be partially involved in condensation

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reactions that lead to the accumulation of acid-insoluble polymeric materials.39 Nevertheless,

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a total glucan recovery of 98.9 wt.% was obtained in this study, with most of it being found

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in the water-insoluble fraction (95.8 wt.%).

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3.2. Enzymatic hydrolysis of steam-treated cane bagasse

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Figure 1 shows the enzymatic hydrolysis profiles of steam-exploded materials that

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were derived from the proposed experimental conditions of the CCRD. The largest glucan

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conversions in 48 h of hydrolysis were obtained from experiments A7, A9 and A10 (Figure

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1a), which corresponded to 57.5, 85.5 and 44.6 g L-1 GlcEq, respectively (Figure 1b).

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Cellobiose was always a minor component in GlcEq measurements. However, cellobiose

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accumulation increased with time in all hydrolysis runs, reaching the highest level when

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high enzyme loadings were used at high levels of substrate TS. In average, cellobiose

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accounted for 1.7 ± 0.9% of GlcEq for all hydrolysis data involved in the CCRD (additional

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data are given in Table S1 and S2 of the Supporting Information) but, for experiments

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carried out at the highest substrate TS and enzyme loadings, cellobiose exceeded the IC50

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inhibitory levels proposed by Teugjas and Väljamäe 39 for Trichoderma reesei

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cellobiohydrolases (2.6 mmol L-1 or 0.9 g L-1). Although not necessarily applicable to this

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study, this information suggests that Cellic CTec3 may need some β-glucosidase

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supplementation to avoid end-product inhibition, particularly at longer reaction times.

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Figure 1

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A good correlation between substrate TS and both glucan conversion [Conversion

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(%) = 0.098 TS2 – 5.157 TS + 115.440; R2 > 0.99] and glucose release [Glc (g L-1) = -0.044

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TS2 + 3.234 TS + 15.570; R2 > 0.99] was observed in experiments A3, A4, A5 (center

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point), A10 and A11, in which the same enzyme loading of 23.1 FPU g-1 glucan was applied

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for hydrolysis. In general, the observed decrease in glucan conversion at high TS was

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attributed to poor mass and heat transfers in viscous slurries, which is connected to the lack

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of free water in the reaction environment.41 Likewise, the applied enzyme loading (EL)

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correlated well with glucan conversion [Conversion (%) = - 0.028 EL2 + 2.951 EL + 7.154,

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R2 > 0.99] and glucose release [Glc (g L-1) = - 0.026 EL2 + 2.657 EL + 6.431; R2 > 0.99] for

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experiments carried out at 15% TS with different enzyme loadings of 1.4 (A8), 23.1 (A3, 4,

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5) and 44.9 (A9) FPU g-1 glucan. The best fit of the experimental data was always obtained

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through a second order polynomial regression, already revealing the non-linear correlation

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that exists among the experimental data. Nevertheless, the observed differences in glucan

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conversion (in %) and glucose release (in g L-1) were strongly related to the

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enzyme/substrate ratio that was used for hydrolysis. Hydrolysis under different conditions

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using almost the same enzyme/substrate ratio, as observed in experiments A9 (44.9 FPU g-1

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glucan at 15% TS) and A10 (23.1 FPU g-1 glucan at 8% TS), resulted in similar patterns of

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glucan conversion but completely different glucose releases. In addition, experiments at the

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highest enzyme/substrate ratio (A7) resulted in both the fastest initial hydrolysis rates

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(Figure 1) and the lowest apparent viscosity observed in this study (Table 4).

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In general, the amount of glucose released at 10 and 15% TS in 48 h (A7 and A9,

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respectively) was higher than those obtained from liquid hot water pretreated olive tree

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prunings at 20% (w v-1) TS using 65.8 FPU g-1 glucan, which produced 52 g L-1 glucose in

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72 h of enzymatic hydrolysis.16 On the other hand, Rosgaard et al. 42 obtained similar sugar


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concentrations (78 g L-1) from steam-treated barley straw at 15% (w w-1) TS using 12.9 FPU

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g-1 glucan but only after 72 h of hydrolysis.

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Experiments A1 and A8 had the lowest glucan conversion due the use of low enzyme

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loadings at high TS. All other conditions achieved glucan conversions of approximately

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70% in 48 h (Figure 1a). According to Arantes and Saddler 1, when 50-70% of the original

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material has been hydrolyzed, a decrease in the reaction rate is observed due the

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accumulation of adverse factors such as the higher recalcitrance of the remaining cellulosic

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material, high levels of end-product inhibition, and enzyme losses due to their irreversible

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and/or unproductive adsorption onto lignin-carbohydrate fragments, which is aggravated by

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the build-up of lignin in the reaction mixture. By contrast, trends of decreasing viscosities at

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longer reaction times are a result of the collapse of the cell wall structure, which also

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involves a decrease in cellulose degree of polymerization and an increase in fiber porosity.42

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Hydrolysis at 20% TS using 38.6 FPU g-1 glucan (A2) resulted in the highest GlcEq

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release of 110 g L-1 after 96 h of hydrolysis. However, around 52 g L-1 GlcEq was already

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achieved in 12 h in several experiments including A2 (Figure 1b). While 110 g L-1 is

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promising for separate hydrolysis and fermentation, 52 g L-1 in 12 h seems to be very good

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for fed-batch hydrolysis or simultaneous hydrolysis and fermentation.

