Accepted Manuscript

Title: Subcritical water hydrolysis of sugarcane bagasse: An

approach on solid residues characterization

Author: D. Lachos-Perez F. Martinez-Jimenez C.A. Rezende

G. Tompsett M. Timko T. Forster-Carneiro

PII: S0896-8446(15)30167-4
DOI: http://dx.doi.org/doi:10.1016/j.supflu.2015.10.019
Reference: SUPFLU 3488

To appear in: J. of Supercritical Fluids

Received date: 21-8-2015

Revised date: 8-10-2015
Accepted date: 28-10-2015

Please cite this article as: D. Lachos-Perez, F. Martinez-Jimenez, C.A. Rezende, G.

Tompsett, M. Timko, T. Forster-Carneiro, Subcritical water hydrolysis of sugarcane
bagasse: An approach on solid residues characterization, The Journal of Supercritical
Fluids (2015), http://dx.doi.org/10.1016/j.supflu.2015.10.019

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 1 Subcritical water hydrolysis of sugarcane bagasse: an approach on solid residues
2 characterization
3 Lachos-Perez D. , Martinez-Jimenez F.1, Rezende C.A.2, Tompsett, G.3, Timko
1

4 M.3, Forster-Carneiro T.1*

1
5 * School of Food Engineering, University of Campinas (UNICAMP), Rua

t
6 Monteiro Lobato, n.80. 13083-862, Campinas- SP, Brazil.

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2
7 Institute of Chemistry, University of Campinas, UNICAMP, 13083-862,
8 Campinas- SP, Brazil.

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9 Department of Chemical Engineering. Worcester Polytechnic Institute. 100
10 Institute Road, Goddard Hall 123. Worcester MA 01609.

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11

12 Abstract an
13 Reducing sugars obtained from sugarcane bagasse, as a waste biomass energy
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14 precursor, can be further transformed into fuel alcohol by fermentation or gaseous fuel

15 by gasification. In this work, subcritical water process was used as an environmentally

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16 friendly solvent for the hydrolysis of sugarcane bagasse with the aim of producing
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17 reducing sugars using residue bagasse from a sugarcane biorefinery. Hydrolysis in

18 subcritical water performance was studied under semi-continuous unit conditions in a

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19 110 mL reactor. Hydrolysis was carried out using different sample loadings (3 and 5 g),

20 flow-rates (9 and 12.5 mL min-1), temperatures (100, 150, 200 and 250 °C) and
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21 pressures (5, 10, 15 MPa). The highest reducing sugar yields wereobtained at

22 temperature above 200 °C, withthe highest reducing sugar yield reaching 15.5%.

23 Scanning electron microscopy was used to analyze the sugarcane bagasse undergoing

24 hydrolysis. Diffuse reflectance infrared spectroscopy was used to characterize the

25 residual solids, with results consistent with the removal of hemicellulose during

26 hydrolysis.

Page 1 of 42
27 Keywords: Sugarcane bagasse, reducing sugars, subcritical water hydrolysis,

28 FESEM, DRIFTS.

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29

30 1. Introduction

31 Increasing energy demands and heavy consumption of nonrenewable fossil fuels

32 have emphasized the development and utilization of lignocellulosic biomass as a

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33 sustainable source for renewable energy production. In particular, conversion of

34 biomass to high-performance biofuels (e.g. bioethanol, biohydrogen and biogas) has

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35 recently received increasing interest [1]. As an agricultural biomass waste, sugarcane

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36 bagasse is abundant, inexpensive and readily available in sugarcane mills. Due to the

37 lack of cost-effective treatment and recycling approaches, most straws are burned or

38
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discarded in the field. As a consequence, environmental pollution, especially high levels

39 of particulate material (i.e. PM2.5), has become a serious issue associated with bagasse
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40 disposal [2].
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41 Sugar and ethanol production is an important sector of the Brazilian economy, and
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42 Brazil is the leading worldwide sugarcane producer. For example, the 2013-2014
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43 sugarcane harvest produced 653 million tons, from which 27 billion liters of ethanol and
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44 37 million tons of sugar were obtained [3]. Sugarcane processing generates a large

45 amount of residue. Sugar mills generate approximately 270–280 kg of 50% moisture

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46 content bagasse per metric ton of processed sugarcane, representing an annual

47 production of approximately 200 million tons of bagasse. Sugarcane bagasse

48 composition is reported as: 19–24% lignin, 27–32% hemicellulose, 32–44% cellulose,

49 and 4.5–9% ash [4]. The potential to convert bagasse cellulosic and hemicellulosic

50 fractions to fermentable sugars has been investigated by several research groups [5-7].

51 However, process optimization is still needed before second generation bioethanol can

Page 3 of 42
52 be produced at an industrial level. The difficulty is production of simple, fermentable

53 sugars from the bagasse, since breaking down the lignocellulosic complex is usually

54 achieved by energy intensive conversion (hydrolysis) of the cellulose and hemicellulose

55 components of the biomass into sugars.

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56 Classical technologies employed for the hydrolysis of feedstock into fermentable

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57 sugars usually rely on acid, alkaline or enzymatic catalysts [8-10]. Unfortunately, all

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58 these technologies have drawbacks. Acid and alkali hydrolysis are corrosive, use toxic

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59 solvents, and require neutralization of the medium after the reaction, generating solid

60 wastes [11]. The main drawbacks associated with enzymatic hydrolysis are the long

61

62
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reaction time needed to achieve full hydrolysis, the cost of the enzymes, and the

quantities of enzymes required.