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Xylan hydrolysis profiles were similar to those of glucans, with higher conversions

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being achieved in experiments A7, A9 and A10 (Figure 2a). The highest xylose

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concentration of 6.6 g L-1 was obtained in A2 and this corresponded to an 85% conversion at

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96 h of hydrolysis (Figure 2b). In general, xylans are more accessible to enzymatic

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hydrolysis than glucans such as cellulose. However, when embedded in the structure of

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pretreated cellulosic materials, xylans are not immediately available for hydrolysis but they

291

are gradually exposed to the concerted action of the enzymes as glucan hydrolysis goes on.


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Therefore, xylans are present in the chemical composition of partially hydrolysed cellulosic

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materials even after most of the cellulose component had been hydrolysed to soluble sugars.

294 295

Figure 2

296 297

The fractal kinetic parameters k and h as well as the R2 values for the regression

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models of Figures 1 and 2 are given in Table 3. The high R2 values for all hydrolysis

299

experiments reveal the ability of the fractal kinetic model to describe both glucan and xylan

300

conversions as a function of time; therefore, both k and h were analyzed in relation to the

301

process variables using second-order model equations and ANOVA to evaluate the

302

adequacy of the fitted models (Table S3 of the Supporting Information). The pure error,

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calculated from the experimental error at the center point of the experimental design, ranged

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from 0.1 to 2.8% according to the total sum of squares, indicating the good reproducibility

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of the experimental data. Based on the F-test, the resulting models are predictive since the

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calculated F-value for all responses are greater than the corresponding tabulated F-value

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within the confidence level of 95%. Also, the high R2 values demonstrate that most of the

308

variance was explained by the fitted models.

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Table 3

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Equations 2 and 3 were generated to fit the fractal kinetic parameters of glucan

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conversion (kGlc and hGlc) while Equations 4 and 5 were used to fit the fractal kinetic

314

parameters of xylan conversion (kXyl and hXyl). The coded models expressed in these

315

equations were subsequently used to generate the response surfaces of Figures 3 and 4,


Energy & Fuels

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316

Page 14 of 38

where EL is the enzyme loading and TS is the substrate total solids.

317 318

kGlc = 0.098 + 0.047 EL - 0.003 EL2 - 0.027 TS + 0.031 TS2 - 0.044 EL x TS

(2)

319

hGlc = 0.287 - 0.040 EL + 0.012 EL2 + 0.059 TS + 0.054 TS2 - 0.060 EL x TS

(3)

320

kXyl = 0.172 + 0.083 EL - 0.004 EL2 - 0.029 TS + 0.020 TS2 - 0.052 EL x TS

(4)

321

hXyl = 0.468 - 0.030 EL + 0.012 EL2 + 0.044 TS + 0.036 TS2 - 0.053 EL x TS

(5)

322 323

Figure 3

324

Figure 4

325 326

As shown in Figures 3a and 4a, the rate coefficient k for both glucan and xylan

327

conversion was strongly affected by changes in the applied enzyme loading (see Figure S1

328

in Supporting Information). However, this effect was more pronounced in reactions that

329

were carried out at lower substrate TS. For instance, at 10% TS (A6 and A7 in Table 1), k

330

increased from 0.083 to 0.299 h-1 for glucan conversion and from 0.105 to 0.386 h-1 for

331

xylan conversion when the enzyme loading was increased from 7.7 to 38.6 FPU g-1 glucan

332

but, at 20% TS, increased enzyme loadings (A1 and A2) gave similar k values for glucan

333

conversion (0.102 and 0.108 h-1) and slightly higher k values for xylan conversion (0.134

334

and 0.194 h-1), respectively. This was so because, at high TS, the mass transport of products

335

and enzymes is much more restricted by the low availability of water in the fiber slurry. The

336

k value decreased from 0.299 to 0.108 h-1 for glucans and from 0.386 to 0.194 h-1 for xylans

337

when the substrate TS increased from 10 to 20% at 38.6 FPU g-1 glucan (A7 and A2,

338

respectively). Also, this effect was observed in smaller proportions at 23.1 FPU g-1 glucan

339

(A10 and A11). By contrast, the negative effect of TS on k is apparently overcome at low


Page 15 of 38

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Energy & Fuels

340

enzyme loadings of 7.7 FPU g-1 glucan (A1 and A6) and this may be associated with higher

341

substrate availability and less enzyme traffic jamming at these hydrolysis conditions. These

342

observations justify the statistical significance of both the TS quadratic term and the binary

343

interaction between TS and enzyme loading, which was also observed in Pareto Charts for

344

the k parameter (see Figure S1 in Supporting Information).

345

Wang and Feng 21 reviewed the effects of enzyme loading and non-ionic surfactants

346

on the enzymatic hydrolysis of lignocellulosic materials using fractal analysis. Changes in

347

the fractal exponent h were associated with substrate accessibility, with good substrates for

348

hydrolysis tending to present low h values.43 However, there is still a lack of studies

349

involving the correlation between h and the initial substrate content at high TS using

350

different enzyme loadings. Response surfaces for the fractal exponent h are shown in

351

Figures 3b and 4b for glucan and xylan conversions, respectively.

352

Regarding the fractal exponent h, substrate TS was the most significant effect for

353

xylan conversion and the only significant effect for glucose conversion (see Figure S1 in

354

Supporting Information). High substrate TS resulted in high fractal exponents in both cases

355

and this can be observed by comparing experiments A1 and A6, in which 7.7 FPU g-1 glucan

356

were applied at 20 and 10% TS (h values of 0.536 and 0.295, respectively), and A10 and

357

A11, in which 23.1 FPU g-1 glucan were applied at 8 and 22% TS (h values of 0.305 and

358

0.475, respectively). In addition to end-product inhibition, the accumulation of lignin in the

359

reaction mixture is higher at high TS and this may have increased the non-productive and/or

360

irreversible binding of enzymes by hydrophobic interactions. By contrast, this was not

361

observed at high enzyme loadings (A2 and A7) because the extent of enzyme adsorption on

362

lignin and lignin-carbohydrate fragments was probably not a critical barrier to achieve

363

relatively good hydrolysis performances.


Energy & Fuels

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364

Using substrate TS not higher than 5%, some studies have shown that the fractal

365

exponent increases with an increase in enzyme loading and this has been attributed to the

366

crowding of cellulases on the cellulose surface, as well as to the formation of aggregates due

367

to protein-to-protein interactions.21, 44 This effect was also observed in experiments A6 and

368

A7 at 10% TS but the use of higher fiber slurries of 15% TS (A8, A3, 4, 5 and A9) and 20%

369

TS (A1 and A2) resulted in the opposite trend.

370

Finally, all experiments showed higher k and h values for xylan conversion when

371

compared to glucan conversion. Such high k values are due the high hemicellulase content

372

of Cellic CTec3, which is able to hydrolyse most of the hemicellulose components of the

373

steam-exploded cane bagasse. By contrast, high h values are probably expressing the low

374

accessibility of residual hemicelluloses that are embedded in and more closely bound to the

375

lignocellulosic matrix, being accessible only after glucans are gradually removed by the

376

concerted action of the cellulolytic enzymes.

377 378

3.3 Viscosity changes during enzymatic hydrolysis

379

Rheological measurements for all CCRD conditions showed that hydrolysis caused a

380

gradual decrease in substrate apparent viscosity and this was primarily associated to changes

381

in substrate TS and fiber morphology. Also, the substrate apparent viscosity decreased with

382

increasing shear rate at all times used for hydrolysis, indicating the non-Newtonian “shear-

383

thinning” behavior that is shown in Figure 5. Previous works with pretreated slurries have

384

reported similar pseudoplastic behavior.12, 14 The shear-thinning nature of the material was

385

explained by Ebeling et al. 45, who reported that the orientation of cotton cellulose

386

microcrystals is dependent on shear rate. Above a certain shear rate value, changes in

387

apparent viscosity are less pronounced because cellulose microcrystals align horizontally


Page 16 of 38

Page 17 of 38

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Energy & Fuels

388

along the shear direction and the resistance to the flow becomes approximately constant.

389

However, this orientation phenomenon is completely reversible. On the other hand, Du et al.

390

46

391

lignocellulose, which are broken by an increase in the shear rate. Thus, the density of the

392

entanglements is reduced and the apparent viscosity decreases.

attributed this pseudoplasticity to the formation of highly entangled structures of

393 394

Figure 5

395 396

The apparent viscosity of fiber slurries also undergoes dynamic and dramatic

397

changes with time due to the progressive action of cellulolytic enzymes. Figure 5 shows that

398

the apparent viscosity is reduced just about one order of magnitude at every 6 h of

399

hydrolysis. This change is due to a loss in lignocellulose structure by the combined effects

400

of cell wall deconstruction and a gradual decrease in the average cellulose and hemicellulose

401

chain lengths.42 Various chemical bonds within the fiber suspension are hydrolyzed while

402

several components are solubilized into the liquid phase, thus reducing the contact between

403

neighboring particles.14, 47

404

Variations in viscosity at shear rates higher than 100 s-1 are probably due to a gradual

405

change from laminar to turbulent flow during the tests. For this reason, changes in apparent

406

viscosity were compared at 10 s-1 and these data are presented in Table 4 as function of