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63 Subcritical water , also known as hydrothermal water, liquid hot water, and hot-

64 compressed water, is defined as hot water at temperature ranging between 100 and 374
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65 °C and maintained under suffiently high pressures to maintain water in its liquid state.
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66 Subcritical water has attracted considerable attention due to its unique and temperature-

67 tunable properties of density, viscosity, dielectric constant, ionic product, diffusivity,

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68 electric conductance, and solvent ability [12]. Moreover, subcritical water is an

69 environmentally innocuous, non-toxic, and safe solvent.

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70 Subcritical water hydrolysis (SWH), also termed hydrothermal liquefaction,

71 hydrothermolysis, and aquathermolysis, has potential for breaking down the cellulose

72 and hemicellulose biopolymers into simple sugars and small molecules for downstream

73 fermentation or gasification. High density of liquid water held at high temperatures

74 promotes the hydrolysis reactions required for conversion of biomass biopolymers into

Page 4 of 42
75 simple sugars. Using water as the reactant reduces some of the drawbacks of

76 conventional methods. Compared to acid, alkali, or enzyme hydrolysis, the main

77 advantages of SWH are that it requires minimal biomass pretreatment (milling, drying,

78 etc.), generates less waste and fewer degradation products, requires shorter reaction

79 times, has fewer corrosion problems, and requires no toxic or expensive solvents.

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80 Given its importance in Brazil and elsewhere, the focus of this work was

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81 valorization of sugarcane bagasse using SWH. We envision SWH as the first stage of a

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82 process to produce reducing sugars that then, on a second stage, could be gasified to

83 generate an energy rich gas. This approach overcomes issues with the continuous feed

84

85
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of solids to the high-temperature and high-pressure gasification reactor. The SWH

reaction can be performed semi-continuously in several parallel reactors to simulate a

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86 continuous flow to the gasifier. The objective of the study was to characterize the

87 composition profile of the liquid hydrolysis products including measurement of liquid-

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88 phase pH and total reducing sugars (TRS). In particular, two reactor protocol were
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89 examined: 1) semi-continuous SWH and 2) semi-continuous SWH coupled with a static

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90 “holding” period. To understand more fully the dynamics of the decomposition process,
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91 we analyzed the solid residues using field emission scanning electron microscopy

92 (FESEM) to investigate cell wall disruption that occurs during SWH and Diffuse
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93 Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) to study composition

94 changes.

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 95 2. Materials and Methods

96 2.1. Raw Material

97 Dry sugarcane bagasse was donated by the Brazilian Bioethanol Science and

98 Technology Laboratory (CTBE, Campinas, SP, Brazil). The material was divided into 6

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99 to 8 parts with internal dimensions of 0.2 x 0.3 m. The thickness of the particles was

100 kept as uniform as possible, with an average size of about 0.005 m. They were extracted

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101 3 servings of each tray; the process was repeated until sufficient mass was obtained for

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102 carrying out the sieving assays considering a mass from 0.020 to 0.030 kg.

103 Approximately 20 to 30 grams of bagasse were prepared using this protocol, sieved, and

104
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the fraction passing a 96 mesh sieve tray was retained. The bagasse was then stored at

105 −18 °C prior to further experiments. Distilled water was used in all experiments.
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106 2.2. Sugarcane bagasse characterization
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107 The sugarcane bagasse was characterized in triplicate according to the

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108 methodologies recommended by National Renewable Energy Laboratory, biomass

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109 laboratory analytical procedure (described in technical report- NREL/TP-510-42618-

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110 42619-42621-42622-42625). Table 1 presents result of CHNS analysis and

111 characterization data of sugarcane bagasse.

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112 2.3. Experimental pilot-plant for hydrolysis and gasification in sub/supercritical

113 water

114 A schematic diagram of the semi-continuous pilot-plant is shown in Figure 1.The

115 pilot-plant was built to hydrolyze and gasify lignocellulosic biomasses using either sub-

116 or supercritical water. The apparatus consists of 2 reactors, termed “reactor 1” (with an

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117 internal volume of 110 mL) and “reactor 2” (790 mL). The maximum working

118 conditions of reactor 1 are 300 °C and 42 MPa and for reactor 2 they are 600 °C and 42

119 MPa. Reactor 1 is constructed of 316 stainless-steel and capped with gasket filters

120 (average pore size 300µm) to retain sample particles. Reactor 1 is heated by an electric

121 heating system type jacket (1500 W) and insulated by ceramic fiber. Reactor 2

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122 (stainless-steel 316; 220 mm e.d × 610 mm length) is a serpentine (4.5 meters of length,

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123 spiral shape) tube. Inconel 625 was used as the material of construction; this material is

124 commonly used in supercritical water gasification (SCWG) [13-15] Inconel material

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125 contains nickel as the major component, which is known to be a good catalyst for

126 methanation [16, 17]. Reactor 2 is heated by an electric heating system with three

127
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resistors 1500 W each, located on the top, bottom and center of the vessel and insulated
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128 by ceramic fiber.

129 A liquid high pressure pump (Dual piston pump, model Prep 36 Pump, Apple
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130 Valley, MN, USA) was used to deliver water to the reactors. The pressure in the system
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131 was controlled by a back-pressure regulator (High-Pressure Piston-Sensing Back-

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132 Pressure Regulators, KHB Series, Lafayette, LA) and measured by two manometers.
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133 The reaction temperature was monitored by thermocouple (type K) outlet of the reactor

134 1 and reactor 2. The hot effluent fluid exiting the reactor 1 was cooled to a temperature
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135 less than 30 °C in a stainless steel shell-and-tube heat exchanger coupled to a

136 thermostatic bath (Marconi, model MA-184, Piracicaba, SP, Brazil) operating at -10 °C

137 (using Ethylene glycol 50% as the coolant.) The rapid cooling assures that the reaction

138 is quenched after reaction. The liquid products were collected in a gas-liquid separator.