407

hydrolysis time. As expected, the initial apparent viscosities were proportional to the

408

substrate TS, providing values at 15, 20 and 22% TS that were approximately two, four and

409

five times higher than that of 10% TS slurries, respectively. For slurries of 15% TS and

410

higher, high viscosities can be due to the lower availability of free-water and greater

411

hydrogen bonding among cellulose chains. Also, as the substrate TS increases, the average


Energy & Fuels

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412

distance between particles decreases, leading to an increase in the particle-to-particle

413

interactions.42 This phenomenon results in more friction and resistance to flow and therefore

414

to higher apparent viscosities. Furthermore, as a result of the pretreatment process, fiber

415

porosity is increased by partial hemicellulose and lignin removal, improving both the

416

accessibility of hydrolases to fiber polysaccharides and the fiber swelling by water

417

absorption, which also reduces the free-water content of the slurry.41

418 419

Table 4

420 421

Different enzyme/substrate ratios led to a considerable reduction in substrate

422

viscosity within the first 3 h of hydrolysis but the most significant changes were observed in

423

experiments where the highest enzyme loadings were used. Table 4 shows that reductions of

424

81%, 97%, and 89% were achieved for runs A2, A7 and A9, respectively. In general, higher

425

substrate viscosities were observed at lower glucan conversions but these values dropped

426

rapidly due to a combination of a gradual reduction in substrate TS and cellulose

427

fragmentation into smaller particles.

428 429

3.4 Effect of Cellic HTec3 on hydrolysis efficiency

430

According to Figures 1 and 2, glucans and xylans followed similar hydrolysis

431

profiles and the latter were present in partially hydrolysed substrates up until 96 h of

432

hydrolysis. For this reason, Cellic HTec3 hemicellulases (10% in relation to Cellic CTec3)

433

were added to the hydrolysis mixture to investigate their boosting effect on glucan

434

conversion by facilitating the removal of xylan oligomers from the surface of cellulose

435

aggregates. These experiments were carried out at 20% TS as part of a typical 22 factorial


Page 18 of 38

Page 19 of 38

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Energy & Fuels

436

design (see conditions at Table 1, Design B). The results reported in Figure 6a were

437

statistically different for different levels of Cellic CTec3 (p < 0.05 in the ANOVA) and this

438

motivated the use of the Tukey’s test to differentiate the individual reaction responses. Until

439

48 h of hydrolysis, there were no differences (p > 0.05) in both response variables when

440

hemicellulases were added to the reaction medium (see experiments B1 and B6; B2 and B7

441

in Figure 6). However, at 72 h, significant differences (p < 0.05) were observed at the

442

highest levels of cellulase activity (see experiments B2 and B7). As a result, the use of Cellic

443

HTec3 at the highest level (10%) caused an increase of 8% in glucan conversion, which

444

corresponds to a GlcEq release of 10.5 g L-1 after 72 or 96 h of hydrolysis. It is also

445

noteworthy that, in 96 h, the combination of Cellic CTec3 and Cellic HTec3 (B3, B4 and

446

B5) produced the same glucan conversion and GlcEq release that was produced by Cellic

447

CTec3 alone at the highest level of the experimental design (B7).

448 449

Figure 6

450 451

Regardless of the hydrolysis time, all xylan conversions and xylose concentrations

452

described in Figure 6b were statistically different in the ANOVA (p < 0.05) for different

453

levels of Cellic CTec3. Likewise, at the highest levels of enzyme loading (B2 and B7),

454

Cellic HTec3 resulted in significant differences (p < 0.05) in all reaction times except 72 h,

455

providing a 9.4% higher xylan conversion and an average increase of 0.7 g L-1 in xylose

456

concentration. However, at the lowest levels of Cellic CTec3 (B1 and B6), supplementation

457

with Cellic HTec3 activity was only statistically meaningful after 96 h of hydrolysis, in

458

which an increase of 8.7% was observed in xylan conversion.

459

Cellic HTec3 had a boosting effect on the enzymatic hydrolysis of cane bagasse


Energy & Fuels

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460

glucans regardless of the low hemicellulose content of the steam-treated material. This

461

unexpected performance is justified by the relatively high recalcitrance of the hemicellulose

462

fraction that withstood pretreatment and fractionation. Being more intimately associated

463

with cane bagasse glucans and/or lignin, these residual hemicellulose fractions are more

464

difficult to access and their hydrolysis progresses slowly as substrate glucans are gradually

465

removed.

466 467

4. CONCLUSION

468 469

Successful ethanol fermentation is only achievable if high sugar concentrations are

470

obtained upon enzymatic hydrolysis. Cellic CTec3 was able to release more than 100 g L-1

471

GlcEq from steam-treated cane bagasse in 72 h at 20% TS using 38.6 FPU g-1 glucan.

472

Biomass slurries exhibited poor rheological properties above 15% TS, however, a

473

remarkable decrease in substrate apparent viscosity was observed after 3 h of hydrolysis.

474

Fractal kinetics provided a good fit of both glucan and xylan hydrolysis data. The rate

475

coefficient k and the fractal exponent h were strongly affected by changes in the applied

476

enzyme loading and these trends were useful to predict substrate accessibility and the ideal

477

enzyme loading for optimal hydrolysis performance at high substrate TS. Finally, Cellic

478

HTec3 improved the total glucan conversion by 8% but only when a relatively high enzyme

479

loading was used for hydrolysis.

480 481

Supporting Information

482

Table S1. Release of glucose equivalents from steam-treated sugarcane bagasse as a function

483

of hydrolysis time under the conditions given in the CCRD experimental design.


Page 20 of 38

Page 21 of 38

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Energy & Fuels

484

Table S2. Release of cellobiose from steam-treated sugarcane bagasse as a function of

485

hydrolysis time under the conditions given in the experimental design of the CCRD.

486

Table S3. Analysis of variance (ANOVA) of the fractal kinetic parameters that were derived

487

from the central composite rotatable design (CCRD, Design A).

488

Figure S1. Pareto chart describing the primary and secondary effects of different total solids

489

and enzyme loadings in: (a) parameter k (h-1) and (b) parameter h for glucan conversion; and

490

(c) parameter k (h-1) and (d) parameter h for xylan conversion.

491

Figure S2. Correlation between the observed and predicted values for the fractal parameters

492

(a) k (h-1) and (b) h for glucan conversion and (c) k (h-1) and (d) h for xylan conversion.

493

Figure S3. Apparent viscosity at 10 s-1 of steam-treated sugarcane bagasse during enzymatic

494

hydrolysis with Cellic CTec3 under the conditions given in Table 1 (CCRD).

495 496

Corresponding author

497

*E-mail: [email protected]. Telephone: +55 4133613175.

498

Note: the authors declare no competing financial interest.

499 500

Acknowledgements

501

The authors are grateful to CNPq (grants 551404/2010-8 and 311554/2011-3) and to

502

the INCT in Energy and Environment for providing financial support to carry out this study,

503

as well as to Novozymes Latin America (Araucária, PR, Brazil) for donating the enzyme

504

preparations used for hydrolysis. Also, the authors wish to thank CAPES for providing

505

scholarships to M.B.U. and D.H.F. and CYTED (Red Provalor, grant 312RT0456) for

506

funding the student exchange between UFPR and UPB.

507 508

References 21 ACS Paragon Plus Environment

Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

509

(1)

Arantes, V.; Saddler, J. N. Cellulose accessibility limits the effectiveness of

510

minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic

511

substrates. Biotechnol. Biofuels 2011, 4:3.

512

(2)

513

W.; Foust, T. D. Biomass recalcitrance: engineering plants and enzymes for biofuels

514

production. Science 2007, 315, 804-807.

515

(3)

516

economic estimates for the production cost of lignocellulosic bio-ethanol. Renewable

517

Sustainable Energy Rev. 2013, 26, 307-321.

518

(4)

519

Bogel-Łukasik, R.; Andreaus, J.; Ramos, L. P. Current Pretreatment Technologies for the

520

Development of Cellulosic Ethanol and Biorefineries. ChemSusChem 2015, 8, 3366-3390.

521

(5)

522

an efficient bioethanol production process based on enzymatic hydrolysis: a review.

523

Bioresour. Technol. 2010, 101, 4851-4861.

524

(6)

525

combined with an intermediate separation of fiber cells-optimization of fermentation of corn

526

straw hydrolysates. Bioresour. Technol. 2012, 121, 100-104.

527

(7)

528

method coupling steam explosion and mechanical carding fractionation. Bioresour. Technol.

529

2013, 139, 59-65.

530

(8)

531

biochemical pathway: a review. Energy Convers. Manage. 2011, 52, 858-875.

Himmel, M. E.; Ding, S.-Y.; Johnson, D. K.; Adney, W. S.; Nimlos, M. R.; Brady, J.

Chovau, S.; Degrauwe, D.; Van der Bruggen, B. Critical analysis of techno-

Silveira, M. H. L.; Morais, A. R. C.; da Costa Lopes, A. M.; Olekszyszen, D. N.;

Alvira, P.; Tomás-Pejó, E.; Ballesteros, M.; Negro, M. Pretreatment technologies for

Zhang, Y.; Fu, X.; Chen, H. Pretreatment based on two-step steam explosion

Wang, N.; Chen, H.-Z. Manufacture of dissolving pulps from cornstalk by novel

Balat, M. Production of bioethanol from lignocellulosic materials via the


Page 22 of 38

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

532

(9)

Lee, J. M.; Shi, J.; Venditti, R. A.; Jameel, H. Autohydrolysis pretreatment of

533

Coastal Bermuda grass for increased enzyme hydrolysis. Bioresour. Technol. 2009, 100,

534

6434-6441.