139 The flow of gas products was measured with a mass flowmeter (Aalborg, model # GFM

Page 7 of 42
140 17, 0-100 mL min-1) and then collected in a stainless steel gas collector with a safety

141 valve and volume of 300 mL.

142 2.4. Hydrolysis apparatus and protocol

143 To validate the new pilot-plant, trial runs were performed at 2.5 MPa and 100 °C;

t
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144 the reaction pressure was kept sufficiently high to prevent formation of a water vapor.

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145 Hydrolysis tests were performed following 2 procedures. In the first step, either 3 g

146 or 5 g of raw material were used and the reactor pressure was set to either 5 MPa or 10

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147 MPa by introducing pressurized water into the system. Complete process conditions are

148

149
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shown in table 2. Prior to hydrolysis, distilled water was delivered to the system at room

temperature to remove air and water-soluble substances. The dynamic period of the
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150 process was started by pressurizing the system and pumping water at the set-point flow

151 rate (9 mL min-1) through the system for 22 min. The temperature at the exit the reactor
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152 1 was set at process temperature (100 °C, 150, 200 °C, and250 °C). Temperature
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153 stabilized after about 10-20 min, and time required to reach temperature stability is
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154 termed the “preheating time”. To assure that there was no hydrolysis of hemicellulose
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155 before reaction commenced; one sample was collected and analyzed during the

156 preheating time. Once the stable operating temperature had been reached, liquid
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157 hydrolysate samples were collected every 2 min for the remainder of the experiment.

158 In the second step of experiments, the amount of raw material was maintained

159 constant, at 5 g and the pressure was set at either 10 MPa and 15 MPa. Prior to

160 hydrolysis, distilled water was delivered to the system at room temperature to remove

161 the air and water-soluble substances. Once the system was pressurized, the pump was

162 stopped, which we called “static time” ; the reaction temperature was increased to the

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163 set point (150°C, 200 °C) and the dynamic period was started by initiating flow (12.5

164 mL min-1, measured at the pump head at laboratory conditions) through the reactor for

165 20 min. The reaction temperature was taken at a thermocouple positioned at the reactor

166 outlet, and the pressure was taken at several locations in the system. At the start of the

167 dynamic process, the temperature required approximately 15-18 min to stabilize. During

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168 the run, hydrolysate samples were collected every 2 min. Table 3 summarizes the

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169 reactor conditions tested during this study.

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170 All experiments were performed in duplicate, and yields are reported as average

171 values, in wet basis, throughout the text. The fluid residence time ( ) was estimated by:

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Equation 1
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172 Where VR is the reactor fluid volume (m3), o is the volumetric liquid flow rate (25
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173 °C), is the density of the liquid feed (distilled water: 1080 kg/m3), and is the
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174 density of the fluid at the reactor 1 temperature and pressure estimated using NIST
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175 steam tables [18]. Equation 1 assumes the fluid stream is at the reactor set-point

176 temperature and pressure and does not account for density changes associated with
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177 temperature non-uniformity inside the reactor. The presence of SWH decomposition

178 products in the reaction mixture was not factored into the residence time calculation, but

179 since more fermentable sugars products are expected to be formed compared to the

180 amount of water consumed, the residence time calculated by Equation 1 could vary

181 from seconds to minutes depending on the pump flow rate.

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182 The severity factor (R0), first proposed by Overend and Chornet [19] and Chornet

183 and Overend [20], has been useful for modeling pseudo first order reaction rates as the

184 combined influence of of temperature and residence time .

Equation 2

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185 Where t is the resident time (min), T is temperature (°C), 100 is the temperature of

186 reference and 14.74 is an empirical parameter related with activation energy, assuming

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187 pseudo first order kinetics. The results are usually represented as a function of log (R0)

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188 2.5. Hydrolyzate analysis

189 2.5.1. Determination of pH

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190 The pH of the hydrolysates was determined using a digital pHmeter (Digimed,
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191 model DM-22, Santo Amaro, Brazil).
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192 2.5.2. Total reducing sugar determination

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193 The TRS content of the hydrolyzate, was determined by the colorimetric method
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194 proposed by Somogyi-Nelson (SN) [21, 22]. Two reactant solutions were prepared, SN-

195 I and SN-II. The hydrolysate was subjected to acid hydrolysis to decompose sugar
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196 oligomers into monomers prior to detection as TRS. After the coloring reaction, sample
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197 absorbance at 540 nm was measured using a spectrophotometer (Hach, modelo

198 DR/4000U, São Paulo, Brazil). The concentration of TRS was calculated using an

199 external calibration curve based on standard glucose solutions (20 mg/L, 60 mg/L, 100

200 mg/L, 140 mg/L, 200 mg/L, 300 mg/L and 400 mg/L), and expressed as glucose

201 equivalents [5]. In some cases, hydrolysis product mixtures were diluted with distilled

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202 water prior to the absorbance measurement to remain within the instrumental calibration

203 range.

204 2.6. Statistical analysis

205 The variance (ANOVA) of the results was evaluated using the software Statistica

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206 for Windows 6.0 (Statsoft Inc., USA). The significant differences at level of 5% (p ≤

207 0.05) were analyzed through the Tukey’s test.

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208 2.7. The solid residue

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209 After SWH, the residual solids contained in reactor 1 were transferred to a Petri

210 an
dish, taking particular care to prevent loss. The amount of solid residue was determined

211 by the difference between the initial amount of raw materials and the final residue
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212 remaining in the reactor at the end of the process, using a digital analytical balance

213 (Mettler Toledo, model: AB204, Switzerland).