535

(10)

536

lignocellulosic biomass through biorefining. Prog. Energy Combust. Sci. 2012, 38, 583-598.

537

(11)

538

Ethanol from Softwood: Comparison of SSF and SHF and Identification of Bottlenecks.

539

Biotechnol. Prog. 2003, 19, 1109-1117.

540

(12)

541

biorefinery applications. J. Rheol. 2009, 53, 877-892.

542

(13)

543

concentration and yield stress of biomass slurries during enzymatic hydrolysis at high‐

544

solids loadings. Biotechnol.Bioeng. 2009, 104, 290-300.

545

(14)

546

slurries at high solids concentrations - effects of saccharification and particle size.

547


548

(15)

549

loadings - A review. Biomass Bioenergy 2013, 56, 526-544.

550

(16)

551

solid loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated olive

552

tree biomass. Process Biochem. 2007, 42, 1003-1009.

553

(17)

554

polymers to constrain water with the potential to inhibit cellulose saccharification.

555

Biotechnol. Biofuels 2014, 7, 159.

Zhu, J.; Zhuang, X. Conceptual net energy output for biofuel production from

Wingren, A.; Galbe, M.; Zacchi, G. Techno‐Economic Evaluation of Producing

Knutsen, J. S.; Liberatore, M. W. Rheology of high-solids biomass slurries for

Roche, C. M.; Dibble, C. J.; Knutsen, J. S.; Stickel, J. J.; Liberatore, M. W. Particle

Viamajala, S.; McMillan, J. D.; Schell, D. J.; Elander, R. T. Rheology of corn stover

Modenbach, A. A.; Nokes, S. E. Enzymatic hydrolysis of biomass at high-solids

Cara, C.; Moya, M.; Ballesteros, I.; Negro, M. J.; González, A.; Ruiz, E. Influence of

Selig, M. J.; Thygesen, L. G.; Felby, C. Correlating the ability of lignocellulosic


Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

556

(18)

Hsieh, C. C.; Cannella, D.; Jørgensen, H.; Felby, C.; Thygesen, L. G. Cellulase

557

inhibition by high concentrations of monosaccharides. J. Agric. Food Chem. 2014, 62, 3800-

558

3805.

559

(19)

560

enzymatic catalysis: Contributions from the fractal and jamming (overcrowding) effects.

561

Appl. Catal. A 2007, 317, 70-81.

562

(20)

563

hydrolysis can be described in terms of fractal-like kinetics. Biotechnol. Bioeng. 2003, 84,

564

254-257.

565

(21)

566

cellulose under different conditions. Bioresour. Technol. 2010, 101, 7995-8000.

567

(22)

568

saccharification of acid pretreated corn stover: empirical and fractal kinetic modelling.

569


570

(23)

571

Ethanol production from sugarcane bagasse using phosphoric acid-catalyzed steam

572

explosion. J. Braz. Chem. Soc. 2016, 27, 1889-1898.

573

(24)

574

whole sugarcane lignocellulosic biomass. Biotechnol. Biofuels 2015, 8:44.

575

(25)

576

C. Influence of mixed sugarcane bagasse samples evaluated by elemental and physical–

577

chemical composition. Ind. Crops Prod. 2015, 64, 52-58.

578

(26)

579

Templeton, D.; Wolfe, J. Determination of total solids in biomass and total dissolved solids

Xu, F.; Ding, H. A new kinetic model for heterogeneous (or spatially confined)

Väljamäe, P.; Kipper, K.; Pettersson, G.; Johansson, G. Synergistic cellulose

Wang, Z.; Feng, H. Fractal kinetic analysis of the enzymatic saccharification of

Wojtusik, M.; Zurita, M.; Villar, J. C.; Ladero, M.; Garcia-Ochoa, F. Enzymatic

Pitarelo, A. P.; da Fonseca, C. S.; Chiarello, L. M.; Gírio, F. M.; Ramos, L. P.

Pereira, S. C.; Maehara, L.; Machado, C. M. M.; Farinas, C. S. 2G ethanol from the

Moraes Rocha, G. J.; Nascimento, V. M.; Goncalves, A. R.; Silva, V. F. N.; Martín,

Sluiter, A.; Hames, B.; Hyman, D.; Payne, C.; Ruiz, R.; Scarlata, C.; Sluiter, J.;


Page 24 of 38

Page 25 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

580

in liquid process samples; Technical Report NREL/TP-510-42621. National Renewable

581

Energy Laboratory, Golden, 2008.

582

(27)

583

Determination of ash in biomass; Technical Report NREL/TP-510-42622. National

584

Renewable Energy Laboratory, Golden, 2008.

585

(28)

586

extractives in biomass; Technical Report NREL/TP-510-42619. National Renewable Energy

587

Laboratory, Golden, 2008.

588

(29)

589

Determination of structural carbohydrates and lignin in biomass; Technical Report

590

NREL/TP-510-42618. National Renewable Energy Laboratory, Golden, 2012.

591

(30)

Ghose, T. Measurement of cellulase activities. Pure Appl. Chem. 1987, 59, 257-268.

592

(31)

Schwald, W.; Chan, M.; Breuil, C.; Saddler, J. Comparison of HPLC and

593

colorimetric methods for measuring cellulolytic activity. Appl. Microbiol. Biotechnol. 1988,

594

28, 398-403.

595

(32)

Kopelman, R. Fractal Reaction Kinetics. Science 1988, 241, 1620-1626.

596

(33)

Ewanick, S.; Bura, R. The effect of biomass moisture content on bioethanol yields

597

from steam pretreated switchgrass and sugarcane bagasse. Bioresour. Technol. 2011, 102,

598

2651-2658.

599

(34)

600

explosion and diluted sulfuric acid pretreatments: Comparative study aiming the sugarcane

601

bagasse saccharification. Ind. Crops Prod. 2015, 74, 810-816.

Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.

Sluiter, A.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D. Determination of

Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D.

Rocha, G.; Gonçalves, A.; Nakanishi, S.; Nascimento, V.; Silva, V. Pilot scale steam


Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

602

(35)

Amores, I.; Ballesteros, I.; Manzanares, P.; Sáez, F.; Michelena, G.; Ballesteros, M.

603

Ethanol production from sugarcane bagasse pretreated by steam explosion. Eletronic J.

604

Energy Eviron. 2013, 1, 25-36.

605

(36)

606

biomass for biorefineries: comparative composition of carbohydrate and non-carbohydrate

607

components of bagasse and straw. Carbohydr. Polym. 2014, 114, 95-101.

608

(37)

609

M. H. L. Enzymatic hydrolysis of steam-exploded sugarcane bagasse using high total solids

610

and low enzyme loadings. Bioresour. Technol. 2015, 175, 195-202.

611

(38)

612

analysis of volatile organic compounds of sugarcane (Cachaça) and fruit spirits. Food Anal.

613

Methods 2013, 6, 978-988.

614

(39)

615

chloride catalyzed dehydration and degradation of glucose. Energy Fuels 2015, 29, 2387-

616

2393.

617

(40)

618

cellulose substrates. Bioresour. Technol. 2013, 6, 104.

619

(41)

620

enzymatic saccharification of pretreated corn stover slurries. Energy Fuels 2009, 23, 492-

621

497.

622

(42)

623

substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw. Appl.

624

Biochem. Biotechnol. 2007, 143, 27-40.

Szczerbowski, D.; Pitarelo, A. P.; Zandoná Filho, A.; Ramos, L. P. Sugarcane

Ramos, L. P.; da Silva, L.; Ballem, A. C.; Pitarelo, A. P.; Chiarello, L. M.; Silveira,

Capobiango, M.; Oliveira, E. S.; Cardeal, Z. L. Evaluation of methods used for the

Zhang, X.; Hewetson, B. B.; Mosier, N. S. Kinetics of maleic acid and aluminum

Teugjas, H.; Väljamäe, P. Product inhibition of cellulases studied with 14 C-labeled

Dasari, R. K.; Dunaway, K.; Berson, R. E. A scraped surface bioreactor for

Rosgaard, L.; Andric, P.; Dam-Johansen, K.; Pedersen, S.; Meyer, A. S. Effects of


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Energy & Fuels

625

(43)

Aguiar, R. S.; Silveira, M. H. L.; Pitarelo, A. P.; Corazza, M. L.; Ramos, L. P.

626

Kinetics of enzyme-catalyzed hydrolysis of steam-exploded sugarcane bagasse. Bioresour.

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Technol. 2013, 147, 416-423.

628

(44)

629

surfactants to eliminate lignin inhibition in enzymatic saccharification of cellulose.

630


631

(45)

632

Shear-induced orientation phenomena in suspensions of cellulose microcrystals, revealed by

633

small angle X-ray scattering. Langmuir 1999, 15, 6123-6126.

634

(46)

635

liquefaction and saccharification of pretreated corn stover at high-solids concentrations in a

636

horizontal rotating bioreactor. Bioprocess Biosyst. Eng. 2014, 37, 173-181.

637

(47)

638

and rheological properties in cellulosic slurries. Appl. Biochem. Biotechnol. 2007, 137, 289-

639

299.

Wang, Z.; Xu, J.-H.; Feng, H.; Qi, H. Fractal kinetic analysis of polymers/nonionic

Ebeling, T.; Paillet, M.; Borsali, R.; Diat, O.; Dufresne, A.; Cavaille, J.; Chanzy, H.

Du, J.; Zhang, F.; Li, Y.; Zhang, H.; Liang, J.; Zheng, H.; Huang, H. Enzymatic

Dasari, R. K.; Berson, R. E. The effect of particle size on hydrolysis reaction rates

640


Energy & Fuels

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641

Table 1. Matrices of the experimental designs that were carried out to investigate the

642

enzymatic hydrolysis of steam-treated sugarcane bagasse (coded values are given in brackets

643

and experiments 3,4 and 5 are the center points of the experimental designs).