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214 2.7.1. Field emission scanning electron microscopy (FESEM)

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215 The microstructure of the surface of sugarcane bagasse was analyzed before and

216 after subcritical water hydrolysis (100 °C and 150 °C). The FESEM equipment was a
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217 scanning electron microscope, equipped with a field emission gun (Quanta 650, FEI,
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218 Hillsboro, Oregon, USA). Prior to analysis, the samples were coated with gold in a SCD

219 050 splutter coater (Oerlikon-Balzers, Balzers, Liechtenstein). Both equipment were

220 available at the National Laboratory of Nanotechnology (LNNano), located in

221 Campinas-SP, Brazil. Analyses of the sample surfaces were performed under vacuum,

222 using a 5 kV acceleration voltage.

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223 2.7.2. Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS)

224 Infrared spectra of sugarcane bagasse residues were collected using a Thermo

225 Nicolet Magna 560 equipped with a SpectraTech DRIFTS cell. Spectra were obtained

226 over the range from 4000 to 400 cm-1, at a resolution of 2 cm-1 and an accumulation of

227 96 scans. Multiple scans were obtained for each sample and averaged spectra are

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228 presented here. The DRIFTS cell was continuously purged with dry nitrogen 10 minutes

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229 prior to measurement to remove atmospheric CO2 and H2O that would interfere with the

230 spectrum.

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231 3. Results and discussions ,

232 3.1. Total reducing sugars yield

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233 Literature [23-25] indicates that reaction temperature is the most important process

234 parameter for SWH, with increasing temperature favoring biopolymer degradation at
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235 the cost of reduced recovery of TRS due to sugar decomposition. Accordingly, we
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236 focused our attention on studying the effects of SWH on TRS yield, with secondary
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237 experiments performed to investigate the influence of reaction pressure.

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238 The TRS yields obtained at different conditions of temperature and pressure are

239 shown in Table 4. A statistical analysis of variance (p < 0.05) was performed to evaluate
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240 the effect of temperature andpressure on TRS yield for step 1. The Tukey test showed

241 that of the highest yield was obtained at 200 °C, and the yields of these operation

242 conditions were statistically different (p ≤ 0.05) compared to those obtained at 5

243 MPa/200 °C and 10 MPa/150 °C. The lowest yield was achieved at 10 MPa and 100 °C,

244 consistent with the low hydrolysis rates expected at this low reaction temperature [26].

245 TRS yield (g glucose/100 g carbohydrate) increased from 100 °C (0.22 %) to 150 °C

Page 12 of 42
246 (3.32 %) to 200 °C until 250 °C (13.01 %) for all pressures tested here [25]. This

247 behavior can be explained by the effects of temperature on hydrolysis rates.

248 Hemicellulose is hydrolyzed at 190–230 °C [27, 28] while modestcellulose hydrolysis

249 takes place below 230 °C[29, 30].

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250 3.2. In general, reaction pressure used for SWH has not been shown to be a key

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251 parameter for determining TRS yield[31, 32]. Consistent with the literature,

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252 pressure had an insignificant effect on the TRS yield, for the conditions

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253 examined here Effects of temperatures on total reducing sugar yields from

254 sugarcane bagasse by Subcritical water hydrolysis

255
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Figure 2 shows that the yield of TRS increases as the temperature increases. As

256 expected [26, 30, 40, 41], negligible sugar yields are obtained at 100 °C and 150 °C.
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257 This is consistent with the literature data which indicate that the hydrolysis of

258 hemicellulose contained in most biomass types does not begin undergoing hydrolysis
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259 until the temperature exceeds 170 °C. The TRS yields reached were 9.15% and 13% at
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260 200 °C and 250 °C, respectively. The increasing sugar yield obtained with increasing

261 temperature can be attributed primarily to thermal effects on hydrolysis kinetics [42, 43]
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262 and secondarily to the increased self-ionization constant of water. The increased self-

263 ionization constant leads to increased concentrations of both H+ and OH in the reaction
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264 mixture, thereby facilitating the acid-catalyzed hydrolysis of cellulose and

265 hemicellulose [44].

266 The highest TRS yields were obtained in step 2, as shown in Figure 3. TRS yields

267 reached 11% and 15.5% after 20 min held at 150 °C and 200 °C, respectively. In the

268 second set of experiments, the reactor 1 jacket (reactor 1 outlet) temperature was

Page 13 of 42
269 adjusted to 150 °C and 200 °C; the dynamic period of the process was started by

270 pumping water at the experimental flow rate through the system for 20 min. After the

271 start of the dynamic period, the temperature at reactor 1 exit slowly increases until

272 stabilization after 15-18 min; the results are expressed throughout the text as the reactor

273 outlet temperature. Figure 3 also shows a slight increase of the TRS yield with

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274 increasing water flow rate (12 mL) as compared with step 1 (9 mL). This is probably

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275 due to the lower residence time in reactor 1, which decreases the time for sugars

276 degradation [5, 45]. Similar behavior was observed during the subcritical water

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277 hydrolysis of corn stover and Citrus junos peel under semi-batch conditions [33, 46,

278 47].

279 3.3. pH of the aqueous solution

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280 Figure 4 shows that the pH of the hydrolyzate recovered after subcritical water

281 reaction decreased as hydrolysis temperature increased, for step 1; obviously,

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282 decreasing pH indicates the presence of acidic materials, probably organic acids in the
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283 aqueous hydrolyzate solution. These organic acids provide acidic protons to catalyze

284 subsequent hydrolysis of monomers and oligomers as an autocatalytic process [42].

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285 Degradation products typically found in the hydrolyzates include acetaldehyde, acetic

286 acid, hydroxyacetone, acetone, acetonylacetone, 2-acetylfuran, acrylic acid, 1,2,4-

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287 benzenetriol, dihydroxyacetone, erythrose, formic acid, furfural, glyceraldehydes,

288 glycolaldehyde, glycolic acid, 5-hydroxymethyl furfural (5-HMF), lactic acid, levulinic

289 acid, levoglucosan, 5-methyl furfural, pyruvaldehyde, solid precipitate (‘‘humic solid’’)

290 and gaseous products [48]. The degradation of monosaccharides into small molecules

291 (e.g. 5-hydroxymethylfurfural (C6H6O3) and furfural (C5H4O2)) proceeds with the

292 stoichiometry shown in the following equations:

Page 14 of 42
 equation 3

equation 4

293 Due to organic acid formation, the pH of the hydrolyzate decreases strongly with

294 increasing temperature and slightly with increasing hydrolysis time, as shown in Figure

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295 4. The trend can be explained because the operational conditions required to break down

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296 the lignocellulosic complex are severe enough to promote the degradation of

297 fermentable sugars simultaneously with the hydrolytic process [5]. Also, degradation of

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298 hemicellulose release acetic acid due to hydrolysis of acetyl side chains. A greater drop

299

300
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in hydrolyzate pH is observed at higher temperatures, consistent with the presence of

higher concentrations of organic acids in the hydrolyzate at these conditions. In terms of

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301 dynamics, the pH of aqueous solution decreases sharply during the first 10 min of

302 hydrolysis and then levels off. The minimum pH achieved here was 3.43, observed for a
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303 hydrolysis temperature of 250 °C [42, 49, 50]. The weakly acidic hydrolyzate pH shown
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304 in Figure 4 for temperatures of 100 °C and 150 °C is consistent with the observation
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305 that these lower temperatures are insufficient for hydrolysis of the lignocellulosic
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306 complex.

307 3.4. Residual solid

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308 The amount of residual solid after steps 1 and 2 of subcritical water treatment was

309 quantified and analyzed. Visually, the residue appears to consist of un-reacted

310 sugarcane bagasse, carbonized sugarcane bagasse, hydrolyzed but still insoluble parts of

311 sugarcane bagasse as well as inorganic compounds. The masses of residual solids

312 remaining after steps 1 and 2 are shown in Figure 5. The amount of residual solids

313 decreased with increasing temperature as expected based on the hydrolysate

Page 15 of 42
314 measurements; by contrast, the TRS yield increased with increasing temperature. The

315 mass of residual solids produced during step 1 decreased drastically from 80% (at 100

316 °C) to 20% (at 250 °C), which parallels the increase in TRS yield shown in Figure 2.

317 The sharp decrease in solids yield is consistent with the ability of subcritical water to

318 decompose insoluble macromolecules of sugarcane bagasse into smaller soluble

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319 compounds in a short residence time [42]. Figure 5 also shows that the mass of residual

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320 solid remaining at the end of step 2 was approximately the same as the one found after

321 step 1, and accounted for approximately 45% of the initial raw material. For sugarcane

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322 bagasse semi-continuous hydrolysis at approximately 200 °C and treatment times

323 ranging from 4-15 min, solid yields ranging from about 40 to 60% have been reported

324
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[27, 28]. There was not a considerable difference between the final amounts of
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325 remaining solids obtained at temperatures of 150 °C and 200 °C following step 2 (43%

326 and 49%, respectively), which suggests that the sugarcane bagasse was not completely
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327 hydrolyzed.
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328
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329 3.4.1. Field Emission Scanning Electron Microscopy

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330
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331 The Figure 6 shows images of FESEM of sugarcane bagasse samples obtained

332 before (column A) and after SWH at 150 °C and 10 MPa (column B) and at 100 °C

333 and 10 MPa (column C) at different magnifications. The morphology of the sugarcane

334 bagasse residue was clearly influenced by the SWH treatment. The surface of the non-

335 treated bagasse presents a morphology is consistent with the native sugarcane cell wall,

336 with visible parallel conducting vessels (row 1-A). After SWH, the bagasse particles

Page 16 of 42
337 appear twisted and disrupted (row 1-B and 1-C). At higher magnification (rows 2 and

338 3), the relatively flat surface observed in the untreated sample appear tobecome more

339 degraded and covered by particulate material. Interestingly, hydrolysis at 150 °C

340 disrupted the lignocellulosic structure more extensively than the treatment at 100 °C,

341 despite the fact that the TRS yield did not increase between 100 and 150 °C. We

t
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342 interpret this observation as suggesting that treatment at 150 °C is sufficient for

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343 disrupting the physical structure of sugarcane bagasse but that the temperature is

344 insufficient for bond hydrolysis at the treatment times studied here.

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345 3.4.2. Diffuse Reflectance Infrared Fourier Transform Spectroscopy

346

347
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The chemical composition of the residual solids was characterized for functional

group content using DRIFTS. The aim of the DRIFTS characterization tests was to
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348 corroborate the TRS yield and pH data and to gain understanding of the composition of

349 the residual bagasse. Figure 7 shows the spectral region from 1650 to 1400 cm-1 that has
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350 been associated primarily with the aromatic C=C bonds present in lignin [30, 51].
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351 Starting at a hydrolysis temperature of 100 °C, bands at 1600 and 1510 cm-1 start
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352 becoming more prominent and distinct. Likewise, minor bands at 1460 and 1430 cm-1,
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353 associated with aromatic C-H bends [30, 51], become more prominent with increasing

354 hydrolysis temperature. Interestingly, the lignin features become less distinct at
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355 hydrolysis temperature of 200 and 250 °C, which may be attributable to deposition of

356 secondary products on the lignin surface.