644

Design A

Design B

CTec3 loading

Total solids

Run

645

CTec3 loading

HTec3

(FPU g-1 glucan)

loading (%)*

Run (FPU g-1 glucan)

(% w v-1)

A1

7.7 (-1)

20 (+1)

B1

7.7 (-1)

10 (+1)

A2

38.6 (+1)

20 (+1)

B2

38.6 (+1)

10 (+1)

A3, 4, 5

23.1 (0)

15 (0)

B3,4,5

23.1 (0)

5 (0)

A6

7.7 (-1)

10 (-1)

B6

7.7 (-1)

0 (-1)

A7

38.6 (+1)

10 (-1)

B7

38.6 (+1)

0 (-1)

A8

1.4 (-1.41)

15 (0)

-

-

-

A9

44.9 (+1.41)

15 (0)

-

-

-

A10

23.1 (0)

8 (-1.41)

-

-

-

A11

23.1 (0)

22 (+1.41)

-

-

-

* Values expressed in relation to the mass of Cellic CTec3.


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Energy & Fuels

646

Table 2. Chemical composition of untreated and pretreated sugarcane bagasse.

647

Component (wt.%)

Untreated

Pretreated

Anhydroglucose1

39.5 ± 0.7

54.5 ± 0.9

Anhydroxylose2

16.7 ± 0.4

3.4 ± 0.2

Anhydroarabinose2

1.5 ± 0.03

bdl8

Acetyl groups2

4.0 ± 0.1

bdl

Unidentified anhydrohexoses3

0.69 ± 0.01

0.89 ± 0.19

Unidentified anhydropentoses4

2.99 ± 0.23

0.45 ± 0.02

Acid soluble lignin

5.5 ± 0.7

3.7 ± 0.2

Acid insoluble lignin

20.6 ± 0.7

30.2 ± 0.7

Ashes

5.1 ± 0.2

5.2 ± 0.5

Extractives in water

3.5 ± 0.1

nd8

Extractives in ethanol

1.7 ± 0.1

nd

Total

101.8

98.3

Total glucan content5

40.2

55.4

Total hemicellulose contet6

25.2

3.8

Total lignin content7

26.1

33.8

648

1

Present as β(1-4)-D-glucans (cellulose);

649

2

Present as heteroxylan components (hemicellulose);

650

3

Dehydration by-product from hexoses measured as hydroxymethylfurfural (HMF);

651

4

Dehydration by-product from pentoses measured as furfural;

652

5

Summation of anhydroglucose and unidentified hexoses that were detected a HMF;

653

6

Summation of anhydroxylose, anhydroarabinose, acetyl groups and unidentified pentoses

654

that were detected as furfural;

655

7

Summation of acid soluble and acid insoluble lignin;

656

8

bdl, below the detection limit of the method; nd, not determined. 29 ACS Paragon Plus Environment

Energy & Fuels

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Page 30 of 38

657

Table 3. Fractal kinetic parameters of the enzymatic hydrolysis data that was obtained from

658

steam-treated sugarcane bagasse under the conditions given in the experimental design of

659

the CCRD.

660

Run

Glucan conversion

Xylan conversion

k (h-1)

h

R2

k (h-1)

h

R2

A1

0.102

0.536

0.995

0.134

0.648

0.968

A2

0.108

0.330

0.999

0.194

0.496

0.991

A3, 4, 5 0.095 ± 0.012 0.272 ± 0.040

0.998

0.166 ± 0.019 0.471 ± 0.028

0.995

A6

0.083

0.295

0.999

0.105

0.443

1.000

A7

0.299

0.387

0.999

0.386

0.525

0.965

A8

0.014

0.352

0.997

0.036

0.555

0.992

A9

0.150

0.246

0.998

0.276

0.445

0.993

A10

0.178

0.305

0.999

0.238

0.499

0.993

A11

0.126

0.475

0.995

0.175

0.591

0.981

661 662


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Energy & Fuels

663

Table 4. Apparent viscosity at 10 s-1 of steam-treated sugarcane bagasse during enzymatic

664

hydrolysis with Cellic CTec3 under the conditions given in Table 1 (CCRD)*.

665

Apparent viscosity (Pa s) after enzymatic hydrolysis for: Run

666

0h

3h

6h

9h

12 h

24 h

A1

90.8

39.3

30.5

21.8

17.6

9.7

A2

97.0

18.1

9.4

4.0

1.8

0.4

A3, 4, 5

53.5 ± 8.1

15.7 ± 1.4

7.4 ± 0.8

2.6 ± 0.5

1.0 ± 0.1

0.4 ± 0.1

A6

26.0

16.8

7.9

2.5

1.1

0.3

A7

20.0

0.7

0.3

0.1

Enzymatic Hydrolysis of Steam-Treated Sugarcane Bagasse: Effect of

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