357 Figure 8 shows the spectral region from 1800 to 1600 cm-1 that is well known to

358 contain information about carbonyls [52]. Here, the carbonyl stretch, originally present

359 as a strong band at approximately 1740 cm-1 decreases in intensity and shifts to lower

360 wavenumbers with increasing hydrolysis temperature. These observations are consistent

Page 17 of 42
361 with hydrolytic stripping of acetyl sidechains present on hemicellulose, preferentially

362 leaving behind primarily ketones and aldehyde groups present in lignin [51]. This

363 should be connected back to the observed drop in pH of the hydrolysate as the acetyl

364 sidechains produce acetic acid, one of the main acids responsible for H+.

t
365 Figure 9 shows the spectral region from 3100 to 2700 cm-1, a region typically

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366 associated with C-H stretching modes [52]. Following assignments from Russo,

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367 Stanzione, Tregrossi and Ciajolo [53], we can assign the individual bands associated

us
368 with CH, CH2, and CH3 stretches; in all cases, the C-H stretching peaksbecome more

369 distinct with increasing hydrolysis temperature, consistent with removal of

370

371
an
hemicellulose and possibly cellulose components that leaves a less complex mixture of

C-H bonds in the residual solid. The aromatic C-H stretch typically present at
M
372 approximately 3040 cm-1 [53] is not observed in any of the samples, and we attribute

373 this to the presence of the broad O-H stretch which obscures the aromatic C-H stretch.
d

Finally, Figure 10 shows the spectral region from 1400 to 800 cm-1 that is
e

374
pt

375 associated with C-O stretches [52] and other important biomass functional groups [30,

376 51, 54]. Again, the individual bands associated with C-O-C linkages at 1160 cm-1 [30]
ce

377 and 1115 cm-1 [55] become more distinct with increasing temperature, consistent with

378 hydrolytic removal of hemicellulose and possibly amorphous cellulose components.

Ac

379 The persistence of the band at 900 cm-1, associated with C-H deformation in cellulose

380 [30], and the bands at approximately 840 cm-1, associated with D-glycosidic bonds [54],

381 suggests that hydrolysis does not remove all cellulose components; based on its known

382 recalcitrance, we suggest that the residual cellulose is primarily crystalline.

383

Page 18 of 42
384 3.5. Severity factor

385 The severity factor was calculated from Equation 2 for step 1. Figure 11 shows the

386 variation in the yields of TRS with log(R0). The single step 1 pretreatment was

387 conducted at four different temperatures ranging from 100 to 250 °C, corresponding to

t
388 severity factors ranging from 1.04 to 5.41. Under moderate conditions, SWH produces

ip
389 long-chain oligomers, which are reduced in length by increasing the severity [40, 50-52].

cr
390 The treatment performed at 200 °C/5 MPa showed severity of 3.9 corroborating

us
391 literature similar data for severity factor in those conditions [27, 53]. The treatment at

392 250 °C showed the highest value of severity 5.9, moreover, the high temperature

393

394
an
collapsed the lignocellulosic structure; the recovery of cellulose generally decreases

with increasing severity [54].

M
395 The severity factor described in the Equation 2 can only be used if the temperature

396 remains constant during the reaction; in the case of the step 2 significant heat-up times
e d

397 were noticed, a modified severity factor was used, which considers integrating the
pt

398 temperature profile during heat up.

ce

Equation 5
Ac

399 where all symbols retain their previous definitions [55].

400 Figure 11 also shows the variation in the yields of TRS with log(R0) for step 2,

401 which makes more obvious that log (R0) does not capture the differences between the

402 two methods. Maybe this is as a result of the severity model used does not include the

403 pH in the equation.

Page 19 of 42
404 Sugar degradation increases with increasing severity factor. Therefore, balancing

405 between hydrolysis efficiency and sugars yield is necessary. When the aim of SWH is to

406 produce fermentable sugars, the factor severity should be decreased to avoid sugars

407 degradation, especially considering that many degradation products are fermentation

408 inhibitors. Here, we are aiming to use SWH to produce a liquid feed for gasification,

t
ip
409 shifting the objectives. Specifically, primary sugar degradation products (furfural-based

cr
410 compounds, organic acids, and similar) can be gasified under supercritical water

411 conditions. However, accumulation of high concentrations of degradation products can

us
412 lead to formation of insoluble secondary products (humins), resulting in downstream

413 operational problems (e.g., clogging of valves, etc.). Therefore, while the motivations

414
an
are different, optimization of the severity factor either as a pre-processing step for
M
415 fermentation or gasification requires balancing biomass hydrolysis with sugar

416 degradation.
d

417
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Page 20 of 42
418 4. Conclusions

419 SWH was studied as a semi-batch technology for valorizing sugarcane bagasse waste

420 biomass. The specific focus of this study was to understand the SWH process as a

421 means to produce a water-soluble carbon-rich product stream amenable for continuous

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422 production of fuel gas via supercritical gasification. The proposed SWH-gasification

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423 technology reduces problems associated with solids feeding into high-temperature/high-

cr
424 pressure gasifier reactors; instead, the solids can be fed under semi-batch conditions into

us
425 the SWH reactor and several SWH reactors can be operated in parallel to simulate

426 continuous feed to a single gasifier.

427
an
A key design challenge is determining the operating conditions of the SWH

428 reactors and the number/size of the SWH reactors required to support the gasifier.
M
429 Accordingly, the studied included examinations of the effects of water flow rates,

430 dynamic temperature profiles, and biomass holding times on TRS yields, hydrolysate
e d

431 pH, and composition of the residual solids. In addition, we tested two reactor protocols,
pt

432 one consisting entirely of dynamic flow, the other consisting of a static period followed

433 by flow. In both cases, we found that increasing the SWH temperature from 100 to 250
ce

434 °C increased the measured TRS yield. For the 1-step process, the highest TRS yield

435 was obtained as 13% after 20 minutes at 250 °C. For the 2-step process, the highest
Ac

436 TRS yield was 15.9%, again after 20 minutes of flow conditions but at 200 °C (the

437 highest temperature examined). The increased TRS yields obtained for the 2-step

438 process were consistent with lower amounts of sugar degradation, as measurements of

439 the residual solid mass indicated that the 1-step process hydrolyzed a larger fraction of

440 the biomass than did the 2-step process. In all cases, hydrolyze pH decreased to a

441 minimum value <4, an important consideration for corrosion-resistant design of

Page 21 of 42
442 downstream process components. This point should be made earlier in the text, as I

443 think it’s probably important.

444 The residual biomass was characterized using electron microscopy and DRIFTS.

445 These tests confirmed structural disruption of the biomass and indicated that SWH

t
446 treatment achieved hemicellulose and (potentially) partial cellulose hydrolysis; bands

ip
447 associated with lignin did not disappear during SWH treatment, consistent with their

cr
448 known stability at the conditions examined here.

us
449 Several different severity factors were used to interpret the SWH data. A particular

450 emphasis was placed at identifying a severity factor to capture TRS yields obtained

451
an
using the two different SWH protocols [Now, I’m making things up…but, I guess it will

452 go something like this.] A simple severity factor calculated strictly based on
M
453 temperature considerations successfully captured the TRS yields observed for either the

454 1-step or 2-step process; however, the simple severity factor did not reconcile the higher
e d

455 TRS yield obtained for the 2-step process. Inclusion of a pH correction term in the
pt

456 severity factor improved its ability to simultaneously capture the performance observed

457 for the two SWH protocols. Further improvements might be possible if the severity
ce

458 factor were modified to include information on the spatially resolved temperature

459 profile within the reactor. [this should be mentioned in the text and By the way, this
Ac

460 could be a cool experiment/analysis…]

461 Based on these analyses, we presented engineering design calculations for the

462 SWH-gasifier process design. Based on the severity analysis and TRS data, we

463 recommend using the 2-step SWH process. We estimated that loading and pre-heating

464 the SWH reactor would require XX minutes and that removing the residual solids would

Page 22 of 42
465 require YY minutes. Based on these estimates, a minimum of Z SWH reactors would

466 be required to simulate continuous flow to the gasifier. These results provide a solid

467 foundation for future work in this area.

468 The equipment for biomass hydrolysis and gasification using water

t
469 sub/supercritical has been assembled and is operating in semi-continuous mode, a great

ip
470 advantage in terms of efficiency compared to conventional processes, it allows

cr
471 integrating in the same device two processes, hydrolysis and gasification; the use of

us
472 subcritical water in processing biomass proves to be interesting, because it offers the

473 possibility of tuning the medium properties just by changing temperature and/or

474

475
an
pressure. The reactions of sugarcane bagasse hydrolysis were highly influenced by

temperature. Nevertheless, in general, pressure has a slight effect on subcritical water

M
476 hydrolysis. Maximum TRS recovered for sugarcane bagasse using subcritical water

477 hydrolysis was 15.5% from total raw material to 250 °C e 15 MPa. To summarize the
d

478 results, DRIFTS spectra are consistent with progressive removal of hemicellulose and
e

479 possibly cellulosic sugarcane bagasse components, leaving behind primarily lignin
pt

480 components.
ce

481 5. Acknowledgments
Ac

482 The authors acknowledge the financial support from Fundação de Amparo à

483 Pesquisa do Estado de São Paulo – FAPESP (2011/19817-1) and the

484 LME/LNNano/CNPEM for technical support during the electron microscopy analysis.

485

Page 23 of 42
485

486 6. References

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548 [25] F.P. Cardenas-Toro, T. Forster-Carneiro, M.A. Rostagno, A.J. Petenate, F. Maugeri Filho,

ip
549 M.A.A. Meireles, Integrated supercritical fluid extraction and subcritical water hydrolysis for the
550 recovery of bioactive compounds from pressed palm fiber, The Journal of Supercritical Fluids,
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552 [26] G. Garrote, H. Dominguez, J. Parajo, Hydrothermal processing of lignocellulosic materials,
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555 compressed liquid water, Industrial & Engineering Chemistry Research, 31 (1992) 1157-1161.
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564 processes, The Journal of Supercritical Fluids, 47 (2009) 373-381.
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568 total mass removal from corn stover, Industrial & Engineering Chemistry Research, 42 (2003)
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576 pretreatment of corn stover, Bioresource technology, 93 (2004) 217-226.

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580 [38] M.Ø. Petersen, J. Larsen, M.H. Thomsen, Optimization of hydrothermal pretreatment of
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597 [45] J.M. Prado, T. Forster-Carneiro, M.A. Rostagno, L.A. Follegatti-Romero, F. Maugeri Filho,
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607 [49] O. Pourali, F.S. Asghari, H. Yoshida, Sub-critical water treatment of rice bran to produce
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cr
610 d-Fructose in Subcritical Water, Industrial & Engineering Chemistry Research, 45 (2006) 2163-
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622 (2006) 253-261.
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626 (2010) 618-626.

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627
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Page 26 of 42
*Graphical Abstract

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Page 27 of 42
*Highlights

Highlight

Subcritical water hydrolysis was effective to recover sugars from sugarcane bagasse.

Reaction temperature showed an important effect on total reducing sugars yield.

The highest yield (15.5%) was obtained at 200 °C and 12.5 mL min-1

Disrupts the cell walls (high temperature) were confirmed by FESEM analyses.

DRIFTS of the residual solids confirmed the removal of hemicellulose during hydrolysis.

t
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Page 28 of 42
Figure 1

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1
2 Figure 1. Schematic diagram of experimental apparatus

Page 29 of 42
Figure 2

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1
2 Figure 2. Effects of reaction temperatures on total reducing sugar yields from
ed

3 sugarcane bagasse by subcritical water hydrolysis, step 1.

4
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5
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1
Page 30 of 42
Figure 3

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Figure 3. Effects of reaction temperatures on total reducing sugar yields from
sugarcane bagasse by subcritical water hydrolysis, step 2.
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Page 31 of 42
Figure 4

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Figure 4. The pH of the hydrolysates of sugarcane bagasse obtained by subcritical
water hydrolysis at different temperatures, step 1.
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Page 32 of 42
Figure 5

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Figure 5. Solid residue left inside the reactor after the subcritical water hydrolysis
of sugarcane bagasse
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Page 33 of 42
Figure 6

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ed

Figure 6. Images obtained by FESEM on the surface of sugarcane bagasse

analyzed before (column A) and after the subcritical water hydrolysis at 150 °C
pt

and 10 MPa (column B) and at 100 °C and 10 MPa (column C) at different

magnifications. Scale bar: 200 μm (row 1); 10 μm (row 2); 3 μm (row 3). .
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Page 34 of 42
Figure 7

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Figure 7. DRIFT spectra analysis of sugarcane bagasse in the aromatic C=C

stretching region (1650-1400 cm-1)
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Page 35 of 42
Figure 8

0.8

0.6 C=O
250 ºC
0.4

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ip
Intensity

0.2 200 ºC

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0.0 150 ºC

-0.2 an 100 ºC
M
-0.4 bagasse
ed

-0.6
1800 1750 1700 1650 1600
pt

-1
wavenumber (cm )
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Figure 8. DRIFT spectra analysis of sugarcane bagasse in the carbonyl C=O

stretching region (1800-1600 cm-1)
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Page 36 of 42
Figure 9

1.0

CH2 (sym)
CH3 (sym)
CH2 (asym)
0.8

CH
250 ºC
0.6

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200 ºC
Intensity

0.4

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150 ºC

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0.2
100 ºC

0.0
an bagasse
M
-0.2
ed

3100 3000 2900 2800 2700

-1
pt

wavenumber (cm )
Figure 9. DRIFT spectra analysis of sugarcane bagasse in the C-H stretching
ce

region (3100-2700 cm-1)

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Page 37 of 42
Figure 10

0.3 C-H
C-H bends C-O & C-O-C (cellulose)
(lignin)

0.2

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Intensity

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0.1

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0.0
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ed

-0.1
1400 1200 1000 800
pt

-1
wavenumber (cm )
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Figure 10. DRIFT spectra analysis of sugarcane bagasse C-O and other important
biomass functional groups stretching region (1400-800 cm-1)
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Page 38 of 42
Table 1

Table 1. Proximate analysis and elemental analysis of sugarcane bagasse

Composition
Proximate analysis %wb
Moisture 9.136 ± 0.529
Ashes 2.023 ± 0.069
Extractives in water 4.110 ± 0.466
Extractives in ethanol 1.640 ± 0.165
Protein 1.530 ± 0.092
Acid soluble in lignin 3.513 ± 0.224

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Acid insoluble in lignin 23.456 ± 0.620
Elemental analysis %wb
Carbon 43.1 ± 0.1

cr
Hydrogen 5.7 ± 0.6
Nitrogen 0.51 ± 0.02

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Oxygen (by difference) 50.69
Dados are expressed as mean ± standard deviation (n=3)

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Page 39 of 42
Table 2

Table 2. Reactor conditions of sugarcane bagasse hydrolysis with subcritical water.

Step 1
Units
1 2 3 4
Mass (g) 5 5 3 3
Reactor outlet temperature (°C) 100 150 200 250
Pressure (MPa) 10 10 5 5
Water flow rate (mL min-1) 9 9 9 9

t
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Process time (min) 22 22 22 22
Water density (kg/m3) 963 922.4 867.4 800.3

cr
Water residence time in the reactor (min) 10.9 10.4 9,8 9

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Page 40 of 42
Table 3

Table 3. Reactor conditions of sugarcane bagasse hydrolysis with subcritical water.

Step 2
Units
1 2
Mass (g) 5 5
Reactor outlet temperature (°C) 150 200
Pressure (MPa) 10 15
Water flow rate (mL min-1) 12.5 12.5

t
ip
Process time (min) 20 20
Water density (kg/m3) 922.4 874,7

cr
Water residence time in the reactor (min) 7.5 7.1

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Page 41 of 42
Table 4

Table 4. Total reducing sugar yield obtained by SWH in different conditions from
sugarcane bagasse.
Temperature (°C) Pressure (MPa) TRS (%)
100 10 0.219±0.040a
150 10 3.318±0.008b
Step 1
200 5 9.2±1b
250 5 13.0±0.4c
150 10 11.0±0.4
Step 2
200 15 15.5±1.0
Results are expressed as mean ± standard deviation of the analysis. Different letters in the same

t
column indicate that the means differ significantly by Tukey test (p≤0.05).

ip
cr
us
an
M
ed
pt
ce
Ac

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